InteractiveFly: GeneBrief

Nitric oxide synthase: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Nitric oxide (NO) is a diffusible, free-radical gas that serves as a multifunctional messenger affecting many diverse aspects of mammalian physiology, such as regulation of vascular tone, macrophage-mediated cytotoxicity, and cell-cell interactions in the nervous system. For example, during the development of the rat central nervous system and the process of neuronal cell differentiation, NO carries messages crucial to synaptogenesis and apoptosis. Nitric oxide synthase (NOS), the enzyme that produces NO, is expressed transiently in the developing rat brain, as one might expect, given its role in neural development (Bredt, 1994). NOS has been found to trigger the switch to the state of growth arrest that occurs during the differentiation of neuronal cells (Peunova, 1995). NO also appears to be involved in long-term potentiation in hippocampal tissue, according to an experimental model that posits neural plasticity in the learning process (Arancio, 1996).

NOS catalyzes the formation of nitric oxide from the terminal guanidino nitrogen of arginine, with the consequent production of citrulline. There are several homologous but separate NOS genes in mammals. Endothelial NOS and neuronal NOS enzymes are produced constitutively and are Calmodulin dependent, with activity regulated by calcium. The calcium dependence of nNOS suggests that nNOS is activated by neural activity. Macrophage NOS is expressed at low levels under basal conditions, is induced by gamma-interferon and lipopolysaccharide, and does not depend on calcium for activity. Inducible NOS is an important component of a host's resistence to infection, especially to parasites such as malaria.

What is the target of NO in signal transduction? NO directly activates the soluble form of guanylate cyclase, the heme protein that catalyzes the formation of cyclic GMP. In addition, NO binds with high affinity to heme moieties in other proteins. NO also affects enzymes that contain iron-sulfur groups, thus affecting iron metabolism. The immediate target of cyclic GMP is PKG (cGMP-dependent protein kinase), known to be the mediator of smooth muscle relaxation and other affector functions of NO signaling (Lincoln, 1995).

What role is played in normal development by developmentally regulated expression of NOS? Drosophila NOS is expressed at high levels in developing imaginal discs. Injection of specific NOS inhibitors into developing larvae at the end of the third instar (several hours before metamorphosis), results in an enlargement of appendages and other structures of the fly body. The changes include (1) hypertrophy of the femur, tibia, and segments of the tarsus; (2) overgrowth of the tissues originating from the genital disc; (3) an increase in the overall surface of the wings; (4) overgrowth of cells of tergites and sternites; (5) hypertrophy of the humerous; (6) occasional duplications of some areas of the eye; (7) occasional malformation of genital structures, legs, and eyes; and (8) occasional ectopic fomation of misplaced body structures. The changes most often affect and are most profound in the legs of adults, where the diameter of certain segments increases 3-4 times (Kuzin, 1996).

The opposite effects occur with the ectopic expression of a mouse NOS transgene. Flies were transformed with a NOS transgene in which NOS was placed under control of a heat-shock promoter. Transgenic larvae were heat-shocked within 1 hour after pupariation to induce ectopic expression of NOS before the final cell divisions took place. This treatment results in a reduction in limb size; distal segments in legs are affected most frequently and to the greatest degree. The segments of the adult leg most often affected by the overexpression of NOS are those that are not affected by the NOS inhibitors and whose precursors exhibit particularly low levels of enzyme staining in the early prepupal stages. It is concluded that ectopic expression of NOS at the late stages of larval development results in a decrease in cell proliferation and a reduction in the size of the adult fly's structures (Kuzin, 1996).

After inhibition of NOS in the eye disc, there is a consistent increase in the number of cells in S phase, but the resultant adult eye usually appears normal. What is the basis for this paradoxical finding? It could be that the apparently normal eye phenotype occurs as a result of programmed cell death, which counteracts excessive cell proliferation induced by NOS inhibition and restores the normal number of cells in the eye during morphogenesis.

To test this hypothesis, p35, a baculovirus inhibitor of apoptosis (programmed cell death) was expressed under the control of a multimerized Glass-binding site, obtained from the Drosophila Rhodopsin1 promoter. This promoter directs expression of the transgene in all cells, both in and posterior to the morphogenetic furrow in the eye imaginal disc. When NOS is inhibited in transgenic larvae, the eyes show numerous changes, reflecting the excessive proliferation of various cell types in the developing eye. After NOS inhibition in transgenic flies, the number of ommatidia increases from a nearly invariant complement of 750 (in wild-type flies and untreated transgenic flies) to nearly 820. This, together with an elevated number of cells per ommatidium, causes an increase in the overall size of the eye. In transgenic flies, the number of secondary and tertiary pigment cells is increased from 12 to 25 cells per sample area as a result of suppressed programmed cell death. Furthermore, the number of bristles in these NOS inhibited transgenic flies is increased in some areas of the eye to five or six in many ommatidia, where normally one would expect only four (Kuzin, 1996).

How general is the phenomenon of NO-mediated growth arrest in organism development? A strong elevation of NOS activity in the developing cerebral cortical plate and hippocampus of prenatal rats at days 15-19 correlates with the time course of cessation of precursor cell proliferation, tight growth arrest, and cell differentiation; notably, NOS activity goes down after proliferation of committed neuronal precursors is completed (Bredt, 1994 and Blottner, 1995). NOS levels are also transiently increased in developing lungs, bones, blood vessels and nervous system (Blottner, 1995, and Wetts, 1995). Elsewhere, NOS activity is greatly elevated in regenerating tissues when cessation of cell division is crucial for prevention of unregulated cell growth (Roskams, 1994, Blottner, 1995 and Hortelano, 1995). In all these cases, a transient elevation of NOS activity might trigger a switch from proliferation to growth arrest and differentiation, thus contributing to the proper morphogenesis of tissues and organs (Kuzin, 1996). nitric oxide/cyclic GMP (cGMP) signaling pathway is thought to play an important role in mammalian vasodilation during hypoxia. Drosophila utilizes components of this pathway to respond to hypoxia. Hypoxic exposure rapidly induces exploratory behavior in larvae and arrests the cell cycle. Exposure of larvae to 1% oxygen (about 5% of normal atmospheric levels) produces a striking behavioral response. Typically, larvae feed on yeast paste with only their posterior ends protruding from the food. This leaves the two posterior spiracles, the openings of the larval tracheal system, exposed to the outside atmosphere. Within seconds of oxygen deprivation, larvae stop feeding, back up slightly, and then exit the yeast. Subsequently, over about a 2 min period, larval motility increases but is largely confined to the surface of the yeast. In a third phase of the response, the larvae leave the yeast and wandered onto a clean agar surface, occasionally escaping the petri dish. After 15 min of 1% oxygen, nearly 75% of the larvae have left the yeast. Prolonged exposure to 1% oxygen (greater than 30 min) results in almost a complete cessation of motility. It is unclear whether the ultimate cessation of motility constitutes another phase of the behavioral response or a depletion of energy reserves (Wingrove, 1999).

These behavioral and cellular responses are diminished by an inhibitor of NO synthase and by a polymorphism affecting a form of cGMP-dependent protein kinase. Osborne (1997) identified the molecular basis for a behavioral polymorphism. Sitters (for^s) are less motile than rovers (for^R) while on food. This difference in foraging behavior is due to the allele state at a single locus, for. The gene dg2, which encodes one of two cGMP protein-dependent kinases (PKG) in Drosophila, is located at the for locus. The for^s stock contains an undefined polymorphism that eliminates expression of at least one dg2 transcript, and these stocks have slightly reduced PKG activity (Osborne, 1997). Thus, a partial reduction in PKG results in a behavioral change detected as decreased movement of larvae on a food source. It was thought that roving behavior might be related to the vigorous exploratory behavior observed during hypoxia. Larvae on a food source (yeast) are likely to suffer a reduction in oxygen availability due to metabolic competition with yeast, and roving might be a response to the reduced oxygen availability. Accordingly, the failure of for^s larvae to rove might result from a reduced behavioral response to hypoxia. The influence of the for alleles on the response to hypoxia were examined. Third instar larvae raised under noncrowded conditions were transferred onto a pile of yeast paste located at the center of a grape agar plate. Larvae were allowed to settle for approximately 10 to 15 min. Under these conditions, no obvious differences in behavior were observed between for^R and for^s larvae. Dishes were sealed and gassed with 1% oxygen through a hole in the lid. The initial response to hypoxia, cessation of feeding and movement to the surface, occurs in both stocks, although for^s larvae respond more slowly. These results indicate that PKG activity contributes to the exploratory response to hypoxia; however, during a prolonged mild hypoxia, for^s larvae eventually left the food (Wingrove, 1999).

NO activates PKG in mammals where it has recognized roles in the response to hypoxia. It was asked whether NO might similarly mediate responses to hypoxia in Drosophila. To do this, the ability of an inhibitor of nitric oxide synthase (NOS) to blunt the behavioral response to hypoxia was examined. Early third instar larvae that had been fed either L-NAME, an inhibitor of NOS, or D-NAME, the inactive isomer of L-NAME, were tested for behavioral responses to hypoxia. After 10 min of 1% oxygen, only 18% of the larvae fed L-NAME clear the yeast pile, compared to 53% of the larvae fed D-NAME. This suggests that NOS activity contributes to the induction of exploratory behavior by hypoxia. An inducible NOS transgene was used to increase NOS activity. Larvae carrying heat shock-inducible NOS (hs-iNOS) were either heat shocked for 20 min at 37°C to induce iNOS expression or left at 25°C as a control. As a second control, wild-type larvae lacking the transgene were subjected to a similar regime. After 60 min of recovery, oxygen was reduced to 10% for 15 min. This modest hypoxia induces only a low percentage of roving in the controls. In contrast, 60% of the larvae in the heat-shocked iNOS stock clear the yeast. Thus, induced iNOS makes the larvae hypersensitive to reductions in oxygen levels. These results suggest that NO and PKG play an important role in the ability of larvae to respond to hypoxia (Wingrove, 1999).

Regions of the larvae that specialize in responding to hypoxia should express significant levels of the activities involved. Two methods were used to define possible foci of function: a histochemical stain (diaphorase staining) for NOS activity, and DAF2/DA, a fluorescein derivative that increases in fluorescence when bound by NO. Diaphorase staining detects higher NOS activity in the imaginal discs. In addition, the CNS (optic lobes and ventral nerve cord; and sections of the gut stained well for diaphorase. The highest amount of diaphorase staining is observed in the spiricular glands of the posterior spiracles and the spiricular pouch of the anterior spiracles. DAF2/DA also stains the pouch of tissue surrounding the anterior spiracles as well as neuronal-like processes within the pouch. This staining near the openings of the tracheal system is interesting because the location is consistent with a possible role in governing the opening (eversion) of the spiracles to increase access to oxygen (Wingrove, 1999).

The behavioral responses observed presumably allow larvae to escape local hypoxia. Embryos, however, that are nonmotile and larvae that are exposed to a more general hypoxia do not have the option to escape. The capacity of embryos and larvae to endure prolonged hypoxia was tested. Early syncytial embryos are quite sensitive to hypoxia, but the ability to survive periods of hypoxia improves during cellularization and gastrulation. Eight-hr-old embryos can survive hypoxia for up to 8 days and larvae can survive for several days if protected from dehydration. Perhaps NO and PKG contribute to the ability of larvae and embryos to endure hypoxia. To test for an involvement of PKG, for^s or for^R embryos (stage 14) were abruptly exposed to severe hypoxia for different periods and the hatching and eclosion was followed under normoxic conditions. Both types of embryos survived 2 to 6 hr of hypoxia; however, while for^s embryos survive 12 hr of hypoxia poorly, for^R embryos survived well. It is concluded that PKG contributes to the survival of embryos exposed to hypoxia. The involvement of NO and PKG in the survival of larvae was also tested. When for^s larvae are kept hypoxic for 6 hr, they show little sign of motility upon return to normoxic conditions, and they rarely pupate or eclose. In contrast, for^R larvae rapidly reacquire motility after 6 hr of hypoxia, and 80% eclose. Larvae fed L-NAME also show diminished viability after hypoxia. It should be noted that the larvae in these experiments were subjected to severe and immediate hypoxia. When less severe conditions are imposed, little difference between for^s and for^R larvae is observed. Thus, it appears that NO and PKG contribute to the ability of Drosophila to endure suddenly imposed hypoxia (Wingrove, 1999).

Drosophila eggs ordinarily require about 24 hr to hatch. Hypoxic embryos, however, can arrest for days and then resume development. Presumably, surviving this arrest requires coordinate blocks to many embryonic processes, including cell proliferation. The influence of hypoxia on the well-defined cell cycles of the Drosophila embryo were examined. Cellularized embryos undergoing S phase of cell cycle 15 (3.5 hr old, stage 8 embryos) were placed in media equilibrated with either 1% or 22% oxygen via a bubbler. After 5 min, BrdU was added, and 5 min later the embryos were fixed and stained for BrdU incorporation, a measure of DNA replication. Hypoxia blocks BrdU incorporation. Since S phase length in cycle 15 (45 min) is longer than the treatment, the arrest indicates that the ongoing S phase is blocked. This block is reversed upon 5 min of reoxygenation. Different cells of the embryo progress through cell cycle 15 according to a strict developmental schedule, giving rise to stage-specific stereotyped patterns of S phase cells. Hypoxia does not appear to disrupt these patterns; upon reoxygenation, S phase resumes in the same domains as when it was blocked. Thus, hypoxia induces a rapid and reversible arrest of S phase (Wingrove, 1999).

Whereas severe hypoxia can arrest development, low oxygen levels can modify it. Development of the tracheal system shares many similarities with the development of the mammalian circulatory system. In both, the terminal branches elongate and ramify as growth increases oxygen demand and creates slight hypoxia. Perhaps NO and PKG contribute to this response to low oxygen. If NO and PKG stimulate tracheal ramification in low oxygen, it might be expected that terminal ramifications will decrease if these components are inhibited and increase if their expression is stimulated. Consistent with this, for^s larvae, which are genetically compromised for PKG activity, and L-NAME-treated larvae, in which NOS activity is inhibited, appear to have less frequent and shorter terminal ramifications. In contrast, induction of iNOS appears to have the opposite effect, increasing the number and the length of terminal branches. These results provide one example in which NO and PKG modulate an aspect of development (Wingrove, 1999).

These results implicate the involvement of NO and PKG in the response to hypoxia in Drosophila. This suggests that the responses observed are induced by a regulatory pathway resembling the one characterized in mammals. In the mammalian nervous system, this signaling process is triggered by calcium release, which stimulates the calcium/calmodulin-dependent activity of neuronal NOS. This results in NO production, which then stimulates cGMP synthesis in adjacent cells containing soluble guanylyl cyclase. In insects, this type of pathway is thought to play a specific role in signaling between specialized cells of the nervous system. Perhaps a similar signaling pathway is utilized in the more global responses observed during hypoxia (Wingrove, 1999 and references).

By virtue of its ability to stimulate vasodilation, NO has been implicated in the mammalian response to hypoxia. There is, however, no known mechanism by which hypoxia can induce a rapid (transcription-independent) increase in NOS activity. Furthermore, the synthesis of NO by NOS requires oxygen as a substrate, and rates of NO synthesis by NOS have been shown to decline under hypoxia. Nonetheless, these results suggest that NOS makes a positive contribution in the response to hypoxia. Perhaps there is sufficient oxygen to support NOS-catalyzed generation of NO during hypoxia. Alternatively, the activity of NOS prior to hypoxia might contribute to the release of NO during hypoxia. The NO produced by NOS under normoxic conditions rapidly reacts with molecular oxygen, resulting in the accumulation of NO2-. It has been suggested that hypoxia stimulates the release of NO from preformed stores of NO2-, which may be established by the earlier action of NOS. It has recently been shown that mammalian xanthine oxidase reduces NO3-/NO2- to NO under hypoxic conditions. Thus, under hypoxic conditions, accumulated NO2- might be reduced through the action of xanthine oxidase or other related enzymatic activities, resulting in the release of NO. Such a mechanism might have a general role in sensing oxygen levels (Wingrove, 1999 and references).

While these findings implicate NO and PKG in the response to hypoxia, their exact roles in the process are unknown. By analogy to the known roles of NO and PKG, they might act as direct transducers of the signal or they might function as facilitators of a separately transduced signal. A model is presently favored in which NO and PKG are involved in a signal tranduction pathway that is related to but perhaps different from the recognized NO signal transduction pathway (Wingrove, 1999 and references).

Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration

Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. This study describes a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This produces both a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. It was demonstrated that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals (Jaszczak, 2015).

During Drosophila development, damage to larval imaginal discs elicits a regeneration checkpoint that has two effects: 1) it delays the exit from the larval phase in development to extend the regenerative period , and 2) it coordinates regenerative growth with the growth of undamaged tissues by slowing the growth rate of distal, undamaged tissues. How regenerarating tissues communicate with undamaged tissues to coordinate growth has been an open question. Damaged tissues may produce signals that directly influence the growth of undamaged tissues or may indirectly influence the growth of undamaged tissues by producing signals that alter the levels of limiting growth factors. Consistent with the latter model, this paper describes an indirect communication pathway for growth coordination during the regeneration checkpoint (Jaszczak, 2015).

An essential component of this growth coordination is the secreted peptide Dilp8, which is released by damaged tissues and is both necessary and sufficient to regulate the growth of distal tissues during the regeneration checkpoint. Dilp8 shares structural similarity to insulin - like peptides, which function to stimulate growth by activating the insulin receptor. However, in contrast to insulin - like peptides , Dilp8 acts to limit growth. A simple model explaining Dilp8 function would be that Dilp8 acts directly as an antagonist to insulin receptor activity, thus reducing growth in undamaged tissues. However, the growth response to checkpoint activation of polyploid larval tissues was shown to differ from imaginal discs. The growth of polyploid larval tissues are very sensitive to changes in insulin signaling, therefore these results are inconsistent with Dilp8 regulating imaginal disc growth by antagonizing systemic insulin signaling (Jaszczak, 2015).

NOS functions in the PG to regulate the growth of imaginal discs during the developmental checkpoint. Growth coordination during the regeneration checkpoint increases NO production in the PG, and is dependent on NOS gene function in the PG. Although constitutive expression of NOS in the PG might produce effects earlier in development that might alter the current interpretations, this study also demonstrated that transient pulses of NOS during the third instar and targeted NOS activation in the PG both produce the same effects: inhibition of imaginal disc growth by limiting ecdysone signaling. NOS activity in the PG reduces ecdysone production through the transcriptional inhibition of the P450 enzymes disembodied and spookier, which are necessary for ecdysone biosynthesis. Although it has been known that NOS activity is capable of regulating growth of imaginal discs (Kuzin, 1996), the experiments described in this study elucidate the mechanism of this growth regulation (Jaszczak, 2015).

The activity of NOS described in this study contrasts with published experiments demonstrating that NO signaling inhibits E75 activity in the PG, thus promoting larval exit (Caceres, 2011) . However, experiments from Caceres demonstrate that earlier NOS expression in the PG during larval development produces small larvae that arrest at second larval instar stage of development. This arrest can be partially rescued by either ecdysone feeding, or by reducing the level of GAL4 - UAS driven NOS expression by raising larvae at a lower temperature. Additionally, previous studies indicated that pharmacological increase of NO levels in larvae can produce larval developmental delays. Together, these observations suggest that NOS activity earlier in larval development might inhibit rather than promote ecdysone signaling during the larval growth period. Finally, this study observed that E75B is not expressed in larvae that have activated the regenerative checkpoint, suggesting that th e NOS dependent pathway that has been described by Caceres is not active during the regeneration checkpoint (Jaszczak, 2015).

This study has focused on the role of NOS during the growth phase of the third larval instar (76-104h AED) and have found that heat-shock mediated pulses of NOS activity during this period of development inhibit growth and ecdysone signaling, while pulses of NOS activity at the end of larval development do not inhibit growth or ecdysone signaling. Based on these results, it is concluded that there are distinct roles for NOS in the PG during different phases in development; NOS activity post-larval feeding promotes ecdysone synthesis through inhibition of E75, whereas NOS activity during the larval growth phase limits ecdysone synthesis and signaling by reducing the expression of ecdysone biosynthesis genes through a yet-to-be defined mechanism. Some intriguing possible mechanisms are through regulation of the growth of the PG, or via activation of cGMP-dependent pathways (Jaszczak, 2015).

Furthermore, this study demonstrated that ecdysone is essential for imaginal disc growth. Most studies have supported a model in which ecdysone acts as negative regulator of growth based on two observations: 1) the final pulse of ecdysone at the end of the third larval instar shortens developmental time and therefore reduces final organ size, and 2) increased ecdysone signaling can antagonize Dilp synthesis in the fat body. However, when measuring the effects of ecdysone on growth, many previous studies have focused on measuring either the growth of the larvae (which as this study observed does not always reflect the growth of the imaginal tissues) or measuring the final size of adults (which is a function of both growth rate and time). When one either examines clones expressing mutant alleles of ecdysone receptor or measures the growth of entire imaginal discs directly following ecdysone feeding as this study has done, ecdysone signaling can be shown to promote imaginal disc growth (Jaszczak, 2015).

During the regeneration checkpoint, both growth coordination and the delay in developmental timing are dependent on reduced ecdysone levels. Therefore, both delay and growth inhibition might be expected to be dependent on the same pathways. However, this study clearly demonstrated that the genetic requirements for these two systemic responses to damage are distinct. NOS is necessary for growth regulation following tissue damage, but is not necessary for the developmental delay. While it was observed that overexpression of NOS in the PG produces developmental delay, the results suggest that this is through a different mechanism than delays produced during the regeneration checkpoint. Therefore, Dilp8 secretion from damaged imaginal discs produce s developmental delay and growth restriction through distinct mechanisms (Jaszczak, 2015).

Finally, these observations suggest that regenerative growth, which is able to proceed despite reduced ecdysone signaling, may have different growth requirements than undamaged tissues. Understanding these differences in growth regulation could provide valuable insights into the mechanistic distinctions between regenerative and developmental growth (Jaszczak, 2015).

Nitric oxide mediates neuro-glial interaction that shapes Drosophila circadian behavior

Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. This study demonstrates that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, this study also demonstrated that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output (Kozlov, 2020).

It is rather surprising that the lack of NOS enzyme is not lethal as NO is part of various developmental processes. NOSΔ mutants are nonetheless strongly arrhythmic in DD and have reduced morning anticipation in LD. The data suggest that both congenital impairments and lack of NO signaling in adulthood contribute to the behavioral phenotype of the mutants. Axonal terminals of the master pacemaker, s-LNvs, in NOS mutants are profoundly disordered, suggesting the wrong or absent synaptic connections with the downstream partners. However, molecular rhythms in the pacemaker neurons are unaffected. During adulthood, NOS activity in the perineurial glia is required for producing free-running locomotor rhythms but not for maintaining PDF rhythms and structure of the s-LNvs. This finding indicates that NO produced in the perineurial glia is necessary for proper the functioning of circadian locomotor output circuits. Taken together, these results demonstrate that NO signaling is essential for establishing and controlling circadian output circuit (Kozlov, 2020).

The functional isoform dNOS1 shows a circadian variation of its RNA levels throughout the day, which suggest that levels of NO could cycle at least in LD. However, dNOS is likely to be regulated by its truncated isoforms in a stage- and cell-type-specific manner, which lays an additional complexity to the regulation of NO production and probably leads to the heterogeneous and context-specific variations of NO. Hyperproduction of NO modulates molecular clockwork, albeit modestly, and is generally detrimental to locomotor rhythms. Therefore, the level and potentially the rhythms of NO production should be tightly controlled in wild-type flies (Kozlov, 2020).

In an NOS RNAi mini screen, two optic lobe-specific drivers, GMR79D04 and GMR85B12, reduced locomotor rhythmicity in DD. This phenotype was observed when NOS was downregulated constitutively in these cells but not when knockdown was restricted to adulthood. These results reinforce the idea that NO is necessary for a proper establishment of neuronal circuits. A low rhythmicity phenotype caused by NOS knockdown with the pan-neuronal driver GMR57C10-GAL4 is congruent with the above findings. Intriguingly, however, in addition to the low rhythmicity, GMR57C10 > NOS RNAi in adulthood resulted in an extended period. What might be the neuronal subsets that produce NO and regulate period length of locomotor activity? A recent study by Aso (2019), NO was shown to act as a co-transmitter in a subset of dopaminergic neurons, specifically in some of the PAMs, PPL1s and PPL2abs. It is thus possible that dopamine signaling modulated by NO is involved in the control of the locomotor activity period. It is also noteworthy that NO-mediated signaling has a profound neuromodulatory effect on spinal motor networks and regulates frequency and amplitude of motor activity in various vertebrate species. NO-mediated regulation of circadian locomotor output in flies might involve a similar mechanism (Kozlov, 2020).

DAR4-M staining showed an enrichment of NO in glial cells, including the surface glia. Targeting glial cells leads to the strongest and most persistent phenotype in locomotor activity both for gain- and loss-of-function of NOS. Among glial subpopulations, the perineurial glia appears to be the major site of NOS activity that regulates locomotor rhythms. The importance of glia in circadian rhythms have been recognized, especially those containing the molecular clocks and exert reciprocal communication with the pacemaker neural circuit. It has been shown that the perineurial glial cells harbor molecular clocks, which drive daily rhythms in the blood-brain barrier permeability but are not required for locomotor activity rhythms. This study is the first to identify NO as a signaling molecule produced in glia and mediates part of the role of glia, independently of their molecular clocks, in Drosophila circadian rhythms (Kozlov, 2020).

It has been shown that in mammals NO mediates light-induced phase-shifts through the cGMP pathway. It is an interesting parallel to note that forced production of NO in the s-LNvs caused phase shift rather than amplitude dampening. Since high levels of NO inhibit E75/UNF dimerization and E75/UNF normally enhances per transcription, it is speculated that NO-induced phase-shift may be partly mediated by the inhibition of E75/UNF (Hormone receptor 51) heterodimerization. It will be interesting to test this hypothesis in future studies. Mammalian clocks contain E75 homologs REV-ERB α/β, which repress Bmal1 transcription. Analogous to the notion in flies, NO is thought to decrease REV-ERB α/β activity. Consistently, in vitro studies in mammalian cell culture showed that excessive presence of NO increases the production of Bmal1 mRNA. These findings altogether point out that NO is an evolutionarily conserved regulator of circadian rhythms (Kozlov, 2020).

In line with recent studies, this research expands the view on the factors that participate in neuronal and molecular mechanisms of circadian rhythmicity. The finding that gaseous messenger NO contributes to the various aspects of circadian rhythmicity emphasizes that non-cell-autonomous, systemic regulation is integral to the circadian circuit operation. These results set a foundation for future studies addressing the mechanism by which NO signaling modulates the state of the pacemaker circuit and its output (Kozlov, 2020).

REGULATION

Soluble Guanylate cyclase acts upstream of Nos in the development of visual system function

A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express a NO-sensitive soluble guanylate cyclase (sGC: Guanyl cyclase alpha-subunit at 99B), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. Inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1) have been examined. This mutation severely reduces but does not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possess a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increases this disorganization in mutants to a greater degree than in the wild type. Adult mutants lack phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude are restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. It is proposed that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior (Gibbs, 2001).

Genetic mutants in Gcalpha1 were produced with EMS mutagenesis. Four alleles of Gcalpha1 were recovered. On the basis of decreases in intensity of GCalpha1 immunoreactivity on a Western blot of adult heads, the allelic series is Gcalpha1⁵ = Gcalpha1¹ = Gcalpha1³ > Gcalpha1². The differences in the levels of GCalpha1 protein are virtually indistinguishable among Gcalpha1⁵, Gcalpha1¹, and Gcalpha1³. Almost all of the following experiments were performed with Gcalpha1¹, the first to be isolated. In a Western blot of protein extract from wild-type adult heads, the anti-GCalpha1 antibody recognized a band of ~76 kDa. In contrast, GCalpha1 antibody staining was greatly reduced in Gcalpha1¹ flies. A transgene containing the wild-type GCalpha1 was expressed with a 1 hr heat shock in the Gcalpha1¹ mutant, which increases the anti-GCalpha1 staining to wild-type levels. The Gcalpha1¹ mutation not only reduces GCalpha1 staining on a Western blot but also greatly attenuates the synthesis of cGMP in the nervous system in response to NO. The activity of sGC in the developing Drosophila nervous system was visualized by exposing the tissue to the NO donor SNP and the phosphodiesterase inhibitor IBMX, followed by immunocytochemistry with an anti-cGMP antibody. At 24 hr APF, the CNS of the Gcalpha1¹ mutants showed cGMP immunoreactivity (cGMP-IR) in only a few neurons of the central brain, suboesphageal ganglion, and ventral nerve cord in response to SNP and IBMX. This represents a small subset of the cGMP-positive population of cells observed in the wild-type CNS at 24 hr APF after similar treatment. Most cells that showed cGMP-IR in Gcalpha1¹ appear to be the same cells that remain cGMP-positive in the wild-type CNS after treatment with an sCG inhibitor and are frequently cGMP-positive in nervous systems that have not been exposed to SNP and IBMX. A receptor-type guanylate cyclase has been cloned from Drosophila (Gibbs, 2001).

The photoreceptors possess NO-sensitive sGC activity during the first half of metamorphic development, after they arrive at their optic lobe targets. cGMP immunoreactivity is observed in the cell bodies of photoreceptors in the developing retina as well as their axons projecting into the lamina and medulla regions of the optic lobe. These represent the axons of both classes of photoreceptors, R1-6 and R7/8. The axons of Bolwig's nerve are also cGMP-positive. In contrast, a very low level of cGMP-IR is observed in the visual system of Gcalpha1¹ after SNP and IBMX treatment at 24 hr APF, primarily in the cell bodies of the eye imaginal disc. This faint cGMP-IR most likely reflects the activity of remaining low levels of GCalpha1 protein in the Gcalpha1¹ mutant. Heat shock-induced expression of a wild-type Gcalpha1 transgene restores the strong response of the photoreceptors to NO in Gcalpha1¹ at 24 hr APF. Two hours after a single 45 min heat shock, cGMP production was observed in the photoreceptor cell bodies and axons and the larval pioneers after treatment with 1 mM SNP and IBMX. The intensity of cGMP-IR induced with heat shock was not observed, however, without SNP and IBMX exposure. Surprisingly, after heat shock expression of Gcalpha1, strong cGMP-IR was also observed in the medulla interneurons of the optic lobe of the Gcalpha1¹ mutants. This was in contrast to the wild-type visual system, in which NO-induced cGMP accumulation is very low in the medulla at 24 hr APF, becoming more prominent by ~48 hr APF. Because both alpha and ß subunits are required for soluble guanylate cyclase activity in response to NO, it is concluded that the GCß1 protein must already be present in the medulla interneurons at 24 hr APF. The delayed onset of NO sensitivity in these cells may then reflect the regulated expression of GCalpha1 rather than GCß (Gibbs, 2001).

Cells of the lamina and medulla express NOS and pharmacological inhibition of NOS or sGC in vitro causes disorganization and overgrowth of the retinal projections in the wild-type visual system. On the basis of these in vitro results, it was predicted that flies mutant for Gcalpha1 would display similar defects in visual system organization. The retinal projection pattern of R7/8 in the medulla of all four Gcalpha1 alleles was examined throughout metamorphosis and in adults, using whole-mount immunocytochemistry with antibodies to chaoptin and fasciclin II, both of which label photoreceptor axons. In general, the mutants did not show dramatic disruption of the projection pattern of R7/8, although minor defects have been noted in some cases. At 48 hr APF, the chaoptin-stained retinal projections of a few Gcalpha1¹ mutants were slightly disrupted when compared with the wild type, producing gaps in the pattern and extension of retinal axons slightly beyond the medullar margin and producing a 'ragged' border. Similarly, staining with a fasciclin II antibody at the same stage revealed what appeared to be a few retinal axons projecting well beyond the medulla in some individuals. Although this phenotype was not widely observed, it was never seen in age-matched wild-type flies and may reflect the variable penetrance of the Gcalpha1¹ mutation (Gibbs, 2001).

The Gcalpha1¹ flies express low levels of GCalpha1 protein, which is also reflected in very low levels of NO-sensitive sGC activity in the developing visual system. sGC is an enzyme; thus, even a small amount is capable of producing a greatly amplified cGMP signaling cascade in the presence of sufficient ligand. It was therefore predicted that although this residual sGC activity normally prevents retinal axon overgrowth in most of the Gcalpha1¹ mutants, it would not be enough to compensate for decreases in NO production in vitro. CNSs and attached eye discs from wild-type and mutant white puparia were placed in culture with the hormone 20-hydroxyecdysone, which promotes metamorphic development of the nervous system in vitro, or hormone plus the competitive NOS inhibitor L-NAME. After 96 hr in culture, the tissue was then processed for chaoptin immunocytochemistry. For analysis, focus was placed on the projection pattern of R7/8 in the medulla, which is easily resolved in whole-mount preparations. Nervous systems were assigned a score of 0-4 based on the severity of disorganization in the projection pattern. The scores for all samples were averaged to obtain a mean score for a given treatment, called the disruption index, and the percentages of nervous systems showing any retinal growth beyond the medulla were determined for mutant and wild-type nervous systems for each treatment (Gibbs, 2001).

A delay in neural development occurs with culturing, so that after 96 hr in vitro, the visual system has progressed to a stage comparable to 48-50 hr APF. In control cultures without inhibitor, a normal, well organized projection pattern is typically seen in the medulla, and no retinal fibers are seen growing past medullar targets in either wild-type or mutant visual systems. Although not significantly different, the disruption index for the mutants under control conditions is slightly higher than that for the wild type, suggesting that the Gcalpha1¹ mutation caused subtle projection pattern defects in a low percentage of individuals, as is observed with whole-mount analysis of noncultured nervous systems. When a low level of L-NAME is added to the cultures, essentially no pattern disruption is observed in the medulla of wild-type nervous systems. However, this concentration of L-NAME causes the growth of many retinal axons beyond the posterior medulla of the Gcalpha1¹ optic lobe. This effect is more severely pronounced in the mutants with a higher level of L-NAME. Under these conditions, the retinal fibers produce a dense, disorganized tangle in the medulla and extend many projections into the lobula. This is not the case for wild-type nervous systems, in which treatment with higher levels of L-NAME produce a slight disorganization of the projection pattern, but no retinal fibers are observed projecting beyond the medulla (Gibbs, 2001).

To examine visual system function in the Gcalpha1 mutant adults, a standard behavioral assay was used for fast phototaxis, using a countercurrent apparatus. In these experiments, a population of ~100 flies was placed in the first of six clear tubes, laid onto a horizontal surface, and transferred through the tubes at 30 sec intervals as they moved toward a light source. An average of 65% of the total population of wild-type flies consistently moved toward the light after each transfer to end up in the last tube (tube 6) by the end of the trial. All four of the Gcalpha1 mutant strains showed a reduction in positive phototaxis at 1 week after eclosion when compared with the claret¹ progenitor strain, but the Gcalpha1¹ mutants were the most severely compromised. A combined 12%-13% of Gcalpha1¹ flies made it to the last two tubes (tubes 5 and 6) of the apparatus in repeated trials, compared with 65% of claret¹ controls. This behavior is not the result of negative phototaxis, as tested by reversing the orientation of the countercurrent apparatus relative to the light source. The profile of Gcalpha1¹ also remains unchanged in the absence of light. Although visually mediated, phototaxis requires the integration of many behaviors. However, Gcalpha1¹ adults show normal performance in a geotaxis assay, demonstrating that these flies do not possess gross motor impairments (Gibbs, 2001).

An investigation was carried out to see whether restoring sGC activity during the period of retinal innervation would improve the phototactic performance of the Gcalpha1¹ mutants, using a heat shock-inducible Gcalpha1 transgene. In this experiment, a 45 min heat shock was given every 8 hr for the first 48 hr of pupal development, approximately encompassing the window of NO-sensitive cyclase activity observed in the photoreceptors. Phototaxis was tested 1 week after adult eclosion, 9 d after the last heat shock. At the completion of this experiment, 51% of the total population of hs-gc⁺;Gcalpha1¹ adults exposed to heat shock during metamorphosis converged in the last two tubes of the countercurrent apparatus. This compares with 13% for the non-heat-shocked hs-gc⁺;Gcalpha1¹ adults and 65% for the claret¹ progenitor strain. In addition, the percentage of flies in the first two tubes is nearly identical between ca¹ controls and heat-shocked hs-gc⁺;Gcalpha1¹ adults. Thus positive phototaxis is restored in the Gcalpha1¹ mutants to near wild-type levels when Gcalpha1 is expressed during the first half of metamorphosis, and the phototactic profile is made to resemble that of the wild type. An improvement in positive phototaxis is not observed when adult hs-gc⁺;Gcalpha1¹ flies are tested 3 hr after a single acute heat shock. In addition, increased positive phototaxis is not seen in hs-gc⁺; Gcalpha1¹ mutants that were exposed to heat shock every 12 hr for 48 hr as adults and tested 24 hr after the last heat shock. These results strongly support the hypothesis that the function of sGC in the phototactic response is developmental, rather than a physiological requirement for sGC activity at the time the behavior is being performed (Gibbs, 2001).

ERGs were perfomed in a further attempt to examine the effects of the Gcalpha1¹ mutation on visual system function. The ERG primarily characterizes the electrophysiological response to light of a subset of photoreceptors, R1-6, and their postsynaptic cells in the lamina. The ERG consists of a corneal positive 'on-transient,' followed by a sustained negative wave that lasts throughout the period of illumination, and then a corneal negative 'off-transient.' The off-transient results from the summation of postsynaptic potentials in the monopolar neurons L1 and L2 of the lamina and can thus be used as an indicator of synaptic efficacy (Gibbs, 2001).

82% of the ERGs from the Gcalpha1¹ flies lack off-transients or have off-transients that are greatly reduced in amplitude. Gcalpha1¹ flies that had Gcalpha1 supplied with heat shock during the first half of metamorphosis express a mean off-transient amplitude that is comparable with that of wild-type controls. The effect of the Gcalpha1¹ mutation appears to be limited to the off-transient amplitude, because both the mean duration of the off-transient peak and the mean amplitude of the sustained component are not significantly different among all three populations. Thus, both positive phototaxis and a normal ERG trace can be restored to adult flies expressing very low levels of sGC activity when Gcalpha1 is supplied developmentally as the retinal growth cones are undergoing target selection in the optic lobe (Gibbs, 2001).

The alpha subunit of a soluble guanylate cyclase has been repeatedly cloned from Drosophila. However, there is controversy as to the expression pattern of Gcalpha1. Using Northern blot analysis, Gcalpha1 mRNA has been demonstrated in wild-type but not eyeless adult fly heads, suggesting that Gcalpha1 is expressed primarily in the adult retina. An antibody was employed to localize the GCalpha1 protein to the retina. Although abundant Gcalpha1 mRNA has been seen in a Northern blot of adult heads, the same probe failed to hybridize to adult retinal tissue in situ, and no retinal staining was found with a GCalpha1 antibody. In this study evidence is presented that Gcalpha1 is expressed in the photoreceptors during development. A genetic mutation that diminishes GCalpha1 protein levels, Gcalpha1¹, reduces NO-induced production of cGMP in developing photoreceptors: cGMP is restored with heat shock-mediated expression of wild-type Gcalpha1. Thus it is concluded that Gcalpha1 is normally expressed in the photoreceptors from ~12 to 48 hr APF, and it is inferred that the ß subunit is also present at this time. It has not been determined whether the observed loss of NO sensitivity in the photoreceptors at ~48 hr APF and into adulthood is attributable to changes in Gcalpha1 expression. However, spatiotemporal changes in alpha and ß subunit expression may provide a molecular basis for regulating the timing of NO-sensitive sGC activity in the photoreceptors and other cells of the developing visual system (Gibbs, 2001).

Reports of Gcalpha1 expression in the adult retina, and cGMP-mediated enhancement of the photoresponse in isolated Drosophila photoreceptors have implicated cGMP as a putative mediator of a phototransduction mediator in flies. Studies show that NO and cGMP can modulate the locust photoresponse and signaling in other insect sensory systems. The Gcalpha1 mutants were initially generated to further establish the role of cGMP in phototransduction; however, both intracellular and extracellular recordings from these flies have revealed a normal response of the photoreceptors themselves to light. In other studies, inositol trisphosphate and diacylglycerol were shown to be primarily responsible for generating the depolarizing potential in the Drosophila retina. The results presented here suggest that the requirement for cGMP in the Drosophila visual system is developmental, rather than physiological, for at least two reasons: (1) cGMP was never observed in the photoreceptors after 48 hr APF, in the presence or absence of NO stimulation; (2) although mutant adults lack positive phototaxis (a basic visually mediated behavior) positive phototaxis is only restored to Gcalpha1¹ adults when Gcalpha1 is supplied with heat shock during the first 48 hr of metamorphosis, encompassing the period of observed NO-sensitive sGC activity in the photoreceptors. In contrast, phototaxis did not improve when Gcalpha1 was expressed acutely or chronically in adult mutants. The ERG results also support the hypothesis that sGC signaling is required during the first half of metamorphosis, when retinal growth cones are selecting postsynaptic partners in the optic lobe and NO-sensitive sGC activity is observed. The sustained depolarization of the photoreceptors was normal in Gcalpha1¹ adults, but the off-transients were frequently undetectable or greatly reduced in amplitude. In addition, some off-transients contained two peaks. The postsynaptic responses of the laminar monoplar cells are responsible for generating the off-transient in Drosophila. Thus, the decreased and aberrant off-transients observed in GCalpha1¹ implicate a defect in the postsynaptic response of laminar monopolar cells to retinal input in these mutants. This abnormal postsynaptic response could arise from disorganized retinal synapses, perhaps as a result of subtle deviations in growth cone behavior in the absence of normal cGMP levels. When Gcalpha1 is expressed with heat shock in Gcalpha1¹ mutants during the first half of metamorphosis, the off-transient shape and amplitude are indistinguishable from those of the wild type. The behavioral and electrophysiological results support the hypothesis that cGMP signaling is required in the photoreceptors to promote the appropriate wiring of first-order retinal synapses during metamorphosis. However, because the Gcalpha1¹ mutation and the heat shock Gcalpha1 expression are global and not eye-specific, the possibility cannot be excluded that formation of downstream connections in the optic lobe and brain also require GCalpha1. The behavioral results in particular may reflect the effects of Gcalpha1 expression on these connections. However, from 12 to 48 hr APF NO-sensitive cGMP production is observed almost exclusively in the photoreceptors and not in other visual centers until later in development. This is also the developmental window during which heat shock Gcalpha1 expression rescues both the phototactic and ERG phenotypes, which strongly suggests that the photoreceptors themselves require GCalpha1 to ensure the appropriate wiring of first-order retinal synapses (Gibbs, 2001).

Despite the profound defects in visual system function, no dramatic and consistent disorganization of the retinal projections is observed in Gcalpha1¹. This contrasts with previous results, in which pharmacological inhibition of NO-sGC signaling caused severe disruption of the wild-type projection pattern in vitro (Gibbs, 1998). There are several possible explanations for these results. (1) The Gcalpha1¹ mutants are hypomorphs and do show low levels of GCalpha1 protein and NO-sensitive sGC activity in the photoreceptors during metamorphosis. Residual enzymatic activity of these low levels of functional sGC could produce adequate levels of retinal cGMP to prevent significant axon overgrowth provided that the ligand, NO, is present at wild-type levels. This is supported by experiments in which nervous systems from Gcalpha1¹ pupae were exposed to low levels of a NOS inhibitor. Under these conditions, the resulting disorganization and overgrowth of the retinal axons were much greater than in wild-type controls. These results suggest that residual GCalpha1 activity and production of cGMP by endogenous NO in Gcalpha1¹ is sufficient to prevent overgrowth of retinal axons in vivo but cannot compensate for suppression of NO signaling in vitro. (2) The Gcalpha1¹ visual system has been examined only at the whole-mount level. A more detailed analysis of the retinal projections and cartridge organization, perhaps using electron microscopic techniques, may reveal further architectural defects in the Gcalpha1¹ mutants. (3) Other signaling pathways have been shown to contribute to formation of the Drosophila retinal projection pattern, and these may compensate for decreased sGC activity during visual system development (Gibbs, 2001).

It is proposed that Gcalpha1expression and subsequent sGC- and NO-induced cGMP activity in the photoreceptors regulate synapse formation between photoreceptors and optic lobe neurons by exerting subtle effects on retinal growth cone behavior during cartridge assembly. Expression of NO-sensitive sGC activity was never seen during retinal axon outgrowth but only in photoreceptors that had arrived at their respective optic ganglia. This makes it unlikely that NO and cGMP are acting in a chemoattractive manner to guide retinal growth cones to the optic lobe. The metamorphic period when NOS expression in the optic lobe and NO-sensitive sGC expression in the photoreceptors was observed (12-48 hr APF) correlates temporally with when retinal growth cones are actively seeking out optic lobe cells with which they will form synaptic cartridges. The results support a model wherein NO from the target acts to stimulate cGMP synthesis in newly arrived retinal growth cones, stabilizing them and preventing further axonal extension but still allowing lateral movement within the target region. When NO production or sGC activity is inhibited pharmacologically, this stabilization is lost, and the photoreceptors resume longitudinal growth. The current results show that although genetically reducing sGC activity leads to more subtle defects in visual system architecture, perhaps at the level of cartridge organization, the overall effects of this mutation on adult visual system function are profound. NO and cGMP have been proposed to regulate vertebrate visual system development by acting as effectors of activity-dependent refinement mechanisms. However, this is not likely to be the case in Drosophila, because the visual system can develop normally in the absence of histamine, the primary visual neurotransmitter. Instead, the effects of NO-induced cGMP production may act to regulate growth cone behavior (Gibbs, 2001).

Nitric oxide pathway interacts with the RB pathway to control growth

Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway, which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger, can reversibly suppress DNA synthesis and cell division. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. Transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors were used to address this issue. Manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. These data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway (Kuzin, 2000).

To regulate ectopic production of NO during development, transgenic lines of Drosophila were generated in which the expression of dNOS1 cDNA was controlled either by the heat-shock-inducible hsp70 promoter (hs-dNOS1 flies) or by the eye-specific GMR promoter, which functions in all cells of the eye imaginal disc in, and posterior to, the morphogenetic furrow [19] (GMR-dNOS1 flies). Examination of scanning electron micrographs of the eyes of, and thin sections of the retinas of, different transgenic lines did not reveal obvious differences among eyes of wild-type flies, transgenic hs-dNOS1 flies with or without heat-shock and GMR-dNOS1 flies. This indicates that a moderate increase in NO production on its own does not noticeably affect eye development.

To investigate the relationship between NOS activity and cell cycle progression, NOS activity was manipulated in transgenic flies ectopically expressing genes of the Rb pathway in the developing eye. Drosophila RBF is structurally related to the mammalian proteins of the Rb family and, like the Rb proteins, RBF is a negative regulator of cell cycle progression. The RBF transgene was placed under control of the GMR promoter and flies with either two (GMR-RBF2) or four (GMR-RBF4) copies of the transgene were used in these experiments. The eyes of adult flies with two copies of the RBF transgenes (GMR-RBF2) appear normal, indicating that at this dosage the RBF transgene does not noticeably disturb cell division in the eye disc. When GMR-RBF2 flies are crossed to hs-dNOS1 flies and the progeny larvae are treated with heat shock before pupariation, however, the resulting adults have multiple defects in the eyes, including missing bristles and pigment cells. Pigment cells, which comprise the boundaries of each ommatidia, appear as a characteristic honeycomb pattern in thin sections of normal eyes. A lack of the regular number of pigment cells in GMR-RBF2 + hs-dNOS1 flies results in the appearance of many fused ommatidia and a rough eye phenotype. Thus, hs-dNOS1 and GMR-RBF2 flies, both of which do not affect the development of eye structure when overexpressed on their own, nevertheless yield eye defects when overexpressed together. This transgenic interaction suggests that NOS and RBF genes interact synergistically during the development of ommatidia. A similar effect was observed employing a different genetic strategy. This time, the overexpression of dNOS1 was restricted to the developing eye by crossing GMR-RBF2 flies with GMR-dNOS1 flies. These double-transgenic flies also display eye defects similar to those of heat-shocked GMR-RBF2 + hs-dNOS1 flies — missing pigment and bristle cells and fused ommatidia. Thus, regardless of the promoter that drives the expression of the dNOS1 transgene, elevated levels of NO and RBF synergize to limit cell number in the developing eye, supporting the notion of interaction between dNOS1 and RBF genes (Kuzin, 2000).

Both RBF and NOS act to suppress cell division. If indeed NOS acts in concert with RBF during eye development, then inhibition of NOS might suppress RBF function and restore the normal number and shape of ommatidia to GMR-RBF4 flies. To test this, endogenous NOS activity was blocked in larvae of GMR-RBF4 flies using a specific NOS inhibitor L-nitroarginine methyl ester, L-NAME (which alone did not affect the eye morphology of the wild-type flies. Remarkably, the eyes of these drug-exposed transgenic flies have an almost normal phenotype as regards the number of photoreceptor and accessory cells and the number and shape of the ommatidia; only a few bristles were still missing. The antiproliferative activity of NO results from its ability to suppress DNA synthesis, as BrdU labeling of the eye imaginal discs showed that the number of cells in S phase is decreased after heat shock in flies carrying the hs-dNOS1 transgene and is increased upon inhibition of NOS activity in GMR-RBF4 flies. Thus, the inhibitory effect of RBF overexpression on cell proliferation is almost completely rescued when endogenous NOS activity is inhibited in the developing larvae (Kuzin, 2000).

In mammalian cells, Rb and Rb-related proteins bind to transcription factors of the E2F family and inhibit E2F-dependent transcription. When phosphorylated by cyclin-dependent kinases, Rb does not bind E2F and E2F-dependent transcription of several genes required for the synthesis of DNA and entry into S phase of the cell cycle is induced. Ectopic overexpression of E2F overcomes the Rb-mediated repression and induces quiescent cells to enter S phase. Similarly, in Drosophila cells, RBF is associated with the E2F transcription factor complex. In transgenic flies overexpressing dE2F and dDP under control of the GMR promoter (GMR-dE2FdDP flies), ommatidia form irregular rows and lack their regular hexagonal shape; in addition, many eye bristles are duplicated. This observation indicates that overexpression of RBF and of E2F have reciprocal effects on cell proliferation in the developing eye (Kuzin, 2000).

To determine whether the antiproliferative activity of NO can counteract excessive precursor cell proliferation caused by E2F overexpression, GMR-dE2FdDP flies were crossed with hs-dNOS1 flies. When progeny larvae are treated with heat shock, a normalized adult eye developed; in some cases a revertant (wild-type) pattern of ommatidial rows, regularly shaped ommatidia, and the usual number of bristles are seen. Similarly, progeny of a cross between GMR-dE2FdDP flies and GMR-dNOS1 flies developed more normal eyes, corroborating a specific dNOS1-E2F interaction. Thus, in contrast to GMR-RBF4 flies, in with which inhibition of NOS was needed to rescue the mutant phenotype (underproliferation of precursor cells), overexpression of dNOS1 is needed to rescue the phenotype of GMR-dE2FdDP flies (overproliferation of precursor cells). This reciprocal effect of NO levels on RBF and E2F function in cell cycle control adds considerable genetic strength to the idea that NO acts in concert with the Rb pathway to suppress cell division during eye development (Kuzin, 2000).

RBF blocks E2F-dependent transcription in cotransfection assays, in accordance with its ability to sequester E2F proteins. When expressed in the eye, GMR-RBF suppresses the rough-eye phenotype of the GMR-dE2FdDP transgenic flies. Thus, overexpression of RBF and E2F have opposing effects on the decision of precursor cells to enter the cell cycle. A test was performed to see whether NO modulates the effects of GMR-RBF2 on E2F function by inhibiting NOS activity in GMR-RBF2 + GMR-dE2FdDP flies. Ectopic expression of E2F in the developing eye increases both cell proliferation and programmed cell death; the net effect is the appearance of more cells in the eye, however. To minimize the E2F-induced augmentation of cell death, an effective inhibitor of apoptosis, the baculoviral p35 gene, under control of the GMR promoter, was used. Whereas the combination of GMR-dE2FdDP and GMR-p35 transgenes produce an even more severe phenotype than the GMR-dE2FdDP transgene alone, the GMR-RBF2 + GMR-dE2FdDP + GMR-p35 flies have a normal eye phenotype, confirming that, in the absence of programmed cell death, RBF suppresses the consequences of E2F over-expression and rescues the E2F phenotype. In contrast, inhibition of NOS activity in these GMR-RBF2 + GMR-dE2FdDP + GMR-p35 larvae prevents RBF from rescuing the E2F phenotype. In particular, when endogenous NO production is suppressed, the arrangement of ommatidia is still abnormal, and many additional bristles and pigment cells are still observed. This suggests that the RBF-E2F interaction involves NOS and that RBF requires NO to antagonize the E2F activity (Kuzin, 2000).

This study of the developing Drosophila eye presents a series of reciprocal genetic interactions that consistently suggest that NO modulates a signaling pathway involved with cell cycle control. Specifically, increased production of NO in the developing eye acts as an antiproliferative signal, whereas inhibition of NOS activity promotes additional rounds of cell division. It is considered that the reciprocal effects of E2F and NOS and complementary effects of Rb and NOS are best explained by the hypothesis that NO affects the Rb signaling pathway, thereby regulating entry into the S phase of the cell cycle (Kuzin, 2000).

NO/cGMP system activates PKA activity during learning in the honeybee

To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals (Müller, 2000).

Associative olfactory conditioning of the proboscis extension response (PER) in the honeybee induces different forms of memory, depending on the number of conditioning trials. The memory induced by a single conditioning trial decays over several days and is sensitive to amnestic treatments. This memory is independent of NO synthase (NOS) blockers and of protein synthesis. In contrast, multiple conditioning trials induce a stable, long-lasting memory. This memory is dissectable into two independent, parallel phases. The first phase is a medium-term memory (MTM) in the hours range, which requires a constitutive PKC activity, and the second phase is an LTM (1 day or more), which requires PKA- and NO-dependent processes. Interestingly, LTM can be divided into an early phase (eLTM, 1-2 days) and a protein synthesis-dependent late phase (lLTM, 3 days or more)(Müller, 2000 and references therein).

Even though it has been demonstrated that the cAMP cascade is important for the induction of long-lasting neuronal and behavioral changes, the findings presented here reveal evidence for a direct connection between conditioning procedure, temporal dynamics in PKA activation, and their contribution to formation of LTM in intact animals. Direct measurement of changes in PKA activity in the ALs induced by in vivo stimulation reveals that multiple conditioning trials that induce LTM also induce an extremely prolonged PKA activation. The latter contributes to LTM formation processes, since imitation of the extended PKA activation in the AL in conjunction with a single conditioning trial induces LTM (Müller, 2000).

Recent findings suggest that a distinct temporal activation of the cAMP cascade, dependent on distinct stimulation parameters, is required for the induction of long-lasting neuronal and behavioral changes. A close connection has been demonstrated between stimulation parameters and the temporal dynamics of changing cAMP levels, adenylate cyclase activity, PKA activity, and CREB phosphorylation (Müller, 2000 and references therein).

During associative conditioning in the honeybee, the temporal dynamics of PKA activation in the ALs depend on both the sequence of CS and US stimulation and the number of conditioning trials. The mechanism underlying the sequence-dependent PKA activation is distinguishable and independent from that underlying multiple trial-induced PKA activation, as demonstrated by selective impairment of the latter by blocking NOS activity. Regardless of the number of trials, US/CS backward pairing and US stimulation induce the same transient PKA activity in the ALs. The US-induced PKA activation in the ALs is mediated by octopamine. The octopamine in the ALs is most likely released by the VUMmx1 neuron, which has been shown to substitute for the US function in associative olfactory conditioning. The extensive aborizations of the VUMmx1 neuron in the ALs suggest that the US-mediated PKA activation occurs within all AL glomeruli. In contrast to this, CS stimulation induces odor-specific changes in Ca2+ levels in distinct subsets of glomeruli. Thus, it is conceivable that the prolonged PKA activation induced by CS/US forward pairing is due to a sequence-dependent interaction between an odor-specific Ca2+-mediated process in distinct glomeruli and a general US/octopamine-mediated process (Müller, 2000 and references therein).

Dually regulated enzymes, like the Ca2+/calmodulin-dependent adenylate cyclase, have been suggested as molecular convergence sites of different inputs important for neuronal plasticity and learning. In membrane fractions of Aplysia neurons, the maximal in vitro activation of the Ca2+/calmodulin-dependent adenylate cyclase is achieved when the Ca2+ stimulus precedes the transmitter stimulus. Although direct evidence is lacking, the dually-regulated adenylate cyclase may be implicated in the sequence-specific prolongation of PKA activity induced by a CS/US forward pairing in the honeybee (Müller, 2000).

Findings from Aplysia and Drosophila assign a critical role for induction of long-lasting changes to the balance of activator and repressor isoforms of CREB. The results from the honeybee, however, show that already during the short conditioning time window a distinct temporal pattern of PKA activation is critical for LTM induction. Assuming a similar connection between the PKA pathway and CREB in the induction of LTM in honeybees, future investigations demand a characterization of whether and how the multiple trial-induced prolonged PKA activation acts on CREB function. But since regulation of CREB isoforms and their function in long-term neuronal and behavioral changes are results of a complex interaction of different second messenger systems, it is very likely that different signaling cascades contribute to LTM formation (Müller, 2000 and references therein).

It is conspicuous that both the formation of multiple trial-induced LTM and the prolonged PKA activation in the ALs require NO-mediated mechanisms. The finding that photorelease of cGMP in the ALs in combination with single-trial conditioning induces LTM supports the idea that the NO/cGMP system within the ALs mediates the prolongation of PKA activation during conditioning. Although the neurons containing the NO-activated guanylate cyclase have not been described in the honeybee, it is most likely that the NO-releasing neurons that modulate cGMP levels in the target cells are located within the ALs. Uncaging NO in the entire AL in combination with single-trial conditioning, however, leads to a significant reduction in conditioned PER as tested at 3 hr and 3 days. Although the reason for this learning impairment is unknown, photolyzing NO in the entire AL probably interferes with a specific function of NO in signal processing during olfactory learning. The latter is very likely, since an odor induces changes in Ca2+ concentrations in a subset of glomeruli only. This in turn results in the activation of the Ca2+-dependent NOS and thus in release of NO in a characteristic subset of glomeruli only. Possibly such a CS-specific release of NO within a subset of glomeruli contributes to aspects of olfactory signal processing required for learning (Müller, 2000 and references therein).

In this context it is interesting to note that multiple conditioning trials lead to more specific responses and thus may be based on more specific synaptic plasticity. The latter may be due to a Hebbian mechanism of pre/postsynaptic activity detection. It has been proposed that such a mechanism may also be essential for invertebrates and that NO may play a central function as a retrograde signaling molecule (Müller, 2000 and references therein).

A series of studies in Drosophila convincingly demonstrate that the mushroom bodies (MBs) are essential for olfactory learning, and that they support context generalization in visual learning, and are required for memory formation of courtship conditioning. These findings not only support the important role of the MBs as multisensory processing centers but also demonstrate that the contribution of the MBs differs, depending on the learning paradigm and the sensory modality used. In contrast to the considerable knowledge with regard to the function of the MBs in Drosophila learning, it was only recently proposed that the ALs contribute to short-term memory in Drosophila courtship conditioning (Müller, 2000 and references therein).

In honeybees, it has been demonstrated that initial olfactory memory (tested 20 min after conditioning) can be induced independently in either the MBs or the ALs. In contrast to Drosophila, however, the majority of studies focused on the function of the ALs in olfactory learning. It has been demonstrated that differential olfactory conditioning causes changes in the neural representation of the rewarded and the unrewarded odor in the ALs for up to at least 30 min after conditioning. Moreover, the requirement of a constitutively active PKC in the ALs for multiple trial-induced MTM suggests that processes located in the ALs contribute to memory maintenance in the range of hours. The results presented here now demonstrate that a prolonged PKA activation in the ALs induced by multiple-trial conditioning is implicated in induction of LTM. However, since imitation of prolonged PKA activation in conjunction with single-trial conditioning does not reach the level of conditioned PER after multiple-trial conditioning, a contribution by other brain areas must be proposed. Collectively, all these findings provide evidence that the ALs are sites that contribute to processes of associative olfactory learning during the conditioning procedure itself and in early phases of memory formation for up to several hours. Moreover, the ALs are possibly also sites of long-lasting structural changes. Activity-dependent changes described for the glomerular volume in the ALs may well be the result of structural plasticity underlying long-term memory (Müller, 2000 and references therein).

Interestingly, in mice and sheep the accessory olfactory system has also been implicated in the formation of olfactory memory. While female mice form a memory of the pheromones of the mating male, sheep learn to recognize the odors of their lambs in the first hours after birth. In both cases, NO has been demonstrated to mediate the formation of this memory. In mice the coincident activation of pheromonal inputs and exogenous administration of NO in the accessory olfactory system can induce a pheromone-specific olfactory memory without mating. Blocking of NOS activity in the olfactory system of sheep prevents the formation of olfactory memory. Although the targets of the NO/cGMP system in the olfactory systems of honeybees, mice, and sheep differ, the conspicuous parallels suggest a conserved function of NO-mediated signaling in the olfactory systems with respect to olfactory memory formation (Müller, 2000 and references therein).

Nitric oxide has been shown to be implicated in neural plasticity that underlies processes of learning and memory. In the honeybee, studies on the role of nitric oxide in associative olfactory learning reveal its specific function in memory formation. Inhibition of nitric oxide synthase during olfactory conditioning impairs a distinct long-term memory that is formed as a consequence of multiple learning trials. Acquisition or retrieval of memory or memory formation induced by a single learning trial is not affected by blocking of nitric oxide synthase. This finding provides a first step toward dissection of molecular mechanisms involved in memory formation, in general, and the special function of nitric oxide synthase in particular (Muller, 1996).

Protein Interactions

Drosophila NOS is dependent on exogenous Ca2+/calmodulin and on NADPH, two cofactors necessary for activity of constitutive mammalian NOSs (Regulski, 1995).

Drosophila nitric-oxide synthase gene encodes a family ofproteins that can modulate NOS activity by acting as dominant negative regulators

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. The only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS) has been cloned and analyzed. It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. Eight new transcripts have been identified that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5'-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene led to premature termination of the open reading frame. As a result, none of the alternative transcripts encoded an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, it has been demonstrated that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. These results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene (Stasiv, 2001).

Drosophila NO synthase combines some of the features of all three mammalian NOS isoforms. Full-length DNOS1 protein (1350 aa) reveals 43%, 40%, and 39% aa identity to rat nNOS, bovine eNOS, and mouse iNOS, respectively. However, the central portion of the coding region shows the highest similarity to the neuronal NOS isoform of mammals. Furthermore, the distribution of exons in regions of dNOS that encode the cofactor-binding sites of the enzyme is highly similar (and for many exons, identical) to that in nNOS. Seven dNOS exons are identical in size and intron type to the homologous exons in human nNOS gene, whereas dNOS exons 10 and 11 appear to be a rearranged fusion of homologous human nNOS exons 11-13. Evolutionary conservation is evident mainly in the regions crucial for catalytic activity of NOS, from the heme-binding site to beyond the CaM-binding site. This region also exhibits the highest level of nucleotide homology with the human nNOS, 61% of aa of the DNOS1 oxygenase domain being identical to the corresponding region in human nNOS. In contrast, much of the reductase domain of the Drosophila enzyme is encoded by a single 1142-nt-long exon 16, whereas the homologous region in the human nNOS gene is dispersed among eight exons (exons 19-26). Together, the structural and sequence homology indicate that dNOS is orthologous to the mammalian nNOS (Stasiv, 2001).

The organization of the region that codes for the DNOS1 oxygenase domain is also highly similar to the organization of the oxygenase-coding region of the mosquito A. stephensi NOS gene. Seven of the dNOS exons are identical in size and intron type to the corresponding AsNOS exons. Exon 11 of the dNOS gene appears to be composed of two homologous exons (exons 8 and 9) in the mosquito gene, whereas two dNOS exons (exons 8 and 9) correspond to exon 6 of the AsNOS gene. The overall homology between the fly and mosquito proteins is 81% (with 69% of the aa identical), whereas the homology within oxygenase domains and CaM-binding sites reaches 88% (with 78% of the aa identical). Interestingly, there is no exon-intron conservation between these two insect NOS genes in their reductase domains. The structural similarity between the mosquito AsNOS and the human nNOS genes within their reductase-coding regions is higher than similarity between corresponding regions of the dNOS gene and its human counterpart. However, additional studies of NOS genes from various invertebrate and vertebrate species are needed to better characterize the evolution of NOS gene (Stasiv, 2001).

The dNOS gene demonstrates a remarkable degree of transcriptional complexity resembling that of mammalian nNOS. Alternative transcription initiation sites combined with the alternative usage of splice sites generate a family of dNOS transcripts. Four alternative variants of the first non-coding exon were found: 1a, 1b, 1c, and 1d. All of them are located within the 6-kb-long 5'-region of the dNOS gene upstream of exon 2. Most likely these exons begin at alternative transcription start sites; using 5'-RACE, it was not possible to extend these exons farther upstream, and using RT-PCR, it was not possible to detect dNOS transcripts in which exon 1 variants are spliced to each other. Exon 1b is common to several dNOS transcripts (dNOS1, 4, 5, and 6), whereas exons 1a, 1c, and 1d are present in the dNOS7, dNOS8, and dNOS3 isoforms, respectively. The fact that dNOS3 and dNOS8 transcripts are expressed only during the larval stage of Drosophila development further supports the notion that alternative promoters are used to direct expression of the different dNOS RNAs in a tissue- and/or development-specific manner. This mechanism of NOS regulation seems to be evolutionary conserved; similarly, complex splicing patterns were found in the 5'-regions of mammalian nNOS genes. For instance, in the human nNOS gene, nine differentially expressed variants of non-coding exon 1 were identified. It is unclear, though, whether these human nNOS transcripts have additional structural alterations (frameshifts, in-frame deletions, or insertions) within their coding region, similar to those found in the dNOS transcripts (Stasiv, 2001).

Alternative splicing affects the coding region as well as the 5'-UTR of the dNOS gene. Transcript dNOS3 is an example of a deleted coding exon (exon 3). This deletion leads to a frameshift and premature termination of the dNOS3 ORF. A transcript similar to the dNOS3 was found in the mosquito A. stephensi, where deletion of exon 2 (translation start codon in the AsNOS is located in exon 1) causes a premature termination of the ORF in the exon 2 mRNA (Stasiv, 2001).

Another transcript, dNOS10, has a novel type cassette in-frame deletion of three consecutive exons (exons 15-17) that composes almost 40% of the dNOS-coding region. Thus, DNOS10 protein retains only 57 carboxyl-terminal aa of the reductase domain. No splicing alterations in this part of NOS gene have been found in other organisms (Stasiv, 2001).

Several dNOS transcripts arise due to the insertion of extra exon(s) in their coding regions, resulting in the premature termination of their ORFs. The dNOS4 and dNOS7 transcripts contain an alternative exon 14a, whereas dNOS5 and dNOS6 RNAs have two extra exons, either 13a plus 14a or 13b plus 14a, respectively. Similar types of exon insertions that introduce premature stop codons have been found in mammalian NOS genes. Two alternative transcripts arise from the human nNOS primary transcript after the unusual splicing of intron 16 (33). In the nNOS+47 RNA, the first 47 nt at the very 5'-end of intron 16 are inserted into the coding region between exons 16 and 17. The nNOS+67 transcript has an extra 67 nt (derived from the central part of intron 16) inserted between exons 16 and 17. Both insertions introduce an in-frame stop codon. Although exon 16 of the human nNOS gene is homologous to exon 14 of the dNOS gene, no dNOS RNA species were detected with insertions of an extra exon(s) downstream of exon 14. Thus, the fruit fly most likely does not have an RNA isoform corresponding to the mammalian nNOSµ transcript, which contains an in-frame 102-nt-long insertion between exons 16 and 17 and is expressed in various rat and human tissues (Stasiv, 2001).

Alternative splicing of the mosquito AsNOS pre-mRNA causes an insertion of additional exon (174 nt long) between exons 11 and 12 in the exon 11⁺ transcript. An alternative AsNOS exon contains a stop codon, which results in a premature termination of translation 16 aa downstream of the novel splice junction. This product of the AsNOS gene resembles the dNOS4 transcript. Finally, transcript dNOS2 has an in-frame cassette deletion of exons 8 and 9 that is identical to the deletion of exons 9 and 10 found in the alternative transcript nNOS-2 of the human nNOS gene (Stasiv, 2001).

Individual dNOS isoforms are differentially expressed in the developing Drosophila according to quantitative RT-PCR results. Although dNOS1 is a predominant RNA product in embryo, larva, and imago, it is important to note that these experiments were performed with RNA pools representing various phases of each developmental stage. It is possible that individual dNOS transcripts are transiently induced at selected steps of the developmental cascade (e.g., before pupariation, when a strong increase in diaphorase staining is observed) (Stasiv, 2001).

The family of dNOS transcripts encodes a variety of DNOS-like proteins. Seven of them, DNOS3, -4, -5, -6, -7, -9, and -10 (but not DNOS2), lack either part of or the entire reductase domain, which leads to a loss of enzymatic activity. However, most of the truncated DNOS proteins (except for DNOS2 and DNOS3) retain almost the entire oxygenase domain, including the sites that are thought to be responsible for NOS homodimerization in mammalian cells. Thus, these truncated forms lack NOS enzymatic activity but may still retain their ability to dimerize. This notion is supported by experiments in which interaction between GAL4-DNOS4 hybrid proteins (DNOS4 was fused to GAL4 binding and to GAL4 activation domain) was detected using a yeast two-hybrid system. Moreover, this implies that the truncated DNOS proteins may not only form dimers with each other but also form heterodimers with the full-length DNOS1. In experiments with purified nNOS and iNOS homodimers, the flow of electrons during catalysis has been shown to occur from the flavins in the reductase domain of one subunit to the heme iron in the oxygenase domain of the other subunit. This suggests that heterodimers between the full-length and truncated NOS polypeptides will have diminished enzymatic activity. Indeed, rat nNOS full-length polypeptide and a synthetic polypeptide lacking the reductase domain can form heterodimers in vitro, but these complexes show drastically decreased enzymatic activity. Furthermore, a fragment of eNOS that lacks the reductase domain can form complexes with the full-length eNOS when co-expressed in cultured cells and exhibits a strong dominant negative effect on eNOS activity. Thus, truncated DNOS proteins capable of forming heterodimers with DNOS1 may act as dominant negative inhibitors of NO production. This notion was confirmed in experiments in which DNOS4, DNOS5, and DNOS6, each of which can form complexes with DNOS1, were able to strongly suppress NOS activity when co-expressed along with the DNOS1. This suggests that formation of heterodimers between the full-length DNOS1 and its truncated isoforms may be a basis for a mechanism of regulation of NO production in Drosophila . It will be interesting to determine whether truncated DNOS proteins are indeed synthesized in the fruit fly and whether they can serve to modulate NOS activity in vivo (Stasiv, 2001).

In summary, these results demonstrate that the dNOS locus in Drosophila generates a large family of transcripts, some of which code for truncated DNOS-like proteins. These proteins are capable of suppressing the enzymatic activity of the full-length DNOS1 protein, perhaps by disrupting the dimerization of DNOS1 molecules. Because such truncated NOS proteins have been postulated to exist in mammals, this novel regulatory function proposed for the truncated Drosophila NOS proteins may apply more widely for NOS regulation (Stasiv, 2001).

Regulation of multimers via truncated isoforms: a novel mechanism to control nitric-oxide signaling

Nitric oxide (NO) is an essential regulator of Drosophila development and physiology. A novel mode of regulation of NO synthase (NOS) function is described that uses endogenously produced truncated protein isoforms of Drosophila NOS (DNOS). These isoforms inhibit NOS enzymatic activity in vitro and in vivo, reflecting their ability to form complexes with the full-length DNOS protein (DNOS1). Truncated isoforms suppress the antiproliferative action of DNOS1 in the eye imaginal disc by impacting the retinoblastoma-dependent pathway, yielding hyperproliferative phenotypes in pupae and adult flies. These results indicate that endogenous products of the dNOS locus act as dominant negative regulators of NOS activity during Drosophila development (Stasiv, 2004).

The dNOS locus of Drosophila is subject to complex transcriptional and posttranscriptional regulation. It produces a large variety of mRNA isoforms through the use of multiple promoters and alternative splice sites. Only one of them, dNOS1, codes for the full-length enzymatically active protein. Another abundant alternative transcript of the dNOS gene is the dNOS4 isoform, which retains the entire intron 13 (this 109-nucleotide-long segment is now referred to as exon 14a of dNOS4). The resulting open reading frame is terminated by a stop codon 63 nucleotides into exon 14a. It encodes a protein containing 757 amino acids with a predicted molecular mass of 84 kDa (cf. DNOS1 is 1350 amino acids, 150 kDa). DNOS4 contains a unique 21-amino acid-long C-terminal peptide encoded by exon 14a, whereas the preceding 736 amino acids are identical to those of DNOS1. Thus, the DNOS4 protein is a truncated version of DNOS1; it lacks the entire reductase domain, while retaining the oxygenase domain. Semiquantitative RT-PCR analysis indicates that dNOS4 mRNA is expressed in the embryo at levels comparable to those of dNOS1 mRNA; dNOS4 levels are lower in larvae and in adult flies, whereas dNOS1 levels do not change appreciably (Stasiv, 2004).

Another variant of dNOS mRNA, dNOS7, encodes a protein identical to DNOS4; however, the transcription initiation site for dNOS7 RNA is different from that of dNOS4 (exon 1a vs. exon 1b, respectively). Unlike dNOS4, dNOS7 is exclusively expressed during the larval stage (Stasiv, 2004).

DNOS4 lacks the C-terminal reductase domain that participates in electron transfer during catalysis, while it retains the catalytic N-terminal oxygenase domain, including the critical heme-binding site. DNOS4 also retains a long stretch of glutamine (Gln) residues at the N terminus; such regions have been shown to promote multimerization of proteins; note that such Gln-rich region is not present in mammalian NOS proteins. These structural features of DNOS4 predict that (1) DNOS4 itself is incapable of producing NO, (2) it may be capable of forming heterodimers with DNOS1, and (3) heteromers between DNOS1 and DNOS4 will have reduced enzymatic activity. To investigate whether DNOS4 is capable of forming a heteromeric complex with DNOS1 and suppressing NOS activity, and to examine which region of DNOS4 may contribute to its effects on DNOS1, expression plasmids were used for DNOS1 and DNOS4 proteins, each with a short peptide tag fused to its C terminus, the influenza virus hemagglutinin (HA) epitope-tagged DNOS1 (pDNOS1-HA), and the synthetic FLAG epitope-tagged DNOS4 (pDNOS4-FLAG). A plasmid was generated, pDNOSoxy-FLAG, that codes for a shorter version of DNOS4 (residues 214-631) and carries the FLAG epitope at its C terminus. It lacks the Gln-rich region at the N terminus, thus representing the "core" oxygenase domain of DNOS, which is highly similar to previously defined oxygenase domains of mammalian NOS proteins (Stasiv, 2004).

To test the effect of DNOS4 and DNOSoxy proteins on enzymatic activity of the full-length DNOS1, recombinant plasmids were transiently coexpressed in cultured human embryonic kidney cells (293 cells) and NOS enzymatic activity was measured in cell-free extracts. NOS activity was undetectable in lysates of untransfected cells and in cells expressing either DNOS4-FLAG or DNOSoxy-FLAG, whereas lysates from cells transfected with pDNOS1-HA showed significant levels of NOS activity. In contrast, enzymatic activity in cell lysates was decreased when a constant amount of the pDNOS1-HA was cotransfected with increasing amounts of the pDNOS4-FLAG. At a 1:1 molar ratio of plasmids encoding DNOS4 and DNOS1, NOS activity was 57% of the activity seen in lysates from cells transfected by the plasmid encoding full-length DNOS1. At a 3:1 ratio, the activity dropped to 36%, and at 10:1 ratio it was 10% of the control level. pDNOSoxy-FLAG, which lacks both the reductase domain and the Gln-rich region, is as effective at suppressing NOS activity (13.5% of control levels when cotransfected with pDNOS1-HA at 10:1 molar ratio) as pDNOS4-HA (which retains the Gln-rich region). Importantly, expression levels of DNOS1 protein were not affected by coexpressed truncated DNOS variants, as determined by immunoblotting using HA-specific antibodies. This observation indicates that a decrease of NOS activity was not simply due to a decrease in expression of the DNOS1 protein, but rather was caused by the presence of coexpressed shorter DNOS polypeptides. Furthermore, removal of the Gln-rich region did not alter the inhibitory effect of DNOSoxy on the enzymatic activity of DNOS1, indicating that the Gln-rich stretch does not appreciably contribute to the inhibitory action of DNOS4. Together, these results indicate that truncated enzymatically inactive forms of DNOS can suppress the activity of the full-length DNOS1 protein, thereby acting as dominant negative inhibitors of NO production (Stasiv, 2004).

To examine the potential of DNOS1 and DNOS4 to form complexes, immunoprecipitation experiments were performed after coexpressing differentially tagged DNOS1 and DNOS4 in 293 cells. Each tagged DNOS protein was immunoprecipitated from extracts of cotransfected cells with antibody specific to its epitope tag (HA for DNOS1 or FLAG for DNOS4). Subsequently, the formation of heteromeric DNOS complexes was examined by immunoblotting using HA-specific antibody for the samples immunoprecipitated with FLAG-specific antibody and vice versa (Stasiv, 2004).

When HA-tagged DNOS1 is expressed in 293 cells, it can be immunoprecipitated with HA-specific but not with FLAG-specific antibody. Conversely, FLAG-tagged DNOS4 can be immunoprecipitated using FLAG-specific, but not HA-specific antibodies. However, if DNOS1-HA and DNOS4-FLAG are coexpressed, each of them can be immunoprecipitated by antibody to either tag. Furthermore, when DNOSoxy-FLAG was coexpressed with DNOS1-HA, complexes between these two proteins were detected using the same combination of immunoprecipitation and immunoblotting. This suggests that even a part of the oxygenase domain lacking the Gln-rich region, but retaining the heme-binding region, is sufficient to form heteromers with the full-length DNOS1 protein. Finally, when lysates of cells transfected separately with either pDNOS1-HA or pDNOS4-FLAG were combined in vitro, the two proteins did not coimmunoprecipitate with each other, indicating that heteromeric DNOS complexes are not formed after cell lysis (Stasiv, 2004).

Together, these data point to a possible mechanism for the observed dominant negative effect of DNOS4 on DNOS1 activity, in which formation of DNOS1-DNOS4 heterodimers inhibits NOS activity by reducing formation of enzymatically active homodimers of DNOS1 (Stasiv, 2004).

To determine whether DNOS4 can act as a dominant negative regulator of NOS activity in vivo, transgenic flies were generated that express FLAG-tagged DNOS4 under the control either of the heat-shock inducible promoter of the hsp70 gene (hs-DNOS4-FLAG flies), or of the GMR promoter, which is active in all cells of the eye imaginal disc within, and posterior to the morphogenetic furrow (GMR-DNOS4-FLAG flies). It was confirmed that DNOS4-FLAG was expressed in transgenic flies; anti-FLAG antibodies detect a protein with an expected mass of 86 kDa in extracts from heads of GMR-DNOS4-FLAG and from hs-DNOS4-FLAG flies, but not in extracts from wild-type flies. The mobility of this protein was identical to that of a protein produced in 293 cells after transfection with pDNOS4-FLAG. Importantly, the expression levels of endogenously produced DNOS1 were not affected by ectopic expression of DNOS4 in transgenic flies. Furthermore, immunochemical detection with FLAG-specific antibodies showed that DNOS4-FLAG was expressed in the expected pattern within, and posterior to, the morphogenetic furrow in the eye imaginal disc of third-instar GMR-DNOS4-FLAG larvae (Stasiv, 2004).

NOS activity was examined in extracts from heads of adult flies and it was found that in hs-DNOS4-FLAG flies and in GMR-DNOS4-FLAG flies activity was 52% and 60%, respectively, of that in control wild-type flies. This indicates that endogenous NOS activity is inhibited by ectopically expressed DNOS4, paralleling observations with cultured cells (Stasiv, 2004).

To determine whether the observed decrease in NOS activity in transgenic flies is accompanied by in vivo formation of DNOS1-DNOS4 heterodimers, coimmunoprecipitation experiments were performed. Using FLAG-specific antibody, proteins were precipitated from head extracts of adult wild-type or hs-DNOS4-FLAG transgenic flies, and then were analyzed by immunoblotting using anti-DNOS1 or anti-FLAG antibodies. Importantly, full-length DNOS1 protein can be immunoprecipitated with anti-FLAG antibody from extracts of hs-DNOS4-FLAG transgenic flies, but not of wild-type flies. This indicates that ectopically expressed DNOS4 can form heterodimeric complexes with endogenous DNOS1 in vivo and suggests that formation of such complexes may explain the dominant negative effect of DNOS4 on NOS activity (Stasiv, 2004).

NO acts as an antiproliferative factor during eye development; pharmacological inhibition of NOS activity results in an increase in the number of cells in the ommatidia. It was asked whether DNOS4 can act to suppress the antiproliferative action of NO and affect cell division in the developing eye. Ectopic expression of DNOS4 driven by an eye-specific promoter in GMR-DNOS4-FLAG flies results in a distorted eye phenotype with visible rearrangements of the ommatidia lattice and extra bristles. To analyze the changes in more detail, the sections of the retina from wild-type and transgenic pupae were compared. GMR-DNOS4-FLAG flies have extra secondary and tertiary pigment cells as well as extra bristle cells; no changes were detected in the number of photoreceptor cells. These extra cells distort the regular hexagonal shape of the ommatidia and the general lattice pattern of the retina (Stasiv, 2004).

To determine whether the increase in the number of cells in pupae was related to increased proliferation in the imaginal discs of larvae, the nuclei of eye-disc cells were labeled in S phase of the cell cycle with 5-bromo-2'-deoxyuridine (BrdU). There was a 2.13-fold increase in the number of BrdU-positive cells in imaginal discs from GMR-DNOS4-FLAG flies as compared with wild-type flies. Extra dividing cells were localized to the region of GMR-driven transgene expression in the morphogenetic furrow and the area posterior to it. The observed increase in the number of dividing cells and the number of cells per ommatidium suggests that introduction of the dominant negative dNOS4 transgene resulted in inhibition of NOS activity and suppression of the antiproliferative effect of NO in the developing eye (Stasiv, 2004).

Because NO interacts with the retinoblastoma (Rb) pathway to control cell division in the developing Drosophila eye, it was of interest to determine whether DNOS4 can act to affect signaling via the Rb pathway in the eye (Stasiv, 2004).

RBF is the Drosophila ortholog of mammalian Rb proteins; like Rb, RBF acts as a negative regulator of cell cycle progression. GMR-RBF flies that carry four copies of the RBF transgene have a profound eye phenotype, due to suppression of cell division by the elevated levels of RBF; some of the pigment cells and bristles are missing and some ommatidia are fused. The RBF phenotype is strongly enhanced by the ectopic overexpression of the dNOS1 transgene. To test the effects of DNOS4 on the action of RBF, GMR-DNOS4-FLAG flies were crossed and then backcrossed to GMR-RBF flies to generate flies bearing one copy of the dNOS4 transgene along with four copies of the RBF transgene. The eyes of the resulting hybrid flies had an almost normal phenotype, without fused ommatidia, and with the usual set of bristles. Thus, the effect of RBF overexpression was counteracted by DNOS4, indicating that this inhibitor of NOS activity counteracts RBF function in the developing eye (Stasiv, 2004).

In mammalian and Drosophila cells, Rb is associated with the E2F transcription-factor complex, whose activity is required for the cells' entry into S phase. Overexpression of E2F overcomes Rb-mediated G1 arrest and induces quiescent cells to enter S phase. Ectopic expression of dE2F/dDP (Drosophila orthologs of mammalian components of the E2F complex) driven by the GMR promoter during development results in generation of extra cells in the adult eye (e.g., multiple extra bristles), particularly when combined with the p35 gene, a baculoviral inhibitor of apoptosis. GMR-DNOS4-FLAG flies were crossed with GMR-dE2F/dDP/GMR-p35 flies and an even more pronounced eye phenotype was found, with a larger eye surface, extra ommatidia, and severe rearrangements of the ommatidia lattice. This indicates that DNOS4 acts to enhance the function of E2F, consistent with the proposed role for DNOS4 as a suppressor of the inhibitory action of NO on cell cycle progression (Stasiv, 2004).

Together, these genetic data confirm that development of the Drosophila eye depends on NO or NO-activated signaling pathways interacting with the Rb pathway. They suggest that, by suppressing the antiproliferative action of NO, DNOS4 acts in vivo to suppress the effect of RBF, and to enhance the effect of E2F, on cell cycle regulation (Stasiv, 2004).

DEVELOPMENTAL BIOLOGY

Nitric oxide (NO) is a membrane-permeant signaling molecule that activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP). The NO/cGMP signaling system is thought to play essential roles during the development of vertebrate and invertebrate animals. The cellular expression of this signaling pathway during the development of the Drosophila melanogaster nervous system has been analyzed. Using NADPH diaphorase histochemistry as a marker for NO synthase, several neuronal and glial cell types have been identified as potential NO donor cells. To label NO-responsive target cells, the detection of cGMP by an immunocytochemical technique was used. Incubation of tissue in a NO donor induces cGMP immunoreactivity (cGMP-IR) in individual motoneurons, sensory neurons, and groups of interneurons of the brain and ventral nerve cord. A dynamic pattern of the cellular expression of NADPHd staining and cGMP-IR is observed during embryonic, larval, and prepupal phases. The expression of NADPH diaphorase and cGMP-IR in distinct neuronal populations of the larval central nervous system (CNS) indicates a role for NO in transcellular signaling within the CNS and as a potential retrograde messenger across the neuromuscular junction. In addition, the presence of NADPH diaphorase-positive imaginal discs containing NO-responsive sensory neurons suggests that a transcellular NO/cGMP messenger system can operate between cells of epithelial and neuronal phenotype. The discrete cellular resolution of donor and NO-responsive target cells in identifiable cell types will facilitate the genetic, pharmacological, and physiological analyses of NO/cGMP signal transduction in the developing nervous system of Drosophila (Wildemann, 1999a).

The grasshopper embryo has been used as a convenient system with which to investigate mechanisms of axonal navigation and pathway formation at the level of individual nerve cells. The developing antenna of the grasshopper embryo (Schistocerca gregaria), where two siblings of pioneer neurons establish the first two axonal pathways to the CNS (Seidel, 2000).

As the embryonic antennae begin to evaginate from the head, they are initially devoid of sensory neurons. Unlike in the thoracic and gnathal segments, where a single sibling pair of sensory cells serve as pioneers to the CNS, in the antenna, two sibling pairs of pioneer neurons are born. These are termed ventral and dorsal pioneers. To examine neurogenesis and peripheral pathfinding in the antenna, the differentiating pioneer neurons were labeled at various stages with a neuron-specific anti-HRP antiserum. This method allowed the reliable identification of the pioneer neurons. The first cells that express HRP-IR at 32% of embryonic development are the pair of ventral pioneer neurons (vPN) at the distal tip of the antenna. At the same time, a single immunoreactive neuronal cell body appears at the base of the antenna. The axon of this base pioneer (BP) is the first peripheral process to reach the CNS from the antenna. During the next few hours two additional dorsal pioneer neurons (dPN) also become immunoreactive. Subsequently, the growth cones of the vPN extended along the ventral inner surface of the antennal epithelium with a migration path that runs straight to the BP. At 34%, the growth cones come into filopodial contact with the BP. Compared with the axons of the vPN the processes of the dPN initiate their outgrowth slightly later, navigating along the dorsal inner surface of the antennal lumen. The dorsal pioneer neurons do not follow a straight trajectory towards the CNS. Rather, after growing for a distance of 100 mm in proximal direction, the axons of the dorsal pioneers performed a prominent turn of about 90°, extending ventrally towards the BP. Remarkably, the BP loses its HRP immunoreactivity after the growth cones of the vPN and the dPN make contact. Subsequently, the processes of vPN and dPN fasciculate with the axon of the base pioneer, which, meanwhile, has established the earliest afferent pathway towards the brain. The axonal projections of the pioneers enter the HRP-stained CNS at a distance of about 50-80 mm from the base pioneer (Seidel, 2000).

At 38% of development, additional HRP-IR neurons differentiate in the antenna. These neurons elaborate prominent apical dendrites indicative of their phenotype as sensory cells. During the period 38%-45% of embryogenesis, the regions of sensory cell formation appear restricted to three circumferential bands. Initially sensory neurons are born at the tip of the antenna. A few hours later another two other regions that initiated sensory cell formation can be discerned. One region is positioned approximately in the middle of the antenna slightly more distal to the pioneer neurons. The third region is found near the base of the antenna but proximal to the BP cell. In this zone, sensory cells of the later pedicellar chordotonal organ are generated. Neurogenesis of numerous sensory cells continues within the three initiating zones. Parallel to the onset of segmentation at 45%, additional sensory cell initiating zones differentiate within the frame of the developing annular segments of the antenna. Taken together, these results indicate that the vPN and dPN axons prefigure two axonal fascicles to the brain, which are joined by later-born sensory neurons to form the bipartite antennal nerve of larval and adult stages (Seidel, 2000).

About midway through insect embryogenesis, the appearance of NO-induced cGMP synthesis in selective neuronal cell types appears to be a common developmental phenomenon. The peripheral neurons of the antenna express cGMP-IR in a rather early stage of development. After stimulation with the NO donor SNP, cGMP-IR can be induced in both pairs of the pioneer neurons in the antenna. The onset of the cGMP-IR becomes visible in the 38% stage, and immunoreactivity persists during the following developmental period. As has been reported for other embryonic grasshopper neurons, strong cGMP-IR is also found in the nuclei of the antennal pioneers. At a 55% of development stage, a second embryonic cuticle is secreted that prevents the access of compounds like SNP or IBMX in embryonic whole mounts. Therefore, with this experimental approach, how long the NO-induced cGMP-IR persists during embryogenesis could not be examined. Occasionally, an additional occurrence of NO induced cGMP-IR was noticed in some distal sensory neurons in preparations of later stages (50%-55%). Embryos that are exposed to 200 mM ODQ for at least 20 minutes or during a culture period of 24-30 hours fail to express cGMP-IR after stimulation with an NO donor. These observations provide evidence that the inhibitors used to study blocking effects of the cGMP pathway are suitable to reduce the sGC activity in situ effectively (Seidel, 2000).

To search for potential cellular sources of NO, NADPH-diaphorase staining of formalin-fixed embryonic whole mounts was used as a histochemical marker for NOS. On transverse sections through the antenna, the blue precipitate of the diaphorase reaction is found concentrated in parts of the epithelial cells facing the basal lamina. The staining of the basal parts of epithelial cells is not very pronounced but there is a striking contrast in staining intensity compared with the mesodermal tissue bordering the basal lamina. Diaphorase staining of the epithelial cells becomes visible during a developmental period ranging from about 32%-35% and disappears at later stages (Seidel, 2000).

To investigate the role of the NO/cGMP signaling system during pathfinding, the pattern of outgrowing pioneer neurons in embryo culture was examined. Pharmacological inhibition of soluble guanylyl cyclase and of NO synthase results in an abnormal pattern of pathway formation in the antenna. Axonogenesis of both pairs of pioneers are inhibited when specific NOS or sGC inhibitors are added to the culture medium; the observed effects include the loss of axon emergence as well as retardation of outgrowth, such that growth cones do not reach the CNS. The addition of membrane-permeant cGMP or a direct activator of the sGC enzyme to the culture medium completely rescues the phenotype resulting from the block of NO/cGMP signaling. These results indicate that NO/cGMP signaling is involved in axonal elongation of pioneer neurons in the antenna of the grasshopper (Seidel, 2000).

Larval and pupal stages

To visualize the expression of Drosophila NOS, histochemical staining for the NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) activity of NOS was used. Beginning in the third instar, NADPH-diaphorase staining is observed in all imaginal discs, imaginal rings, histoblasts, and the larval brain. Staining becomes more intense as development proceeds, and by late third instar (larvae and early pupae), a highly specific and reproducible pattern of very intense staining is evident. In the leg imaginal disc, staining is initially seen at the very beginning of the third instar. Staining is confined to the center of the disc, corresponding to the presumptive distal tip of the leg. As the discs matured, staining intensifies. Later, when the discs begin to evert in the prepupae, staining of the forming leg becomes less intense. Wing, eye, haltere, and genital discs in the third instar have distinct and reproducible patterns of intense staining which gradually decreases in a specific spatial pattern during early pupal development (Kuzin, 1996).

During the development of retinal projections into the optic lobe lamina and medulla, the Nitric oxide synthase produced by the optic ganglia of these optic lobe structures produces NO, which serves to regulate the growth of individual retinal axons. Nitric oxide synthase produces NO through the conversion of arginine to citrulline, using NADPH as a cofactor. This activity can be detected histochemically. From the time of puparium formation and continuing for about forty hours, Drosophila brains show strong staining in regions of the optic ganglia and the lamina and medulla, as well as in Bolwig's nerve (see Bolwig's organ). In the visual system at 24 hours after puparium formation, an anti-NOS antibody labels projections in the neuropilar regions of both the lamina and the medulla, although the staining is most evident in the medulla. No staining is evident in the cell body layers. Double labelling with the NOS antibody and an antibody to chaoptin that recognizes photoreceptor cell bodies and axons shows that the axons of photoreceptors R7 and R8 terminate within a region of the medulla containing processes that stain strongly with the NOS antibody. These patterns of staining strongly suggest that NOS is present in retinal targets of photoreceptor axons (Gibbs, 1998).

Nitric oxide sensitive guanylate activity is present in subsets of photoreceptors during metamorphosis. The isolated CNS with intact eye imaginal discs was incubated with a NO donor and an inhibitor of phosphodiesterases. Many neurons within the brain and optic lobes show the appearance of cGMP after such incubation. At the onset of metamorphosis (at the time of puparium formation) and at least ten hours afterwards, this response is limited to the central brain and does not include the photoreceptor axons. Photoreceptors begin to respond shortly after this time. At 16 hours after puparium formation and continuing for more than 24 hours, cGMP is evident in the photoreceptor cell bodies and along the total length of their axons. cGMP synthesis is most dramatic in photoreceptors R1-R6 that project to the lamina, while R7 and R8 show slightly less cGMP synthesis (Gibbs, 1998).

The isolated CNS of Drosophila has been shown to undergo morphological changes in culture mirroring thos observed during metamorphosis. In culture, visual systems continue to develop. When stained with the chaoptin antibody, the segregation of R1-R6 and R7/8 into the lamina and medulla, respectively, is observed, as is the distinctive retinotopic patterning of R7 and R8 terminals in the medulla. After 96 hours in vitro, R7 and R8 have also segregated into two separate layers of the medulla. Although the retinal axons have arrived at their approximate targets and assembly of an organized optic lobe has commenced by 50 hours after puparium formation, the formation of synaptic connections between the photreceptors and optic lobe neurons occurs during the second half of metamorphosis. Thus, synapse formation has most likely not occurred even after 96 hours in vitro. Nervous system cultured in the presence of a competitive NOS inhibitor displays a disrupted pattern of photoreceptor projections in the medula. Individual axons are observed growing in an abnormal fashion beyond the borders of the medulla into the brain. The disruption of retinal axon projections observed with NOS inhibitior does not appear to be a result of severe degeneration or disorganization of targets in the medulla. The effects of NOS inhibition can be prevented with 8-bromo-cGMP, which appears to antagonize the disruptive effects of the NOS inhibitior. Methylene blue, which inhibits guanylate cyclase by binding to the heme groups and which also inhibits NOS activity through a similar mechanism, affects retinal patterning in the medulla, producing nervous systems with significantly greater disruption indices than does the specific NOS inhibitor (Gibbs, 1998).

What is the function of the NO produced by optic lobe neurons? Photoreceptors grow into the optic neuropil over a period of about 40 hours beginning in the third larval instar. Here, the axons arrange themselves retinotopically within their appropriate neuropils but do not immediately begin the process of assembly of the final connections. The first axons to reach their targest may remain in an arrest period for over a day prior to the time that the photoreceptor axons begin to seek their synaptic partners. Subsequent growth cone spreading and activity thus necessitate a mechanism permitting maintenance and flexibility of the growth cone while preventing outgrowth of the axon beyond the target. It is during this time that the retinal axons beome responsive to NO. When NO/cGMP signaling is disrupted, the retinal axons, rather than expanding to associate with their final targets, resume longitudinal growth to deeper layers of the medulla or into the brain. This aberrant growth suggests that NO provides an arrest signal that is required once the axons emerge from their dormant period. At this time, they are switching from a behavior pattern of responding to pathfinding cues to another pattern of forming interactive associations with potential synaptic partners. It is proposed that NO may serve as a stabilizing influence on the maneuvering growth cone, preventing further extension of the axons beyond the vicinity of appropriate post-synaptic neurons during period before the establishment of permanent connections (Gibbs, 1998).

Two types of cyclic GMP regulated neuronal maturation models exist in the grasshopper. One type, termed ecdysis regulated, is not mediated via NO. In this type, neurons that contain crustacean cardioactive peptide (see Cardioacceleratory peptide) develop in the subesophageal ganglion and in segmental arrays of cells in the CNS. These neurons exhibit an ecdysis-related expression of cGMP that is unresponsive to NO donor compounds. A second type of cGMP regulated maturation, termed developmentally regulated, is found in certain motoneurons, interneurons and sensory neurons late in embryonic development. For example, the RP2 motorneuron is the first in any segment to become responsive to NO generating compounds. Subsequently, within a segment, the motoneurons show a stereotyped order in which they become NO responsive. RP2 is followed by the acquisition of competence in the aCC motoneuron; the motor axons that innervate certain muscles become responsive after that. It would seem that every muscle group in the abdomen receives innervation from an axon that is NO responsive. Most of these muscles also receive innervation from other motor axons that do not exhibit NO sensitivity. In each case, neuron NO sensitivity appears after the growth cone has arrived at its target but before it has started to send out branches. NO sensitivity typically ends as synaptogenesis is nearing completion (Truman, 1996b).

Data from interneurons and sensory neurons are also consistent with the hypothesis that NO sensitivity appears as a developing neuron changes from axonal outgrowth to maturation and synaptogenesis. A well defined interneuron, the H cell, becomes NO response well after it is born, at a time when axon production is finished and the neuron begins maturation as characterized by soma enlargement, changes in membrane currents and elaboration of dendritic arbors. Mechanoreceptor sensory neurons that become NO sensitive include those of the dorsal body wall chordotonal organs, the segmental and wing-hinge stretch receptors, the abdominal 'ear,' campaniform sensilla and mechanosensory bristles. The photoreceptor neurons of the compound eye also become responsive to SNP. Some proprioceptors, like the dBw chordotonal neurons and the wing-hinge stretch receptors, are the first mechanoreceptors to become responsive to NO donors. Other proprioceptors, the afferents that supple external sensory organs (such as bristle and dome sensilla) are born late and develop NO sensitivity late. Associated glial cells also show a weak response to NO (Truman, 1996b).

Cyclic GMP likely constitutes part of a retrograde signalling pathway between a neuron and its synaptic partner. NO sensitivity also appears in some mature neurones at times when they may be undergoing synaptic rearrangement. Comparative studies on other insects indicate that the association between a NO-sensitive guanylate cyclase and synaptogenesis is an ancient one, as evidenced by its presence in both ancient and more recently evolved insect groups. Although cGMP responses are relatively poorly developed in embryos of Drosophila, a cGMP response to NO donors is very prominent when the larval nervous system goes into its second developmental period at metamorphosis. Although diaphorase staining, indicative of NOS presence, shows developmental regulation, the same kind of dramatic modulation in diaphorase staining is not observed as is observed for NO responsiveness. Double staining of the nervous system for cGMP (after supplying exogenous NO) followed by diaphorase staining to reveal NOS positive cells, shows that most, but not all, of the cell bodies that are diaphorase positive are not responsive to SNP (Truman, 1996b).

It is not known what causes the abrupt appearance of NO responsiveness in these neurons although the most obvious possibility is contact with target. An intriguing feature of soluble guanylate cyclase is that it is inhibited by elevated levels of intracellular Ca2+. Ca2+ transients and Ca2+ spikes are characteristic of extending axons and growth cones in many types of developing neurons, and a reduction in Ca2+ currents often accompanies the transition from the axonal outgrowth phase into the maturational phase. This reduction in intracellular Ca2+ might serve to unmask the guanylate cyclase, thereby allowing it to respond to extracellular signals (Truman, 1996b and references).

NOS function in neural precursor proliferation and nervous system morphogenesis in Manduca

Proliferation of neural precursors in the optic lobe of Manduca sexta is controlled by circulating steroids and by local production of nitric oxide (NO). Diaphorase staining, anti-NO synthase (NOS) immunocytochemistry and the NO-indicator, DAF-2, show that cells throughout the optic anlage contain NOS and produce NO. Signaling via NO inhibits proliferation in the anlage. When exposed to low levels of ecdysteroid, NO production is stimulated and proliferation ceases. When steroid levels are increased, NO production begins to decrease within 15 minutes independent of RNA or protein synthesis and cells rapidly resume proliferation. Resumption of proliferation is not due simply to the removal of NO repression though, but also requires an ecdysteroid stimulatory pathway. The consequence of these opposing pathways is a sharpening of the responsiveness to the steroid, thereby facilitating a tight coordination between development of the different elements of the adult visual system (Champlin, 2000).

In both Drosophila and Manduca, ingrowing photoreceptor axons stimulate proliferation in a subset of optic anlage (OA) cells. This process provides one way of coordinating development of the eye with the optic lobe. In addition, ecdysteroids provide a means to coordinate development throughout the entire visual system including developing layers that are not in direct contact. For example, ecdysteroid is required to sustain the movement of the morphogenetic furrow and, hence, the progressive formation of rows of photoreceptors in the imaginal eye disc. The same ecdysteroid requirements are found for proliferation of precursors for the medulla and lobula as well as the lamina optic lobe neurons. In this context, the function of NO signaling within the OA appears to be to sharpen the proliferative responsiveness of cells to the steroid so that optic lobe neuron production is tightly coordinated along the entire length of the OA and progresses in unison with photoreceptor production in the eye disc. When NO signaling is inhibited, a five-fold increase in the 20E titer (20 to 100 ng/ml) is needed to go from the first signs of proliferation to full proliferation. In the presence of NO signaling, this range is reduced to just two-fold. This may be of particular importance in Manduca since metamorphic development of the visual system is reversibly interrupted during pupal diapause when environmental cues act to suppress the ecdysteroid titer (Champlin, 2000).

In controlling proliferation within the OA, the ecdysteroid and NO pathways intersect at two levels. They both appear to act at the target cell level in controlling entry into mitosis with ecdysteroid promoting entry and NO inhibiting it. However, ecdysteroid also acts upstream by suppressing NO synthesis. NO production decreases rapidly (within 15 minutes) in response to an increase in 20E and even if RNA or protein synthesis is blocked. Therefore, ecdysteroid suppression of NO production appears to occur through a rapid, non-genomic signaling mechanism (Champlin, 2000).

NO has been shown to inhibit proliferation in a variety of cell types, the best characterized of which are the endothelium and smooth muscle of vertebrate blood vessels. NO produced by endothelial cells inhibits proliferation both in the endothelium itself and also the overlying smooth muscle cells. Although it is well established that endothelial-derived NO acts as a vasodilator on smooth muscle cells via a cGMP-mediated pathway, conflicting reports have been published for both endothelial cells and smooth muscle cells as to whether NO also acts through cGMP to inhibit proliferation. In the OA, no evidence has been found for an involvement of cGMP in the inhibition of proliferation by NO. Ecdysteroid-dependent entry into mitosis is blocked by incubation with a NO donor even when the soluble guanylate cyclase inhibitor, ODQ, is included to block production of cGMP. Furthermore, the cGMP analog, 8-bromo-cGMP, is not able to mimic the inhibitory effect of NO. Consistent with this, cGMP has not been detected in the OA during development or in response to NO treatment. An alternative target for NO signaling within the OA may be through the nitrosylation of target cell proteins (Champlin, 2000).

The exact relationship between NO repression and steroid regulation of the cell cycle is not yet known in either vertebrates or invertebrates. In principle, these opposing signals could interact at any level between the steroid receptor and the regulated cell cycle factors. In any case, NO repression in the OA of Manduca does differ from that seen in vertebrate in at least one respect. In the latter, the arrest typically occurs during the G1 phase of the cell cycle while in the OA it is late in the G2 phase (Champlin, 2000).

Nitric oxide synthase is present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. The possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis has been investigated. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, that grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins (Gibson, 2001).

Key cellular interactions must occur for the antennal lobe of M. sexta to develop normal cellular architecture. The axons of olfactory receptor cells (ORCs) arriving from the antennae form a template of protoglomeruli and induce glial cells in the antennal lobe to migrate to surround these protoglomeruli. The glial cells, in turn, act to stabilize the protoglomeruli and possibly confine subsequent axonal and dendritic branching to the glomerulus. Animals in which the antennae are removed to prevent ORC innervation of the antennal lobes never develop glomeruli. Glial cells remain in a rim around the perimeter of the neuropil, and the dendrites of multiglomerular antennal-lobe neurons branch in a diffuse pattern, rather than in glomerular tufts. The dendrites of uniglomerular projection neurons, which grow into protoglomeruli soon after their formation, are less affected. In animals treated to reduce severely the number of glial cells, ORC axons still grow to the developing antennal lobe, and their terminals coalesce to form protoglomeruli, but, in the absence of a sufficient number of glia to surround these protoglomeruli, the structure is lost and both axon terminals and multiglomerular neurons again form diffuse arborizations throughout the lobe. In short, the ORC axons establish the glomerular architecture via an interaction with antennal-lobe glial cells and possibly, also, via interactions with antennal-lobe neurons. Although intercellular interactions are essential, until the current study, no candidate signaling molecules had been identified (Gibson, 2001).

The profound effects resulting from blockade of NO signaling in the current study suggest that NO is indeed produced by the ORCs during development. It is an important component of the signaling by which the ORCs influence the antennal-lobe glial cells to migrate to surround protoglomeruli, and it also appears to restrict the outgrowth of dendrites of the 5-HT⁺ neuron to the basal (i.e., ORC-axon-free) half of each glomerulus. These specific effects of NO do not appear to be mediated by sGC, but may be mediated in part by ADP-ribosylation (Gibson, 2001).

When ingrowing ORC axons are prevented from signaling via NO by either of two pharmacological manipulations, the neuropil-associated glial cells of the antennal lobe fail to migrate. The effects of blocking NO signaling closely resemble those of removing ORC axons, suggesting that NO signaling may be a critical mode of communication between ORC axons and glia early in development. The results of L-NAME or CPTIO treatment on migration of peripheral glial cells down the antennal nerve provide additional support for this view. The decreased size of the antennal nerves in many treated animals is highly unlikely to be responsible for the similarity in appearance of treated animals to deafferented animals because as few as 12% of the normal complement of ORC axons are sufficient to produce protoglomeruli and induce the formation of mature glomeruli. Furthermore, the axons in treated animals often form a protoglomerular template (Gibson, 2001).

The simplest explanation for these effects on glia is that NO, produced in ORC axons, diffuses to nearby glia and stimulates them to migrate. Another formal possibility is that NO acts indirectly, causing the early-invading dendrites of uniglomerular projection neurons or the ORC axons themselves to alter expression of some factor necessary for glial cell migration. Involvement of uniglomerular projection neurons appears unlikely, because glomeruli form, with a glial envelope, even when the projection neurons are surgically removed prior to glomerulus development. Dendrites of local interneurons arrive at the glomeruli too late to be likely candidates. Thus, the effect of NO on glial cell migration is probably either direct or via an effect on the ORC axons themselves, enabling them to provide the migration signal to glia (Gibson, 2001).

Injections or infusions of NO-signaling blockers have to be started no later than late stage 2 to produce a strong effect on glial migration. Because antennal-lobe glia do not begin to migrate until stage 5, the results may indicate that NO initiates, at stage 2-3, a developmental program that is necessary to allow glia to migrate several days later. NO has been shown to inhibit the transcription factors AP-1 and NF-kB p50 by S-nitrosylation, and to promote cell migration as well as cell-surface expression of integrin alpahvß3, known to mediate cell migration in gliomas (Gibson, 2001).

It is interesting to note that, while the neuropil-associated glia of the antennal lobe and the glia of the antennae fail to migrate following blockade of NO signaling, the sorting zone glia, which migrate distally from the developing antennal lobe, appear to have migrated normally in all cases. The current results could indicate that the sorting zone glia are induced to migrate by a signal other than NO. They have been shown to differ from neuropil-associated glia of the antennal lobe in other ways; for example, sorting-zone glia require the presence of ORC axons for proliferation while antennal-lobe glia do not. Previous research indicates that sorting-zone glia have responded, via proliferation and migration, to ORC axon arrival by stage 3. It is therefore possible that initiation of the sorting-zone glial response to ORC axons would have occurred prior to the establishment of effective levels of anti-NO-signaling drugs in the test animals. This will be difficult to test, as earlier injections of L-NAME cause developmental arrest (Gibson, 2001).

L-NAME-treated animals in which glomeruli form provide a useful insight into the role of NO in determining outgrowth of the dendrites of the 5HT⁺ neuron. The 5HT⁺ dendrites normally ramify in areas of the glomerulus not occupied by the ORC axons, but when NO signaling is blocked, the dendrites are able to arborize throughout the region occupied by the ORC axons. The ability of NO to cause growth-cone collapse and thus halt process extension has been documented and attributed to blockade of growth-cone protein acylation. An attractive hypothesis is that, in M. sexta, such a process serves to prevent the majority of 5HT⁺ dendrites from entering ORN territory within the glomerulus. An alternative possibility is that an effect of NO on glial cells alters some growth-limiting effect on 5HT⁺ dendrites by the glia, although this seems less likely because glial cells outline the glomeruli and are not present at the equator, where 5HT⁺ dendritic branches normally terminate (Gibson, 2001).

Several lines of evidence reported here indicate that the NO-cGMP signaling pathway is not involved in either the glial cell migration or 5HT⁺ dendrite-outgrowth responses to NO. Previous studies indicate that NO does not induce detectable cGMP production in antennal-lobe glia at the relevant stages. Double labeling for 5HT and cGMP in NO-stimulated control brains at stage 7, when the 5HT⁺ neuron dendrites extend into the glomeruli, indicates that the 5HT⁺ neuron does not exhibit cGMP production either. Furthermore, the sGC inhibitor ODQ produces antennal lobes in which the glomeruli appear normal, with typical arborization of the 5HT⁺ neuron dendrites and the normal arrangement of neuropil-associated glia (Gibson, 2001).

Although sGC is the best characterized target of NO, several other downstream effectors of NO signaling are known. Direct NO-mediated nitrosylation of proteins, a feature common to these other mechanisms, can in turn lead to direct gating of cyclic nucleotide-gated channels, to modification of properties of extracellular matrix proteins, and to covalent attachment of ADP-ribose or NAD. ADP-ribosylation has been shown to affect the targeting or attachment of proteins to the membrane, the adhesion properties of fibronectin, and polymerization of actin and tubulin, mediated (at least in part) by Ras superfamily GTPases.

When ADP-ribosylation was blocked using Novobiocin, it was found that the neuropil-associated glia do not migrate, yet they extend processes to surround glomeruli. The animals were given only a single Novobiocin dose early in development (late 2 or early 3), well before the time at which glia normally extend processes and migrate. Therefore, these results most likely indicate an uncoupling of glial cell process extension and cell-body migration by Novobiocin. Because L-NAME and CPTIO treatment appear to have blocked both process extension and cell-body migration in these glia, it is possible that NO promotes process extension by one mechanism (not yet identified in this system), while mediating migration via an effect on ADP-ribosylation. Novobiocin was found also to mimic the effect of L-NAME and CPTIO on arborization of the dendrites of the 5HT⁺ neuron, suggesting that ORC axons restrict the outgrowth of 5HT⁺ dendrites via an NO-mediated increase in ADP-ribosylation of one or more growth cone proteins (Gibson, 2001).

In summary, this work suggests that NO serves as an important messenger in communication between the ORC axons and the antennal-lobe glia and the 5HT⁺ neuron of the antennal lobe. The fact that blockade of glial migration at stage 5 requires blockade of NO signaling no later than late stage 2 may indicate that NO works in part by initiating critical developmental programs. Moreover, the ability of Novobiocin to mimic some effects of NO-signaling blockade may indicate a role for NO-induced ADPribosylation (Gibson, 2001).

Adult

In both Drosophila and Apis (the honeybee), crude brain tissue contains the highest activities of NOS by far, whereas non-neuronal tissues, like thoracic tissue, show less than 5% of total NOS. Within the brain of adult animals, the dissected antennal lobes show the highest levels of NOS activities; the central brain and intermediate and visual neuropils show the lowest levels by far. The chemosensory neuropil in both Drosophila and Apis shows the highest activity levels. The antennal nerve and the contralateral projecting sensory fibers in the antennal commissure are strongly stained and the glomeruli exhibit a strong degree of labelling, with an non-homogeneous distribution within any given glomerulus. Some of the glomeruli are devoid of staining. The mechanosensory fibers show no labelling. In the central complex, the fan-shaped body and the nodule are stained. Unlike Apis, in which the mushroom bodies display a clearly compartmentalized pattern, in Drosophila no staining is detectable in the calyces (Müller, 1994).

The Malpighian tubule of Drosophila, a model for epithelial fluid transport, is an excellent system in which to study the roles of signal transduction processes in the control of fluid transport. The nitric oxide/cGMP signaling system activates epithelial fluid secretion by Drosophila Malpighian tubules. Exogenous cGMP stimulates fluid secretion through action of apical electrogenic Vacuolar H+ ATPase, a multisubunit complex that functions as a proton pump. V-ATPase was originally characterized as an endo-membrane component that pumps protons from the cytoplasm to the internal space of organelles. It also plays a role in energizing plasma membrane ion transport. V-ATPase drives one or more alkali metal/H+ exhangers. By this function, active transport of alkali metal cations (i.e. Na+ or K+) takes place (Davis, 1997 and references).

Nitric oxide stimulates fluid secretion with an associated elevation in intracellular cGMP levels. It is probable that the NO-induced increase in intracellular cGMP levels is due to an activation of soluble guanylate cyclase NO. The neuropeptide cardioacceleratory peptide (CAP2b) is expressed in Drosophila and has been shown to stimulate fluid secretion in Malpighian (renal) tubules. CAP2b acts to raise intracellular cGMP in tubules but not cAMP. As Malpighian tubules have been shown to exhibit NADPH diaphorase activity, implying the existence of Nitric oxide synthase, CAP2b is implicated in the stimulation of fluid transport via the activation of NOS and thence downstream components of the NO/cGMP signaling pathway. Treatment of Malpighian tubules with methylene blue, an inhibitior of soluble guanylate cyclase, attenuates CAP2b-enhanced secretion of fluid by Malpighian tubules. NOS activity was determined in unstimulated and CAP2b-stimulated tubules. After CAP2b treatment NOS activity rises to 144% of basal levels, and this CAP signal is sensitive to known inhibitors of NOS. This is the first demonstration of neuropeptide activation of a transport process via the NOS/cGMP signaling cascade in an epithelium (Davis, 1997)

Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells. Calcium mobilization in identified cell types within an intact renal epithelium (the Drosophila melanogaster Malpighian tubule) was studied by GAL4-directed expression of an aequorin transgene. Aequorin is a Ca2+ sensitive liminescent protein isolated from the coelenterate Aequorea victoria. It is a complex of apoaequorin, a 21 kDA polypeptide, and coelenterazine, a hydrophobic luminophore. Aequorin is used for monitoring Ca2+ changes. CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. The data suggest a role for calcium signaling in the modulation of the nitric oxide signaling pathway in this epithelium. CAP2b must act to increase fluid secretion rates solely by an initial rise of [CA2+]i in principal cells. CAP2b stimulates tubule Nitric oxide synthase activity. It is probable that the CAP2b induced rise in [CA2+]i is sufficient to trigger the activation of Drosophila calcium sensitive Nos. The maximal CAP2b concentrations employed elevate principal cell calcium levels from 87 to 255 nM, a value close to the EC50 of Drosophila NOS. This implies that Drosophila Nos is responsive over the range of the CAP2b concentrations employed. This may account for the observation that thapsigargin treatment results in increased basal cGMP levels that are not further increased on CAP2b stimulation. Thus the data provide strong evidence for a calcium-mediated link between CAP2b and NOS/cGMP activation of fluid secretion. The GAL4-targeting system allows general application to studies of cell-signaling and pharmacology that does not rely on invasive or cytotoxic techniques (Rosay, 1997).

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that have postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae was sought. Application of NO donors induce cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity is sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals that have been surgically isolated from the central nervous system are capable of synthesizing cGMP, it is suggested that a NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, it has been demonstrated that NO donors and membrane permeant cGMP analogs cause vesicle release at the NMJ. Moreover, the NO-induced release can be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggests that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae (Wildemann, 1999b).

Naturally occuring polymorphisms in behavior are difficult to map genetically and thus are refractory to molecular characterization. An exception is the Drosophila melanogaster foraging (for) gene, which has two naturally occurring variants relating to food-search behavior: rover and sitter. Molecular mapping placed foraging mutations in the dg2 gene, which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), a target of NOS signaling. Rovers have higher PKG activity than sitters, and transgenic sitters expressing a dg2 complementary DNA from rover show transformation of behavior to rover type. Thus, PKG levels affect food-search behavior, and natural variation in PKG activity accounts for a behavioral polymorphism (Osborne, 1997).

Individuals with a rover allele (for^R) move greater distances while feeding than do those homozygous for sitter alleles (for^s). This difference in foraging behavior is observed during both the larval and adult stages. Rovers and sitters do not differ in general activity in the absence of food. Both rovers and sitters are wild-type forms that exist at appreciable frequencies. Several mutations of the locus map with the naturally occurring alleles in the 24A3-5 region of the D. melanogaster polytene chromosomes. This region contains dg2, one of two cGMP-dependent protein kinase (PKG) genes in Drosophila. The dg2 gene has three major transcripts, T1, T2, and T3, and the for mutations are localized to this region. The P[GAL4] transposable element in 189Y was inserted in the 5' end of the dg2 T2 transcript. This homozygous viable insertion identified a new for allele, because P-element excision reverts larval foraging behavior from the sitter to the rover phenotype. As is the case with other sitter alleles, locomotion of the 189Y larvae is not reduced in the absence of food, indicating that the change in behavior is foraging-specific (Osborne, 1997).

To determine whether PKG is directly responsible for the foraging polymorphism in Drosophila, dg2 was overexpressed in sitter larvae. This results in a change of behavior to the rover phenotype. The transgenic strain contains four copies of a heat shock-driven dg2-cDNA. The basal level of PKG expression in this transgenic strain is sufficient to rescue rover larval behavior, thus eliminating the lethal and sublethal effects of heat on the dg2-transgenic larvae. As expected, the PKG enzyme activities of the dissected larval central nervous systems (CNSs) show that without heat shock, the dg2-cDNA transgenic strain have levels of PKG similar to those of for^R and significantly higher than those of the sitter control strain (Osborne, 1997).

The basis for the dg2 activity difference between for^R and for^s was further addressed by measurement of RNA levels and PKG protein. Northern (RNA) analysis revealed that for^s and for^s2 show a small but consistent reduction in the abundance of T1 RNA relative to that in for^R. T2 and T3 RNA are also reduced in these strains, but to a lesser extent. To assess protein levels, extracts of adult heads were subjected to protein immunoblot analysis by probing with an antibody to bovine PKG, or the extracts were affinity-purified by chromatography on cGMP-sepharose, labeled, and electrophoresed. In both experiments, a prominent band at a molecular mass of 80,000 Daltons was found. This is the only band strongly induced by heat shock in the dg2-cDNA transgenic strain, and it is less intense in for^s than for^R. (This band is also somewhat less intense in for^s2 and nearly absent in 189Y homozygotes). Taken together, these results argue that the difference between the naturally occurring alleles for^R and for^s is in the level of expression of the enzyme (Osborne, 1997).

The assignment of mutations in the for gene to the dg2 locus not only establishes the identification of PKG mutations but also implicates the cGMP signal transduction pathway in the regulation of food-search behavior in D. melanogaster. Small but significant differences in the levels of this kinase affect the naturally occurring behavioral polymorphism. These small differences in PKG are even detectable in homogenates, indicating that the differences in PKG level in rovers and sitters might be larger in cells relevant to the expression of the foraging behavior. These results suggest that the amount of kinase activity affects larval food-search behavior. Indeed, even modest quantitative changes in kinase activity affect behavior. Induced mutations that affect behavioral phenotypes often lie in signal transduction pathways. For example, the cyclic adenosine monophosphate (cAMP) system influences associative learning in flies, and genetic variants in two other serine/threonine kinases: the calcium/calmodulin-dependent protein kinase II and protein kinase C affect learning and behavioral plasticity in flies and mice. The finding that for encodes a PKG shows that a naturally occurring genetic polymorphism in behavior involves these pathways. PKG has a variety of pleiotropic cellular regulatory functions that are also typical of signal transduction components. Electrophysiological studies have shown that injected kinase affects neuronal membrane conductance in snails and mammals; that inhibitors of PKG block long-term potentiation in mammalian hippocampus and that PKG is involved in presynaptic long-term potentiation in cultured hippocampal neurons. Outside the nervous system, PKG has also been implicated in controlling proliferation of smooth muscle cells and neutrophil degranulation. These findings assign behavioral functions to this relatively scarce member of the serine/threonine kinases and show that subtle differences in PKG can lead to naturally occurring variation in behavior (Osborne, 1997 and references).

EVOLUTIONARY HOMOLOGS

NOS protein isoforms

Alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle , since its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family (Silvagno, 1996).

A new constitutive nitric oxide synthase (NOS) that utilizes both L-arginine and bradykinin (BK) as substrates has been purified from rat cerebellum. This NOS is calmodulin-dependent with L-arginine, but calmodulin-independent with BK. Peptide products obtained using BK and a related nonapeptide as substrates were isolated and sequenced. Both N- and C-terminal arginines of BK are oxidized to citrullines by this enzyme. With BK as substrate NOS activity is competitively inhibited by NG-methyl-, NG-nitro-L-arginines, and a BK receptor antagonist. These results suggest that oligopeptide-utilizing NOS may occur in other tissues, and shows that oligopeptides can function either as substrates or as inhibitors of NOS (Chen, 1996).

NOS knockouts

Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in mice that lack the neuronal nitric oxide synthase (NOS) gene. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus (Huang, 1993).

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by a NOS inhibitor and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants that are blocked by the nNOS inhibitor, indicating normal functioning of endothelial NOS. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO (Irikura, 1995).

Mice lacking expression of the endothelial isoform of NO synthase (eNOS), a gene that is distinct from nNOS, develop larger infarcts after middle cerebral artery occlusion. Possible hemodynamic differences in the peri-ischemic zone of eNOS-deficient and wild-type mice after middle cerebral artery occlusion were examined using functional CT scanning techniques. Hemodynamic deficits were more severe in mutant than wild-type mice. When expressed as a percentage of the total insult, core areas were significantly increased in mutant mice, as compared with wild types. Conversely, areas of the hemodynamic penumbra were significantly smaller in mice deficient in eNOS activity than in wild-type. Furthermore, the calculated relative perfusion index within the hemodynamic penumbra was significantly lower in the mutant group with eNOS gene deletion. These data indicate that mice lacking eNOS expression show a greater degree of hemodynamic compromise after middle cerebral artery occlusion and suggest that NO, a product of eNOS activity, may protect brain after focal cerebral ischemia, possibly by improving blood flow within the penumbral zone (Lo, 1996).

In addition to its role in blood vessel and macrophage function, nitric oxide (NO) is a neurotransmitter found in high densities in brain regions known to regulate emotion. Mice with targeted disruption of neuronal NO synthase (nNOS) display grossly normal appearance, locomotor activity, breeding, long-term potentiation and long-term depression. A substantial increase in aggressive behaviour and excess, inappropriate sexual behaviour is seen in nNOS- mice (Nelson, 1995).

NOS protein interactions

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. The N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93. nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore appear to mediate synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes (Brenman, 1996).

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (see Drosophila Calmodulin). The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains, termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin. Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." Co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS, expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin (Michel, 1997).

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95 (see Drosophila Discs large), through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. A novel nNOS-associated protein, CAPON, is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes (Jaffrey. 1998).

Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins (Fang, 2000).

A selective interaction has been identified for the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. CAPON directly interacts with the nNOS PDZ domain. Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced. There has been little characterization of Dexras1, and its downstream targets are not definitively established. Members of the Rap subfamily of Ras-like G proteins transmit growth factor signals to MAP kinase; signaling cascades through B-Raf preferentially over A-Raf. Dexras1 may have similar effectors as other Rap subfamily members, since its effector loop (residues 53-61) is 78% identical to the effector loop in Rap2b (residues 32-40). Indeed, in preliminary experiments, Dexras1 has been transfected into HEK293 cells and activation of MAP kinase activity has been detected, monitored by the phosphorylation of myelin basic protein. Conceivably, Dexras1-specific effectors may exist that mediate the effects of NO in the central nervous system (Fang, 2000).

Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signaling proteins, but its precise role in regulating specific cellular targets is not known. Hsp90 associates with endothelial nitric oxide synthase (eNOS) and is rapidly recruited to the eNOS complex by agonists that stimulate production of nitric oxide: vascular endothelial growth factor, histamine and fluid shear stress. The binding of Hsp90 to eNOS enhances the activation of eNOS. Inhibition of signaling through Hsp90 attenuates both agonist-stimulated production of nitric oxide and endothelium-dependent relaxation of isolated blood vessels. These results indicate that Hsp90 facilitates signaling mediated by growth-factor, G-protein and mechanotransduction pathways that lead to the activation of eNOS. These observations indicate that in addition to its role as a molecular chaperone involved in protein folding and maturation, Hsp90 may also be recruited to cellular targets depending on the activation state of the cell (Garcia-Cardena, 1998).

PDZ domains function in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, affinity chromatography was performed of brain extract with the nNOS PDZ domain. The carboxyl-terminal-binding protein (CtBP) was identified as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, these data suggest a new function for nNOS as a regulator of CtBP nuclear localization (Riefler, 2001).

The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. CtBP1 is SUMOylated (see Drosophila SUMO) and its SUMOylation profoundly affects its subcellular localization. SUMOylation occurs at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacks the SUMOylation site and is not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifts CtBP1 from the nucleus to the cytoplasm, while it has little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolishes the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 is inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression (Lin, 2003).

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. A novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078) has been identified and characterized that increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consists of 392 amino acid residues and possesses five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggests that the C-terminal tail region of NIDD specifically interacts with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS are enriched in synaptosome and synaptic plasma membrane fractions and are present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS are co-localized in the brain, although the colocalizing regions are restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increases NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane (Saitoh, 2004).

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain (Song, 2007).

Nitric oxide synthase - enzyme biology

Nitric oxide synthase binds arginine and NADPH as substrates, and as cofactors, it binds FAD, FMN, tetrahydrobiopterin, heme and calmodulin. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a heme-binding region, analogous to cytochrome P-450. On the C-terminal side it is flanked by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolysis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produces three fragments: that of Mr approx. 135000 (N-terminus Gly-221) results from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (heme region) and 80000 (reductase region, N-terminus Ala-728) are produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species are shown to be transient, and three smaller, highly stable fragments are formed: Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain). The species of Mr approx. 60000 represents a domain retaining heme and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet is cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain, present in inducible nitric oxide synthase. Prolonged proteolysis treatment leads to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion are similar to those formed with trypsin, although the pathway of intermediates differs. The heme domain is smaller, starting at residue 275, yet still retains the arginine binding site (Lowe, 1995).

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine, then converting the N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer. This cofactor is thought to mediate both partial reactions. Electron paramagnetic resonance spectroscopy shows that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction from that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS (Salerno, 1996).

Calmodulin binding to NOS triggers reduction of its heme groups, leading to NADPH oxidation and NO synthesis. In the absence of bound calmodulin, ferric NOS exhibits a Kd of 0.6 microM for L-arginine. L-Arginine binding reduces 8-fold the affinity of the ferric NOS heme for cyanide. Carbon monoxide binding to substrate-free ferrous NOS occurs at a rate of 2 x 10(5) M-1 S-1; this rate is decreased 12-fold when L-arginine is bound. In contrast, bound calmodulin does not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor does it alter NOS binding affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH leads to incomplete reduction of the heme iron; full reduction is achieved only in the presence of added L-arginine. These data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: it slows ligand interactions by binding in the distal pocket very near the heme and it also appears to increase the reduction potential of the iron. In contrast, bound calmodulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass from the flavin domain onto the heme iron (Matsuoka, 1994).

NO has recently been proposed to autoinhibit NOS. Does a NOS heme-NO complex form during aerobic steady-state catalysis? The majority of enzyme (70-90%) is present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS forms only in the coincident presence of NADPH, L-arginine, and O2. Its level remains constant during NO synthesis until the NADPH is exhausted, after which the complex decays to regenerate ferric resting NOS. The buildup of the ferrous-NO complex is rapid (< 2 s) and causes a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay can occur several times with no adverse affect, either on its subsequent formation or on NOS catalytic activity. It is concluded that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrous-nitrosyl complex during NO synthesis, independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS (Abu-Soud, 1995).

In the absence of haemin, NOS is expressed to a very high level but remains predominantly insoluble. Purification of the soluble fraction of the expressed protein shows that it has poor activity and is heme-deficient. Supplementing the culture medium with haemin results in pronounced solubilization of the expressed enzyme, which has a higher specific activity and contains 0.95 equiv. of heme per monomer under these conditions. The amount of H(4) biopterin endogenously present in the different NOS preparations positively correlates with the amount of enzyme-bound heme. Heme-deficient enzyme preparations containing 30-40% of the holoenzyme bind only 40% of H4biopterin. These results suggest that the prosthetic heme group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin (List, 1996).

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identifies Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions are tolerated, and normal binding of heme, calmodulin, and NADPH mitigates against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues (Cho, 1995).

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO-) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-D-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals whereas NO- is undetectable. In the presence of L-arginine (L-Arg) and cofactors, NOS generates prominent NO signals. This NO generation does not require SOD; it is blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester. Isotope-labeling experiments with L-[15N]Arg further demonstrate that NOS-catalyzed NO arises from the guanidino nitrogen of L-Arg. The time course of NO formation parallels that of L-citrulline. The conditions used in the prior study are shown to result in potent superoxide generation; this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from L-Arg (Xia, 1997).

eNOS-dependent superoxide formation may play an important role in the pathology of conditions like diabetes, hypertension, aging, and atherosclerosis. Alterations of NOS activity in favor of superoxide formation are proinflammatory and proatherogenic. Redox-cycling compounds like lucigenin and adriamycin shift eNOS activity from a -NO synthase to an NADPH oxidase. Evidence is presented for the generation (by a calcium/calmodulin-dependent mechanism) of superoxide by eNOS. In particular, the effect of BH4, in the presence and absence of L-arginine, reveals that changes in BH4 concentration can regulate the formation of superoxide relative to -NO. The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Superoxide generation is inhibited by heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical. Superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex; occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. These data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. These data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS (Vasquez-Vivar, 1998).

The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems. The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme. Replacement of Asp-314 by Ala alters the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogates the ability of nNOS to form catalytically active dimers. Replacement of Thr-315 by Ala reduces the protein stability and alters the geometry around the heme site, especially in the absence of bound (6R)-5,6,7, 8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS (Iwasaki, 1999).

Activation of the NO pathway

Within the embryonic mammalian cerebellum, the NO synthesizing enzyme, NO synthase (NOS), is expressed exclusively by granule cells and stellate/basket neurons. In the adult cerebellum, levels of NOS expression can be used to define discrete clusters of granule cell populations. Differential expression of NOS by granule cells temporally coincides with the establishment of afferent innervation of granule cells. In primary cerebellar cultures that comprise a functional network of glutamatergic and GABAergic cerebellar neurons, blockade of electrical activity by tetrodotoxin induces the expression of the neuronal isoform of NOS (nNOS) in granule cells. Conversely, direct depolarization of cultured neurons with K+ completely downregulate nNOS expression. Suppression of NMDA receptor- and AMPA receptor-mediated spontaneous synaptic signaling in cultured cells results in a drastic upregulation of nNOS expression in granule neurons. In contrast, blockade of GABAA receptor-mediated intercellular communication does not affect nNOS expression by granule cells. Blocking N-, P-, and Q-type voltage-dependent Ca2+ channels results in a graded upregulation of NOS expression, whereas manipulations of the cAMP-dependent signal transduction pathway induces no changes. It is concluded that nNOS expression in developing cerebellar granule cells is regulated by excitatory neurotransmission, and that calcium is an important signal transduction molecule involved in this regulatory process (Baader,1996).

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (See Drosophila PKC) was investigated. Incubation of rat cerebellar slices with a specific metabotropic glutamate receptor agonist increases cyclic GMP concentration two-fold. The increase is dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol ester, a PKC activator, increases cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by phorbol are independent of extracellular calcium blocked by a specific NOS inhibitor, and are not additive. These results suggest that a metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices has similar activities at 100 nM free calcium, whereas at 25-70 nM free calcium, NOS in PMA-treated slices is more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations (Okada, 1995).

Response of PC12 cells to nerve growth factor (NGF) involves a proliferative phase that is followed by growth arrest and differentiation. The cytostatic effect of NGF is mediated by nitric oxide. NGF induces different forms of nitric oxide synthase in neuronal cells. Nitric oxide acts as a cytostatic agent in these cells: inhibition of NOS leads to reversal of NGF-induced cytostasis and thereby prevents full differentiation; the capacity of a mutant cell line to differentiate can be rescued by exogenous NO. It is suggested that induction of NOS is an important step in the commitment of neuronal precursors and that NOS serves as a growth arrest gene, initiating the switch to cytostasis during differentiation (Peunova, 1995).

During development, neuronal differentiation is closely coupled with cessation of proliferation. Studying nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells, a novel signal transduction pathway was found that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) . The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter (See Drosophila Dacapo) and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21(WAF1) is required for neurite extension, PC12 line was prepared with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restores this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, a signal transduction pathway has been identified that is activated by NGF; proceeds through NOS, p53, and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells (Poluha, 1997).

Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in both a time and concentration-dependent manner. Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [3H]arginine to [3H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase; this constitutes a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities (Baltrons, 1995).

Relaxin (RLX), a peptide hormone of ovarian origin, inhibits growth and promotes differentiation of MCF-7 breast adenocarcinoma cells. RLX stimulates the production of nitric oxide (NO) in several cell types. NO has been reported to have antitumor activity by inhibiting proliferation, promoting differentiation, and reducing the metastatic spread of some tumor cell types. MCF-7 cells were grown in the absence or presence of RLX at different concentrations, and cell proliferation, constitutive and inducible NO synthase activities, nitrite production, and intracellular levels of cyclic GMP were investigated. The results obtained indicate that RLX increases inducible NO synthase activity and potentiates NO production. This was accompanied by an elevation of intracellular cyclic GMP, which is known to mediate the cell response to NO. The RLX-induced activation of the L-arginine-NO pathway in the MCF-7 cells is inversely related to the rate of cell proliferation. These results suggest that the cytostatic effect of RLX on MCF-7 breast cancer cells may rely on its ability to stimulate endogenous production of NO (Bani, 1995).

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunofluorescence imaging approaches, the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cells was investigated. In unstimulated endothelial cells, eNOS is predominantly localized at the cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nucleus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 pM, and a maximal estradiol effect is seen at a concentration of 1 nM. Neither progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca2+ concentration precedes eNOS translocation. Both the Ca2+-mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antagonist ICI 182,780, and the intracellular Ca2+ chelator BAPTA prevents estradiol-induced eNOS translocation. Use of the nitric oxide-specific dye diaminofluorescein shows that estradiol treatment increases nitric oxide generation by endothelial cells; this response is blocked by ICI 182,780 and by the eNOS inhibitor Nomega-nitro-L-arginine. These results show that estradiol induces subcellular translocation of eNOS by a rapid, Ca2+-dependent, receptor-mediated mechanism, and they suggest a nongenomic role for estrogen in the modulation of NO-dependent vascular tone (Goetz, 1999).

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. The serine/threonine protein kinase Akt (protein kinase B), a kinase whose activity is dependent on phosphatidylinositol (PI) 3-kinase, can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO (Fulton, 1999).

NO and Iron metabolism

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites consisting of iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches (Hentze, 1996).

There are IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In two mammalian species, an IRE is present in the 5' untranslated regions of mitochondrial aconitase mRNAs. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. These results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis (Gray, 1996).

Iron-regulatory protein (IRP) is a master regulator of cellular iron homeostasis. Expression of several genes involved in iron uptake, storage, and utilization is regulated by binding of IRP to iron-responsive elements (IREs), structural motifs within the untranslated regions of each gene's mRNAs. IRP-binding to IREs is controlled by cellular iron availability. Recent work reveals that nitric oxide (NO) can mimic the effect of iron chelation on IRP and on ferritin mRNA translation, whereas the stabilization of transferrin receptor mRNA following NO-mediated IRP activation can not be observed in gamma-interferon/lipopolysaccharide-stimulated murine macrophages. NO is a signaling molecule to IRP and a regulator of mRNA translation and stabilization. Fibroblasts with undetectable levels of endogenous NO synthase activity have been stably transfected with a cDNA encoding murine macrophage inducible NO synthase. Synthesis of NO activates IRE binding, which in turn represses ferritin mRNA translation and stabilizes transferrin receptor mRNA against targeted degradation. Furthermore, iron starvation and NO release are shown to be independent signals to IRP. The posttranscriptional control of iron metabolism is thus intimately connected with the NO pathways (Pantopoulos, 1995).

NO and apoptosis

Although nitric oxide (NO) induces neuronal cell death under some conditions, it also can prevent apoptosis resulting from growth factor withdrawal. The molecular mechanism by which NO protects undifferentiated and differentiated PC12 cells from trophic factor deprivation-induced apoptosis has been investigated. After 24 hr of serum withdrawal, PC12 cells undergo apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and cytochrome c release. The apoptosis of PC12 cells is inhibited by the addition of NO-generating donor (SNAP) and the specific caspase-3-like protease inhibitor (Ac-DEVD-cho) but not a YVADase (or caspase-1-like protease) inhibitor (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevent the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppression of DEVDase activity is only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO does not S-nitrosylate caspase-3-like proteases in PC12 cells. NO inhibits the proteolytic activation of caspase-3. A cGMP analog (8-Br-cGMP) blocks apoptotic cell death, caspase-3 activity and activation, and cytochrome c release. A soluble guanylyl cyclase inhibitor (CODQ) significantly attenuates NO-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death, PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, a protein kinase G inhibitor reverses both SNAP- and 8-Br-cGMP-mediated anti-apoptotic events. All these apoptotic phenomena are also suppressed by NO production through neuronal NO synthase gene transfer into PC12 cells. Furthermore, similar findings are observed in differentiated PC12 cells stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting the activation of caspase proteases through cGMP production and activation of protein kinase G (Kim, 1999).

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, it was determined if NO-induced cell death in neurons is dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), a NO donor, elevates caspase activity and induces death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminishes caspase induction and protects SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk does not provide significant protection. A role for p38 MAP kinase is further substantiated by the observation that SB203580 blocks translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promotes Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons are resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway (Ghatan, 2000).

Transcriptional regulation of NOS genes

Understanding transcription initiation of the endothelial nitric-oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present study therefore focused on a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of constitutive expression of the human eNOS gene in vascular endothelium. The use of deletion analysis and linker-scanning mutagenesis identified two tightly clustered cis-regulatory regions in the proximal enhancer of the TATA-less eNOS promoter: positive regulatory domains I (-104/-95 relative to transcription initiation) and II (-144/-115). Analysis of trans-factor binding and functional expression studies reveal a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contain Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells reveal examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-base pair region of the human eNOS promoter in vascular endothelium (Karantzoulis-Fegaras, 1999).

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. A cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA contains multiple NF-kappa B elements. The role of the IFN-Jak kinase-Stat 1 pathway for regulation of hiNOS gene transcription is described in this study. In A549 human lung epithelial cells, a combination of cytokines TNF-alpha, IL-1 beta, and IFN-gamma function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner (Ganster, 2001).

Post-transcriptional regulation of NOS genes

Cytokine stimulation of human DLD-1 cells results in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there is a basal transcription of the NOS II gene, which does not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR show a significantly reduced luciferase activity. The Elav-like protein HuR binds with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduces the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiates the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA (Rodriguez-Pascual, 2000).

Miscellaneous NO targets

p21ras is a direct target of NO. p21ras is singly S-nitrosylated and this modification takes place on Cys118. NO stimulates guanine nucleotide exchange on wild-type p21ras, resulting in an active form, but not on p21ras with a mutated amino acid #118 residue. NO does not stimulate mitogen-activated protein kinase activity in cells transfected with p21ras with a mutated amino acid #118 residue (Lander, 1997).

Nitric oxide inhibits the activation of transcription by NFkappaB (Drosophila homolog: Dorsal), a transcription factor implicated in regulation of immunologic NOS (iNOS). Neuronal NOS (nNOS) is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits transcriptional activation by the transcription factor NFkappaB, as evidenced by the fact that NOS inhibitors enhance transcriptional activation by NFkappaB. In astrocytes, an NO scavenger dramatically induces the NFkappaB-dependent enzyme iNOS, supporting the physiologic relevence of endogenous NO regulation of NFkappaB. These data suggest that nNOS-generated NO in astrocytes regulates NFkappaB activity and consequently iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NFkappaB-regulated genes (Togashi, 1997).

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. Incubation of endothelial cell lysates or purified eNOS with caveolin-1 results in the direct interaction of the two proteins. Utilizing a series of caveolin-1 deletion mutants, two cytoplasmic domains of caveolin-1 were identified that interact with eNOS, the scaffolding domain (amino acids 61-101) and, to a lesser extent, the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, results in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. The site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocks the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, these data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely to be temporally and spatially distinct events that regulate NO production in endothelial cells. The inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are most likely subject to negative regulation by this muscle-specific caveolin isoform (Garcia-Cardena, 1997).

Arterial baroreceptors are mechanosensitive nerve endings in the aortic arch and carotid sinus that play a critical role in acute regulation of arterial blood pressure. Nitric oxide (NO) or NO-related species suppress action potential discharge of baroreceptors. The effects of NO were investigated on Na+ currents of isolated baroreceptor neurons in culture. Exogenous NO donors inhibit both tetrodotoxin (TTX) -sensitive and -insensitive Na+ currents. The inhibition was not mediated by cGMP but by NO interaction with channel thiols. Acute inhibition of NO synthase increases the Na+ currents. NO scavengers (hemoglobin and ferrous diethyldithiocarbamate) increase Na+ currents before but not after inhibition of NO synthase. NO synthase was identified in baroreceptor neurons. These results indicate that NO/NO-related species function as autocrine regulators of Na+ currents in baroreceptor neurons. Modulation of Na+ channels may represent a novel response to NO (Li, 1998).

It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. This study shows that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. Both S-nitrosocysteine and cytokine-activated NOS2 inhibits NF-kappaB in human respiratory cells or murine macrophages. This inhibition is reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity does not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro results in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect is reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 is detected in cells, and the level is modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation (Marshall, 2001).

NO regulates angiotensin II receptors in vascular smooth muscle cells (Cahill, 1995).

NO delays programmed cell death in mature B cells, regulating the expression of the protooncogene bcl-2 (Genaro, 1995).

NO activates CFTR chloride current in human T lymphocytes (Dong, 1995).

NO selectively amplifies FGF-2-induced mitogenesis in primary rat aortic smooth muscle cells (Hassid, 1994).

NO increases expression of the immediate early genes c-fos and zif/268 in the cultured neurons (Morris, 1995).

NO inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid (Seilicovich, 1995).

NO can directly inhibit the DNA binding activity of NF-kappaB family proteins (Matthews, 1996)

NO attenuates IFN-gamma-induced VCAM-1 expression primarily by inhibiting basal constitutive NF-kappa B activity in smooth muscle cells (Shin, 1996).

NO directly activates calcium-dependent potassium channels in vascular smooth muscle (Bolotina, 1994).

NO and guanylate cyclase

The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. NO increases the Vmax of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme displays rhombic symmetry and is indicative of a high-spin heme. The ferric heme binds cyanide to form a 6-coordinate low-spin complex. Unlike the ferrous form of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a Kd of 26 +/- 4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of approximately 57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon oxidation of the heme iron (Stone, 1996).

The cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) share a strong primary sequence homology within their respective active site regions. Not surprisingly, these enzymes also exhibit overlapping substrate specificities, a feature that often interferes with efforts to elucidate their distinct biological roles. PKA and PKG exhibit dramatically different behavior with respect to the phosphorylation of alpha-substituted alcohols. Although PKA will phosphorylate only residues that contain an alpha-center configuration analogous to that found in L-serine, PKG utilizes residues that correspond to both L- and D-serine as substrates. The PKG/PKA selectivity of these substrates is very high (Wood, 1996).

Consider the process undergone by a newly hatched vermiform larva on its way to becoming a grasshopper: the egg from which the larva hatches is laid in soil. The larva continues the stereotyped hatching behavior as it digs through the egg pod, which provides a passageway to the soil surface. Once at the surface, ecdysis or shedding of the vermiform cuticle is initiated. When this process is complete, the first-instar cuticle is expanded to assume the form of the first-instar hopper.

These behaviors are associated with dramatic increases in intracellular levels of cyclic GMP in sets of identified neurons in the ventral nervous system. The most prominent cyclic-GMP-expressing cells are 34 neurons that appear to contain crustacean cardioactive peptide (see Drosophila Cardioacceleratory peptide). These neurons are distributed from the subesophageal ganglion to A7. These CCAP cells show no detectable cGMP at hatching or while the vermiform larva digs through the soil. Within approximately one minute of reaching the soil surface and freeing itself, the larva initiates ecdysis and associated air swallowing and tracheal filling. These changes are immediately preceded by the appearance of cGMP in the CCAP cells. cGMP levels in these neurons peak by 5 min and then decline back to basal levels by 20-30 min. Conditions that cause ecdysing animals to resume digging prolong the elevation of cGMP levels. Once animals have assumed their 'hopper' form, however, external stimuli can no longer affect the time course of the cGMP response. The neurons containing elevated cGMP levels probably influence the air-swallowing, tracheal filling and circulatory changes that are associated with ecdysis. Pairs of descending midline neurons in abdominal segments 2-4 also become cGMP-immunoreactive, but they show peak expression after cGMP levels in the CCAP cells have declined. Also, neurons in the caudolateral region of the abdominal ganglia often become cGMP-immunoreactive when ecdysing animals are forced to resume digging for an extended period. Curiously, neurons involved in ecdysis-related response are unresponsive to NO donor compounds, indicating that cGMP production during ecdysis is not mediated via NO (Truman, 1996a).

Hydrogen peroxide (H2O2) has a concentration-dependent effect on endothelial permeability and F-actin distribution. Endogenous production of nitric oxide (NO) is involved in the effect of H2O2. The involvement of NO was demonstrated by examining the endothelial permeability to sodium fluorescein (MW 376 Da, Na-F) and to different-sized fluorescein-isothiocynate-labeled dextrans (FITC-dextrans) and by staining F-actin with rhodamine-labeled phalloidin in cultured bovine aortic endothelial cells growing on filters. A low concentration of H2O2 (10(-5) M) has no effect on either dense peripheral bands of F-actin (DPBs) or permeability. When an inhibitor of NO production is coadministrated with H2O2, DPBs are disrupted and the permeability to FITC-dextran 40 and FITC-dextran 70 is increased, however, permeability to Na-F and FITC-dextran 20 does not increase. Combining of H2O2 with l-arginine, a substrate for nitric oxide synthase, causes an increase in DPBs and a decrease in permeability to FITC-dextran 40 and FITC-dextran 70. Neither l-arginine nor the NOS inhibitor alone has any effect on either F-actin structure or endothelial permeability. A 10-fold higher concentration of H2O2 causes a disruption of DPBs and an increase in permeability; this can be prevented by adding l-arginine. An analogue of cGMP, 8-Br-cGMP, maintains DPBs and abolishes the increase in permeability induced by the treatment with either 10(-4) M H2O2 or a combination of H2O2 and the NOS inhibitor. These results suggest that the endogenous production of NO is involved in maintaining endothelial junctions in H2O2-treated cells; this involvement occurs via a cGMP-dependent mechanism (Liu, 1997).

All cellular signaling pathways currently known to elevate cGMP involve the activation of a guanylyl cyclase to synthesize cGMP. An exception to this rule is described. In the vertebrate parietal eye, the photoreceptors depolarize to light under dark-adapted conditions, unlike rods and cones but like most invertebrate photoreceptors. The signaling pathway for this response involves a rise in intracellular cGMP resulting from an inhibition of the phosphodiesterase that hydrolyzes cGMP. Furthermore, this phosphodiesterase is driven by an active G protein in darkness. These results indicate an antagonistic control of the phosphodiesterase by two G proteins, analogous to the Gs/Gi control of adenylyl cyclase. These findings demonstrate an unusual phototransduction mechanism and at the same time indicate that signaling involving cyclic nucleotides is more elaborate than previously thought (Xiong, 1998).

Unlike many neuron populations, supraoptic nucleus (SON) neurons are rich in both nitric oxide synthase (NOS) and the NO receptor-soluble guanylyl cyclase (GC) (the activation of which leads to cGMP accumulation). Elevations in cGMP result in increased coupling among SON neurons. The effect of NO was investigated on dye coupling in SONs from males, proestrus virgin females, and lactating rats. In 167 slices, 263 SON neurons were recorded; 210 of these neurons were injected intracellularly (one neuron per SON) with Lucifer yellow. The typically minimal coupling seen in virgin females is increased nearly fourfold by the NO precursor, L-arginine, or the NO donor, sodium nitroprusside (SNP). L-Arginine-induced coupling is abolished by a NOS inhibitor. In slices from male and lactating rats who have a higher basal incidence of coupling, SNP increases coupling by approximately twofold over control (p < 0.03). SNP effects are prevented by the NO scavenger hemoglobin and by the selective blocker of NO-activated GC, ODQ. These results suggest that NO released from cells within the SON can expand the coupled network of neurons and that this action occurs via cGMP-dependent processes. Because increased coupling is associated with elevated SON neuronal excitability, the effects of 8-bromo-cGMP on excitability were also studied. In both phasically and continuously firing neurons, 8-bromo-cGMP (1-2 mM), but not cGMP, produces membrane depolarizations accompanied by membrane conductance increases. Conductance increases remain when depolarizations are eliminated by current-clamping the membrane potential. Thus, NO-induced cGMP increases SON neuronal coupling and excitability (Yang, 1999).

The genome of the nematode Caenorhabditis elegans encodes seven soluble guanylate cyclases (sGCs). In mammals, sGCs function as alpha/ß heterodimers activated by gaseous ligands binding to a haem prosthetic group. The principal activator is nitric oxide, which acts through sGCs to regulate diverse cellular events. In C. elegans the function of sGCs is mysterious: the worm genome does not appear to encode nitric oxide synthase, and all C. elegans sGC subunits are more closely related to mammalian ß than alpha subunits (Cheung, 2004 and references therein).

Wild races of C. elegans feed on bacteria either alone or in groups. The behavioral variation between 'solitary' and 'social' feeding races is associated with two natural isoforms of the G protein-coupled neuropeptide receptor NPR-1, which have different ligand responses. Null mutants of npr-1 aggregate strongly on bacterial food, and NPR-1 acts partly in neurons exposed to the body fluid of the animal to inhibit social feeding. Two of the seven C. elegans sGCs, GCY-35 and GCY-36, promote aggregation behavior. gcy-35 and gcy-36 are expressed in a small number of neurons. These include the body cavity neurons AQR, PQR, and URX, which are directly exposed to the blood equivalent of C. elegans and regulate aggregation behavior. GCY-35 and GCY-36 act as alpha-like and ß-like sGC subunits and their function in the URX sensory neurons is sufficient for strong nematode aggregation. Neither GCY-35 nor GCY-36 is absolutely required for C. elegans to aggregate. Instead, these molecules may transduce one of several pathways that induce C. elegans to aggregate or may modulate aggregation by responding to cues in C. elegans body fluid (Cheung, 2004).

Cyclic GMP targets

• A cGMP-dependent protein kinase I pathway activates transcription from AP-1-responsive promoters, mediated by the AP-1 (Jun/Fos) transcription factor complex (Pilz, 1995).
• A cGMP-dependent protein kinase I pathway relays the death of trophic factor-deprived PC12 cells and sympathetic neurons (Farinelli, 1996).
• A cGMP-dependent protein kinase I pathway increases the number of endothelin receptors by upregulation of transcription (Redmond, 1996).
• A cGMP-dependent protein kinase I pathway inhibits intracellular calcium ([Ca2+]i) mobilization in vascular smooth muscle cells by phosphorylation of the inositol 1,4,5-triphosphate receptor (Komalavilas, 1994 and Ruth, 1993). and
• A cGMP-dependent protein kinase I pathway reverses G-protein-coupled inhibition of smooth muscle myosin light chain phosphate (Wu, 1996).
• A cGMP-dependent protein kinase I pathway activates protein phosphatase inhibitor 1 resulting in inhancement of smooth muscle contraction (Tokui, 1996).
• A cGMP-dependent protein kinase I pathway negatively regulates phosphoinositide hydrolysis and intracellular Ca2+ release responses (Clementi, 1995).
• NO functions to inhibit the skeletal muscle ryanodine receptor calcium release channel (Meszaros, 1996).
• A cGMP-dependent protein kinase I pathway inhibits vascular smooth muscle cell growth (Cornwell, 1994).

An automatic system was designed to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. The egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-ß and insulin pathways, respectively (Hirose, 2003).

Lifespan extension in egl-4 (ks61) mutant is suppressed by a daf-16 mutation, suggesting that egl-4 controls lifespan through the insulin-like signaling pathway. However, daf-16;egl-4 double mutants are larger than either single mutant, and daf-2;egl-4 doubles have an intermediate size between those of the single mutants. Both these results are interpreted to mean that egl-4 functions to control body size independently of the insulin-like signal pathway that includes daf-16 and daf-2 (Hirose, 2003).

As to the genetic interaction of egl-4 in the body size control, large body size of an egl-4 mutant is not suppressed by a daf-3 mutation either, suggesting that the pathway in which egl-4 functions to control body size is different from the TGFß branch of the dauer control pathway to which daf-3 belongs. However, suppression of body size of an egl-4 mutant by a sma-6 or dbl-1 mutation suggests that EGL-4 functions upstream of DBL-1 and SMA-6 that act in another TGFß pathway as a ligand and a receptor, respectively. This result seems reasonable because this pathway is known to control body size, and is also important to elucidate mechanisms for the control of body size by egl-4. Thus, functions of egl-4 are related to at least three signaling pathways. These multiple functions of egl-4 in multiple pathways can be explained if EGL-4 kinase phosphorylates multiple substrates. Because EGL-4 requires cGMP for effective kinase activity, a guanylyl cyclase must be a component upstream of EGL-4 (Hirose, 2003).

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform of this enzyme mediates the ability of cGMP to relax contracted vascular smooth muscle preparations. Vimentin as a high-affinity and specific binding protein for cGMP kinase. The site of high-affinity binding between cGMP kinase and vimentin does not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase do not compete for high-affinity binding. Neither the cGMP kinase proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment binds vimentin with high affinity, suggesting that the cGMP kinase dimer is necessary for the interaction. Vimentin is readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment, even though the monomeric 69-kDa fragment is catalytically active toward other substrates, such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur (MacMillan-Crow, 1994).

The elaboration of distinct cell types during development is dependent on a small number of inductive molecules. Among these inducers is Sonic hedgehog (Shh), which, in combination with other factors, patterns the dorsoventral (DV) axis of the nervous system. The response of a cell is dependent in part on its complement of cyclic nucleotides. cAMP antagonizes Shh signaling, and the influence of cGMP on the Shh response was examined. Cells in chick neural plate explants respond to Shh by differentiating into ventral neural-cell types. Exposure of intermediate-zone explants to cGMP analogs enhances their response to Shh in a dose-dependent manner. The Shh response is also enhanced in dorsal-zone explants exposed to chick natriuretic peptide (chNP), which stimulates cGMP production by membrane-bound guanylate cyclase (mGC). Addition of chNP to intermediate-zone explants does not enhance the Shh response, consistent with a reported lack of mGC in this region of the neural tube. Finally, the presence of a nitric oxide (NO)-sensitive guanylate cyclase (GC) was established by demonstrating cGMP immunoreactivity in neural tissue following NO stimulation of whole chick embryos. Intracellular levels of cGMP and cAMP may thus provide a mechanism through which other factors modulate the Shh response during neural development (Robertson, 2001).

NO expression in the heart

NO is a free radical that modulates heart function and metabolism. A neuronal-type nitric oxide synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and endogenous NO produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline is observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous nitric oxide is generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrates labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles exhibit no nNOS immunoreactivity. The nNOS immunoreactivity also displays a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrates that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection is observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, is inhibited by nitric oxide produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevents this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart (Xu, 1999).

NO and cell cycle

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway were investigated. The induction of p21 by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) seems to be due to p21 transactivation because SNAP elevates the activity of the p21 promoter but does not stabilize p21 mRNA and protein. Because SNAP does not stimulate the deletion mutant of the p21 promoter that lacks p53 binding sites, the involvement of p53 was investigated. The expression level of p53 is down-regulated after mitogenic stimulation, whereas it is sustained in the presence of SNAP. SNAP markedly stimulates DNA binding activity of p53. Furthermore, SNAP fails to induce p21 in VSMCs of p53-knock out mice and in A431 cells that contain mutated p53. The antiproliferative effect of SNAP also is attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However a guanylate cyclase inhibitor does not prevent SNAP-induced p21 expression. 8-bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination does not induce p21. Although 8-bromo-cGMP has a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP is little throughout the G(1) phase. The antiproliferative effect of SNAP is not attenuated by an inhibitor of cGMP-dependent protein kinase. These results suggest that NO induces p21 through a p53-dependent but cGMP-independent pathway (Ishida, 1999).

Nitric oxide (NO) regulates the expression of p21(Waf1/Cip1) in several cell types. The present study examined the role of both the extracellular signal-regulated kinase (ERK) and p70 S6 kinase [p70(S6k): see Drosophila RPS6-p70-protein kinase] in the NO-induced increase in p21 expression that occurs in adventitial fibroblasts during the cell cycle. Both ERK and p70(S6k) are phosphorylated in response to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the activation is rapid, transient, and precedes increased p21 expression under defined conditions where serum is present. Addition of a selective inhibitor of ERK phosphorylation (PD98059) prevents the subsequent phosphorylation of p70(S6k) and the increase in p21 protein. Both cGMP and cAMP activate both ERK and p70(S6k), whereas only selective inhibitors of protein kinase G prevent the activation of the kinases by SNAP. A complex between ERK and p70(S6k) was documented by immunoprecipitation procedures. Rapamycin blocks p70(S6k) phosphorylation induced by NO and also inhibits p53 phosphorylation and p21 expression whereas PD98059 only prevents the NO-induced increase in p21 protein without influencing either p53 activation or p21 mRNA expression. The studies show a unique relationship between NO, ERK, and p70(S6k) and also provide evidence for a novel role of p70(S6k) in the activation of p53 (Gu, 2000).

Temporal order of gene activation induced by NO in mammalian cells

Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. This study has determined he temporal order of gene activation induced by NO in mammalian cells and the signaling pathways that mediate the action of NO have been examined. Using microarrays to study the kinetics of gene activation by NO, it was determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. The results were verified using quantitative real time PCR and the conclusions about the effects of NO were further validated by using cytokines to induce endogenous NO production. Pharmacological and genetic approaches were appled to determine the signaling pathways that are used by NO to regulate gene expression. Inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, were used to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. The results demonstrate that NO utilizes several independent signaling pathways to induce gene expression (Hemish, 2004).

One conclusion of this study is that there are distinct waves of gene induction events initiated by NO in mammalian cells. The first wave activates genes that are immediate targets of the NO signals. These genes (group I) include several of the known immediate-early genes, such as c-fos and egr-1. Several group I genes code for transcription factors; this is consistent with the fact that this initial wave of gene activation is followed by a second wave (activation of group II genes). Group II genes may include direct targets of transcription factors activated in the first wave. Finally, a distinct third wave of gene activation can be detected that starts at ~12 h after the addition of the NO donor. These genes may represent the targets of the group II genes; they may also reflect changes inherent to the cell cycle arrest status induced by NO. It will be interesting to determine whether there are any key regulatory genes in these groups required for the transition to the next stage (Hemish, 2004).

Genes in group I are especially interesting because they represent immediate targets of NO, and their activation may reflect changes in the transcription machinery (e.g., S-nitrosylation of some transcription factors). Most of these genes are activated within 30 min after addition of the NO donor; using Q-PCR it was also found that some of them are activated as early as 10-15 min after addition of the donor. The regulatory regions of these genes may be good candidate sites to search for putative NO response elements; they may also lead to identification of transcription factors affected by NO (Hemish, 2004).

The findings were validated by quantitating the NO-induced changes using Q-PCR technique. Furthermore, it was found that the tested genes induced by exogenous NO donor were also induced by the mixture of cytokines, which gives rise to endogenously produced NO. The degree of contribution of the NO signaling pathways varies widely from fully underlying the action of cytokines on gene expression (e.g., in the case of HO-1 and mdm2) to mediating only a part of the signaling cascades that lead to gene activation (e.g., BNIP3 and gly96). The overlap between the sets of genes activated in NIH3T3 cells by NO and by cytokines may reflect an important role for NO in the response of fibroblasts to cytokines in vivo during inflammation and tissue repair. It will be interesting to compare these results with the transcriptional profiles of cells exposed to individual cytokines to estimate the relative contribution of NO in the action of these effectors (Hemish, 2004).

Specific groups of genes were identified that require the activity of PI 3-kinase, PKC, or NF-kappaB to be induced by NO. These data correspond well to reports of the involvement of these proteins in the physiological changes induced by NO or changes in the enzymatic activity of these proteins induced by NO. A distinct group of genes was found whose activation by NO was prevented by the lack of p53. These data show that the p53 protein is up-regulated in response to NO and plays a role in the antiproliferative function of NO. This provides further support for the relevance of the profiling data in explaining the long term biological effect of NO (Hemish, 2004).

NO and neurons

The stomatogastric ganglion (STG) of the crab Cancer productus contains ~30 neurons arrayed into two different networks (gastric mill and pyloric), each of which produces a distinct motor pattern in vitro. The functional division of the STG into these two networks requires intact NO-cGMP signaling. Multiple nitric oxide synthase (NOS)-like proteins are expressed in the stomatogastric nervous system, and NO appears to be released as an orthograde transmitter from descending inputs to the STG. The receptor of NO, a soluble guanylate cyclase (sGC), is expressed in a subset of neurons in both motor networks. When NO diffusion or sGC activation are blocked within the ganglion, the two networks combine into a single conjoint circuit. The gastric mill motor rhythm breaks down, and several gastric neurons pattern switch and begin firing in pyloric time. The functional reorganization of the STG is both rapid and reversible, and the gastric mill motor rhythm is restored when the ganglion is returned to normal saline. Finally, pharmacological manipulations of the NO-cGMP pathway are ineffective when descending modulatory inputs to the STG are blocked. This suggests that the NO-cGMP pathway may interact with other biochemical cascades to partition rhythmic motor output from the ganglion. Because citrulline accumulation was restricted to the distal axons and terminals of the NO-producing neurons, it was not possible to identify their somata in anterior ganglia. It is therefore unclear whether NO is released as a cotransmitter, as has been demonstrated in Aplysia, or whether the NOS-containing neurons are coactivated with parallel descending inputs to the STG. NO may also be released from another source (Scholz, 2001).

The STG neurons that contain a NO-sensitive sGC include the core neurons of the pyloric circuit (PD, PY, and LP) as well as the four GM neurons that can switch between the gastric mill and pyloric networks. An interneuron with an ascending axon (either AB or Int1) is likely to be the last member of the NO-responsive group. Suppression of intrinsic NO-cGMP signaling, using either ODQ or PTIO, speeds up the core pyloric rhythm and causes the GM neurons to pattern switch and burst in pyloric time. Therefore, in the context of the 12 identified sGC-expressing cells, the suppression of the NO pathway results in a ganglion that is dominated by the pyloric motor rhythm. It is important to stress, however, that PTIO- and ODQ-induced changes in firing activity are not limited to neurons that express the receptor of NO. For example, the activity of NO-insensitive neurons (e.g., DG, VD, and IC) also changes when NO-cGMP signaling is inhibited. These indirect effects of NO are likely to reflect synaptic interactions between NO-sensitive and NO-insensitive cells. The nature of these interactions has yet to be determined (Scholz, 2001).

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. NO/cGMP signalling cascade plays a key role in regulating migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, potential sources were identified of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting (Haase, 2003).

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous systems of many vertebrates and invertebrates. The mechanism of NO action at an identified synapse between a mechanoafferent neuron, C2, and the serotonergic metacerebral cell (MCC) was studied in the cerebral ganglion of the mollusc Aplysia californica. Stimulation of C2 produces a decreasing conductance and very slow EPSP in the MCC. C2 is thought to use histamine and NO as cotransmitters at this synapse, because both agents mimic the membrane responses. Evidence is provided that treatment with NO donors stimulates soluble guanylyl cyclase (sGC) in the MCC, and as a result cGMP increases. S-Nitrosocysteine (SNC, an NO donor) and 8-bromo-cGMP (8-Br-cGMP) both induce membrane depolarization and increase input resistance, two responses that are characteristic of the very slow EPSP. Inhibitors of sGC suppress both the very slow EPSP and the membrane responses to SNC but not the histamine membrane responses. NO-induced cGMP production has been determined in the MCC using cGMP immunocytochemistry (cGMP-IR). In the presence of 3-isobutyl-1-methylxanthine (IBMX), 10 microM SNC is sufficient to induce cGMP-IR, and the staining intensity increases as the SNC dose is increased. This cGMP-IR is suppressed by inhibitors of sGC. The results suggest that NO stimulates sGC-dependent cGMP (Koh, 1999).

In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate synaptic transmission at the level of the spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated. Exposure to a depolarizing concentration of KCI evoked (respectively) 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner. NO generator sodium nitroprusside (NP) causes a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it fails to affect either a basal or an evoked release of Glu. NP causes a concentration-dependent increase in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels (Kamisaki, 1995).

The presence of nitric oxide synthase in spinal cord and dorsal root ganglia was investigated by immunohistochemistry using antibodies against the constitutive neuronal form of nitric oxide synthase (NOS). NOS immunoreactivity is present in both man and rat with similar distribution, being present in primary sensory neurons of dorsal root ganglia and their afferent terminals in the dorsal horn of spinal cord. NOS immunoreactive interneurons are found in the superficial layer of the dorsal horn, around the central canal and in the intermediolateral cell column. NOS immunoreactivity is also present in numerous motoneurons in the ventral horn. The widespread distribution of NOS in both the sensory and motor nervous system is indicative of the involvement of nitric oxide in different neural functions (Terenghi, 1993).

Neuronal nitric oxide synthase (NOS), visualized immunohistochemically or with NADPH diaphorase histochemistry, is transiently expressed in discrete areas of the developing rat nervous system. In the brain, transient NOS expression occurs in the cerebral cortical plate. At E15-E19, the majority of cells in the plate stain, with their processes extending through the corpus striatum to the thalamus. This staining decreases after birth and vanishes by the 15th postnatal day. Neurons in olfactory epithelium also express NOS from E15 till early postnatal life. In embryonic sensory ganglia virtually all neuronal cells are NOS positive, whereas by early adulthood only 1% express NOS. By contrast to these areas of transient NOS expression, in other neuronal sites NOS staining appears after cell bodies cease dividing and cells extend processes; the staining persists into adult life. The transient expression of neuronal NOS may reflect a role in developmental processes such as programmed cell death (Bredt, 1994).

The presence of nitric oxide synthase (NOS) in CA1 pyramidal cells of the rat hippocampus was demonstrated by single-cell PCR. The sequence of the major amplification-product obtained is identical to that of the constitutively expressed brain-isoform of NOS. These results confirm immunocytochemical data that NOS is present in CA1, and, therefore, nitric oxide could function as a retrograde messenger in long-term potentiation (Chiang, 1994).

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction is 6.7, a characteristic value for nNOS. No difference in nNOS levels is seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy reveals for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN (Chen, 1997).

The neurotransmitter glutamate plays an important role in the control of secretion of gonadotropin-releasing hormone (GnRH). Recent evidence suggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamate effects. NOS cell body and fiber staining occurs in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are located. Other major GnRH cell body sites such as the median preoptic nucleus (MPN) and medial preoptic area (MPOA) display moderate staining of NOS cell bodies and fibers. Intense NOS staining is also observed in the median eminence, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus of the hypothalamus. While no GnRH neurons are found to double stain for NOS in the hypothalamus, GnRH neurons are frequently surrounded by NOS neurons in the OVLT, MPN and MPOA; there are potential contacts between NOS and GnRH neurons in these areas. In addition, there is significant overlap of GnRH and NOS fibers in the lateral portion of the internal zone of the median eminence, where GnRH fibers and terminals converge. Double-staining studies for NADPH-diaphorase and the NMDA R1 receptor subunit show that many NOS neurons in the OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus co-localize the NMDA R1 receptor subunit. Central administration of a nitric oxide synthase inhibitor blocks the ability of NMDA to induce LH secretion, demonstrating the functional importance of co-localization of NMDA R1 receptor subunit immunoreactivity in B-NOS neurons. These studies provide evidence that supports a role for nitric oxide as an important regulator of GnRH neurons in the female. They also suggest that hypothalamic NOS neurons are targets for glutamate regulation as evidenced by co-localization of the NMDA R1 receptor subunit (Bhat, 1995).

NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons (Roskams, 1994).

Neuronal differentiation requires a coordinated intracellular response to diverse extracellular stimuli, but the role of specific signaling mechanisms in regulating this process is still poorly understood. Soluble guanylate cyclases (sGCs), which can be stimulated by diffusible free radical gasses such as nitric oxide (NO) and carbon monoxide (CO) to produce the intracellular messenger cGMP, have recently been found to be expressed within a variety of embryonic neurons and implicated in the control of both neuronal motility and differentiation. Using the enteric nervous system (ENS) of the moth, Manduca sexta, the roles of NO and NO-sensitive sGCs were examined during the migration and differentiation of an identified set of migratory neurons (the EP cells). EP cells arise en masse from a neurogenic placode in the foregut, forming a packet of postmitotic but immature neurons on the superficial gut musculature. The subsequent development of the EP cells is noteworthy in that it involves three distinct phases of motile behavior: a period of cell migration (during which the neurons disperse along a set of preformed muscle bands); a period of axon elongation (when the neurons cease migrating but continue to elaborate axons along the pathways), and a period of terminal outgrowth (during which the neurons sprout terminal synaptic branches onto the adjacent visceral musculature). Shortly after the onset of their migration, a subset of EP cells begins to express NO-sensitive sGC activity (visualized with an anti-cGMP antiserum). Unlike many neurons in the central nervous system, the expression of sGC activity in the EP cells is not transient but persisted throughout subsequent periods of axon elongation and terminal branch formation on the gut musculature. In contrast, nitric oxide synthase activity (visualized using NADPH-diaphorase histochemistry) is undetectable in the vicinity of the EP cells until the period of synapse formation. Manipulations designed to alter sGC and NOS activity in an in vivo embryonic culture preparation had no discernible effect on either the migration or axonal outgrowth of the EP cells. In contrast, inhibition of both of these enzymes resulted in a significant reduction in terminal synaptic branch formation within the postmigratory neurons. These results indicate that while NO-sensitive sGC activity is expressed precociously within the EP cells during their initial migratory dispersal, a role for this signaling pathway can only be demonstrated well after migration is complete, coincident with the formation of mature synaptic connections. Although no local source of putative NOS expression could be identified within the target muscles of the midgut EP cells, an intensely stained band of NADPHd activity gradually develops in the visceral mesoderm at the foregut-midgut boundary. In the developing ENS, therefore, it is possible that NO is released from this diaphorase-positive band of visceral mesoderm only after the stages of migration and axonal elongation are complete, thereby initiating the third phase of EP cell motility (terminal branch formation and synaptogenesis) in an sGC-dependent manner (Wright, 1998).

The adult pattern of axonal connections from the eye to the brain arises during development through the refinement of a roughly ordered set of connections. In the chick visual system, refinement normally results in the loss of the ipsilateral retinotectal connections. Inhibition of nitric oxide synthesis reduces the loss of these transient connections. Because nitric oxide is expressed by tectal cells with which retinal axons connect, and because reduction of nitric oxide synthesis by tectal cells results in a change in the connections of retinal axons, nitric oxide probably serves as a messenger from tectal cells back to retinal axons during development (Wu, 1994).

The ferret retinogeniculate projection segregates into eye-specific layers during the first postnatal week and into ON/OFF sublaminae, which receive inputs from either on-center or off-center retinal ganglion cells, during the third and fourth postnatal weeks. The restriction of retinogeniculate axon arbors into eye-specific layers appears to depend on action potential activity but does not require activation of NMDA receptors. The formation of ON/OFF sublaminae is also activity-dependent and is disrupted by in vivo blockade of NMDA receptors. To investigate a possible mechanism whereby blockade of postsynaptic NMDA receptors in the lateral geniculate nucleus (LGN) results in changes in the size and position of presynaptic axon arbors, the role of the diffusible messenger nitric oxide (NO) was tested in the development of the retinogeniculate pathway. NO synthase (NOS) is transiently expressed in LGN cells during the refinement of retinogeniculate projections. During the third and fourth postnatal weeks, treatment with NG-nitro-L-arginine (L-NoArg), an arginine analog that inhibits NOS, results in an overall pattern of sublamination that is significantly reduced when compared with normal and control animals. Single retinogeniculate axon arbors are located in the middle of eye-specific layers rather than toward the inner or outer half as in normal or control animals. The effect of NOS inhibition is not a consequence of the hypertensive effect of L-NoArg. In contrast to the effect of L-NoArg on the formation of ON/OFF sublaminae, treatment with L-NoArg during the first postnatal week does not disrupt the formation of eye-specific layers. Biochemical assays indicate significant inhibition of NOS during both treatment periods. These data suggest that NO acts together with NMDA receptors in activity-dependent refinement of connections during a specific phase of retinogeniculate development (Cramer, 1996).

The role of nitric oxide (NO) as a mediator of synaptic plasticity is controversial in both the adult and developing brain. NO generation appears to be necessary for some types of NMDA receptor-dependent synaptic plasticity during development, but not for others. Recent studies of three different visual projections reveal a role for NO as an activity-dependent retrograde messenger during some but not all NMDA receptor-dependent developmental processes. In the ferret LGN, after retinal axon termination within eye-specific laminae, the afferents further segregate into ON and OFF sublaminae. This sublamination is NMDA receptor-dependent and is significantly reduced by NOS inhibitors. However, in the mammalian visual cortex, shifts in ocular dominance columns induced by monocular deprivation are disrupted by NMDA receptor blockade but are not sensitive to inhibition of NOS activity. Furthermore, ocular dominance column formation itself is not disrupted by NOS inhibition. Finally, in the chick, the normal complete elimination of the ipsilateral retinotectal projection is partially disrupted by systemic NMDA receptor blockade and by systemic NOS inhibition (Renteria, 1999 and references).

Xenopus laevis retinal ganglion cell axons stop growing in response to NO exposure. The same response occurs in tectal neuron processes bathed in the NO donor S-nitrosocysteine (SNOC) and in RGC growth cones to which SNOC is very locally applied. NO synthase (NOS) activity is present in the Rana pipiens optic tectum throughout development in a dispersed subpopulation of tectal neurons, although effects of NO on synaptic function in a Rana pipiens tectal slice were varied. NOS was chronically inhibited in doubly innervated Rana tadpole optic tecta using L-N^G-nitroarginine methyl ester in Elvax. Despite significant NOS inhibition as measured biochemically, eye-specific stripes remain normally segregated. This suggests that NOS activity is not downstream of NMDA receptor activation during retinotectal synaptic competition because NMDA receptor activation is necessary for segregation of retinal afferents into ocular dominance stripes in the doubly innervated tadpole optic tectum. It is concluded that NO has some signaling function in the retinotectal pathway, but this function is not critical to the mechanism that refines the projection and causes eye-specific stripes (Renteria, 1999).

Noxious stimulation can trigger persistent sensitization of somatosensory systems that involves memory-like mechanisms. Noxious stimulation of the mollusc Aplysia produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway. Injection of cGMP induces LTH, whereas antagonists of the NO-cGMP-PKG pathway prevent pinch-induced LTH. Co-injection of calcium/cAMP-responsive-element (CRE) blocks both pinch-induced LTH and cAMP-induced LTH, but antagonists of protein kinase A (PKA) fail to block pinch-induced LTH. Thus the NO-cGMP-PKG pathway and at least one other pathway, but not the cAMP-PKA pathway, are critical for inducing LTH after brief, noxious stimulation (Lewin, 1999).

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increases cell proliferation and decreases differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibits NPC proliferation and increases neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduces NPC proliferation and increases the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC is blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibits proliferation and induces neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis (Cheng, 2003).

NOS is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). This study examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS in muscle cells (cell-autonomously) reduces AChR aggregation at embryonic Xenopus NMJs by 50%-90%, whereas overexpression of NOS increases AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely block agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induces AChR clustering in the absence of agrin. These results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells (Schwarte, 2004).

These findings are consistent with the idea that an increase in NO production is part of the agrin/MuSK signal transduction pathway. However, the mechanism of NOS activation and the subsequent steps leading to aggregation of AChRs and other postsynaptic proteins are unclear. Several possible signaling mechanisms both upstream and downstream of NOS have been considered that could mediate agrin activity in muscle cells. The effects of NO on AChR aggregation are likely be mediated, at least in part, by stimulation of a typical NO effector, soluble guanylate cyclase (sGC). This enzyme is stimulated by NO to increase its synthesis of cyclic GMP, activating cGMP-dependent protein kinase (PKG, 1995). Like NOS, both sGC and PKG are concentrated postsynaptically at the NMJ. A cGMP analog, 8-bromo-cGMP, which stimulates PKG, increases AChR aggregate area at embryonic Xenopus NMJs by 100%-200%, similar to its effect on aggregation in chick muscle cells. Furthermore, overexpression of sGC and PKG in Xenopus embryos increased AChR aggregation to a similar extent as overexpression of agrin and NOS. It is unclear how sGC and PKG might interact with other components involved in agrin signal transduction, but defining the protein targets of PKG phosphorylation might reveal connections with other molecules known to be involved in postsynaptic differentiation (Schwarte, 2004).

NO and behavior

A role for the NO-cGMP pathway in mediating chemosensory activation in snail feeding is suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase. In in vitro preparations reduced to isolated lips and CNS, intracellular recordings were made from motoneurons driven by the interneurons of the central pattern generator (CPG) for feeding. Fictive feeding in such preparations can be recorded from these motoneurons following the application of sucrose to the lips. Sucrose activation of fictive feeding is inhibited by the NO scavenger hemoglobin, a NO synthase inhibitor and by methylene blue, an inhibitor of guanylyl cyclase. Fictive feeding in isolated lip-CNS preparations can be activated without sucrose by superfusion of NO donor molecules and by a nonhydrolyzable analog of cGMP. The feeding CPG can also be activated centrally by depolarizing a modulatory interneuron, the slow oscillator (SO). When the CPG is activated in this way, fictive feeding is not susceptible to inhibition by hemoglobin, the most potent of the inhibitors of sucrose-activated fictive feeding. Behavioral experiments on intact snails confirm the findings from in vitro experiments and show that hemoglobin prevents feeding and methylene blue significantly delays the onset of feeding. These results indicate (1) that NO is a putative chemosensory transmitter in the snail L. stagnalis, (2) that the NO-cGMP pathway can mediate chemosensory activation of specific patterns of centrally generated behavior, (3) that NO is not involved in transmission within the central network of neurons responsible for the behavior, and more generally (4) that a freely diffusing and highly reactive gaseous signalling molecule can have restricted and specific behavioral functions (Elphick, 1995).

Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produce lightlike phase shifts of circadian rhythms. The SCN exhibits calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways block Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo block the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN, mediating the phase resetting of the biological clock (Ding, 1994).

NO and LTP and memory

Temporal correlation between pre- and postsynaptic activities is an important mechanism that regulates synaptic connectivity during development and synaptic plasticity in the adult. In developing neuromuscular junctions, postsynaptic activity is critical in functional suppression and, ultimately, elimination of the synapses. Although repetitive postsynaptic firing asynchronous to the presynaptic activity results in a persistent synaptic suppression, the underlying molecular mechanism remains unknown. Evidence that nitric oxide (NO), a free radical implicated in several forms of synaptic plasticity, may serve as a retrograde signal for activity-dependent suppression in the neuromuscular synapse. NO donors and activators of the cyclic GMP pathway suppress spontaneous and evoked synaptic currents. Moreover, the synaptic suppression induced by repetitive postsynaptic depolarization is prevented by the NO-binding protein hemoglobin and by inhibitors of NO synthase. Thus, synaptic suppression may be triggered by NO released from a postsynaptic myocyte that fires asynchronously to the presynaptic terminal (Wang, 1995).

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, this preparation was used to investigate the role of NO. LTP was examined following intra- or extracellular application of a NO scavanger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presyaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP (Arancio, 1996).

Sheep learn to recognize the odors of their lambs within two hours of giving birth; this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odor, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. NO is involved in olfactory learning. Neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odor. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. It is proposed that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories (Kendrick, 1997).

High-frequency stimulation (HFS) of corticostriatal glutamatergic fibers induces long-term depression (LTD) of excitatory synaptic potentials recorded from striatal spiny neurons. This form of LTD can be mimicked by zaprinast, a selective inhibitor of cGMP phosphodiesterases (PDEs). Biochemical analysis shows that most of the striatal cGMP PDE activity is calmodulin-dependent and inhibited by zaprinast. The zaprinast-induced LTD occludes further depression by tetanic stimulation and vice versa. Both forms of synaptic plasticity are blocked by a selective inhibitor of soluble guanylyl cyclase, indicating that an increased cGMP production in the spiny neuron is a key step. Accordingly, intracellular cGMP, activating protein kinase G (PKG), also induces LTD. Nitric oxide synthase (NOS) inhibitors block LTD induced by either HFS or zaprinast, but not that induced by cGMP. LTD is also induced by the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine. SNAP-induced LTD occludes further depression by HFS or zaprinast. Intracellular application of PKG inhibitors blocks LTD induced by HFS, zaprinast, and SNAP. Electron microscopy immunocytochemistry shows the presence of NOS-positive terminals of striatal interneurons forming synaptic contacts with dendrites of spiny neurons. These findings represent the first demonstration that the NO/cGMP pathway exerts a feed-forward control on the corticostriatal synaptic plasticity (Calabresi, 1999).

Postsynaptic injection of Ca(2+)/calmodulin [Ca(2+)/CaM] into hippocampal CA1 pyramidal neurons induces synaptic potentiation, which can occlude tetanus-induced potentiation. Because Ca(2+)/CaM activates the major forms of nitric oxide synthase (NOS) to produce nitric oxide (NO), NO may play a role during Ca(2+)/CaM-induced potentiation. Extracellular application of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or postsynaptic co-injection of L-NAME with Ca(2+)/CaM blocks Ca(2+)/CaM-induced synaptic potentiation. Thus, NO is necessary for Ca(2+)/CaM-induced synaptic potentiation. In contrast, extracellular perfusion of membrane-impermeable NO scavengers N-methyl-D-glucamine dithiocarbamate/ferrous sulfate mixture (MGD-Fe) or 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) does not attenuate Ca(2+)/CaM-induced synaptic potentiation, even though MGD-Fe or carboxy-PTIO blocks tetanus-induced synaptic potentiation. This result indicates that NO is not a retrograde messenger in Ca(2+)/CaM-induced synaptic potentiation. However, postsynaptic co-injection of carboxy-PTIO with Ca(2+)/CaM blocked Ca(2+)/CaM-induces potentiation. Postsynaptic injection of carboxy-PTIO alone blocks tetanus-induced synaptic potentiation without affecting basal synaptic transmission. These results suggest that NO works as a postsynaptic (intracellular) messenger during Ca(2+)/CaM-induced synaptic potentiation (Ko, 1999).

Long-term potentiation, a persistent increase in synaptic efficacy, may require a retrograde signal originating in the postsynaptic cell that induces an increase in presynaptic neurotransmitter release. A mouse homozygous for a targeted null mutation in the endothelial isoform of nitric oxide synthase has been constructed. Long-term potentiation in the CA1 region of these mice is entirely absent under weak stimulation conditions. Application of a membrane-permeant guanosine-3',5'-cyclic monophosphate analog during tetanus fails to compensate for this deficit, suggesting that nitric oxide produced by endothelial nitric oxide synthase may affect long-term potentiation through a cascade that does not include guanylyl cyclase. Strong tetanic stimulation can induce robust long-term potentiation in these mice. This potentiation is not blocked by pharmacological inhibitors of nitric oxide synthase. Furthermore, mice lacking endothelial nitric oxide synthase show no shift in the frequency-response curve for the induction of long-term potentiation. Basal synaptic transmission, paired-pulse facilitation and the electrical properties of CA1 cells in these mice are similar to controls. These results support a selective role for endothelial nitric oxide synthase in long-term potentiation, but also demonstrate that nitric oxide synthase is not always involved in this process under all conditions (Wilson, 1999).

Long-term potentiation (LTP) is a potential cellular mechanism for learning and memory. The retrograde messenger nitric oxide (NO) is thought to induce LTP in the CA1 region of the hippocampus via activation of soluble guanylyl cyclase (sGC) and, ultimately, cGMP-dependent protein kinase (cGK). Two genes code for the isozymes cGKI and cGKII in vertebrates. The functional role of cGKs in LTP was analyzed using mice lacking the gene(s) for cGKI, cGKII, or both. LTP is not altered in the mutant mice lineages. However, LTP is reduced by inhibition of NO synthase and NMDA receptor antagonists, respectively. The reduced LTP was not recovered by the cGK-activator 8-(4 chlorophenylthio)-cGMP. Moreover, LTP was not affected by a sGC inhibitor. In contrast, it is effectively suppressed by nicotinamide, a blocker of the ADP-ribosyltransferase. These results show that cGKs are not involved in LTP in mice and that NO induces LTP through an alternative cGMP-independent pathway, possibly ADP-ribosylation (Kleppisch, 1999).

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction (Kantor, 1996).

Nitric oxide (NO) has been implicated in hippocampal long-term potentiation (LTP), but LTP is normal in mice with a targeted mutation in the neuronal form of NO synthase (nNOS-). LTP is also normal in mice with a targeted mutation in endothelial NOS (eNOS-), but LTP in stratum radiatum of CA1 is significantly reduced in doubly mutant mice (nNOS-/eNOS-). By contrast, LTP in stratum oriens is normal in the doubly mutant mice. These results provide the first genetic evidence that NOS is involved in LTP in stratum radiatum and suggest that the neuronal and endothelial forms can compensate for each other in mice with a single mutation. They further suggest that there is also a NOS-independent component of LTP in stratum radiatum and that LTP in stratum oriens is largely NOS independent (Son, 1996).

Body size and behavioral state of C. elegans and cGMP-dependent protein kinase

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. Defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. This analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. Genes likely to be required for these functions downstream of sensory inputs have been identified. One of these genes has been characterized as egl-4; it encodes a cGMP-dependent protein kinase. This cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states (Fujiwara, 2002).

Despite having a simple nervous system of 302 neurons, C. elegans is capable of perceiving and responding to a wide variety of environmental stimuli such as odorants, mechanical stimuli, food, osmotic, and ionic changes and pheromones. In C. elegans, environmental cues are detected through specialized sensory neurons. Sixty of the 302 neurons in C. elegans are ciliated sensory neurons, which are thought to be responsible for most sensory perceptions. Like many sensory neurons in other animals, the sensory cilia of these neurons are specialized structures where environmental cues, including odorants and pheromones, interact with receptor proteins (Fujiwara, 2002).

A class of mutants including che-2, osm-6, and che-3 lack a normal sensory cilium structure. The structural defects in these mutants are readily assessed because several ciliated sensory neurons take up vital dyes through the cilia, and these mutants fail to do so. As expected, mutants lacking cilia show diminished sensory responses to soluble and volatile chemicals. They have diminished responses to dauer pheromone, which induces a transition to an alternative nondeveloping dauer larva stage. It has also been reported that the cilium-defective mutants exhibit a longer life span than wild-type animals (Fujiwara, 2002).

Mutants with defects in cilium structure also exhibit abnormalities in the regulation of growth to a normal body size and in the expression of alternative states of locomotory behavior. The changes in body size in the cilium-defective mutants are not due to an inability to locate food. The results suggest that sensory perception can regulate neuroendocrine functions that determine the growth and ultimate body size of an organism. Locomotory behavior of C. elegans in the presence of food is characterized by alternating behavioral states. In one state, the animal traverses widely separated regions of the plate (roaming), and in the other state, the animal restricts its activity to a confined region (dwelling). Analysis of cilium-defective mutants reveals that defects in sensory perception result in a relative decrease in the time spent roaming. Hence, the relative time spent roaming versus dwelling may be regulated by sensory perception (Fujiwara, 2002).

A genetic analysis has been persued to determine how such changes in development and behavior are regulated by sensory perception. To identify neuronal mechanisms acting downstream of sensory perception in the regulation of these processes, a screen was performed for suppressor mutations of the che-2 small body size phenotype (chb). A subset of these suppressors also suppress the defect in locomotory behavior of che-2. One of these suppressors, chb-1 (which is allelic to egl-4), encodes a cGMP-dependent kinase. A homologous cGMP-dependent protein kinase is expressed in vertebrate brain, although the physiological functions of this kinase in the nervous system have been controversial. These results suggest that cGMP-dependent kinase is required for the processing of sensory information that is essential to multiple behavioral and developmental circuits in C. elegans (Fujiwara, 2002).

In Drosophila, strains having less cGMP-dependent protein kinase activity move less during foraging (Osborne, 1997. Sokolowski, 1998). Although the same kinase influences the movement of both species, the mechanisms may be different. In Drosophila, the general locomotory activity level during foraging is different in distinct strains. In C. elegans, transitions between alternative active and inactive states are affected in a single strain. The effects in Drosophila are also opposite in character; a decrease in this protein results in increased roaming in C. elegans but less movement in Drosophila. The expression of a cGMP-dependent protein kinase in Drosophila is observed in neuronal and nonneuronal tissues. It is not yet known where expression is required for the regulation of foraging behavior of Drosophila. It is not yet known (Osborne, 2001) where expression is required for the regulation of foraging behavior of Drosophila (Fujiwara, 2002 and references therein).

The data presented in this study suggest that EGL-4 acts in the nervous system and particularly in sensory neurons in C. elegans. This represents a novel demonstration of the role of a cGMP-dependent protein kinase in sensory neurons for modulation of sensory information. egl-4 mutant phenotypes including enhanced dauer formation at high temperature, large body size, egg-laying defects, and even chemotaxis defects are suppressed by daf-3 mutations, suggesting that daf-3 functions downstream of egl-4. daf-3 encodes a member of the SMAD protein family acting downstream of the daf-7 TGF-ß cascade and is expressed in many neurons, the intestine, the pharynx, and hypodermi. Additional genetic interactions are observed with dbl-1 and lon-1, suggesting that egl-4 may act upstream of the dbl-1 TGF-ß cascade, which is a separate cascade from the daf-7 TGF-ß cascade. Although it is unclear whether egl-4 directly regulates dbl-1 and how daf-3 may interact with the dbl-1 TGF-ß cascade, the results suggest that the EGL-4 cGMP-dependent kinase may link sensory perception to the dbl-1TGF-ß cascade (Fujiwara, 2002).

A model is proposed in which sensory perception, acting through modulation of a cGMP-dependent kinase, regulates the growth and locomotory behavior of the animal. Normally, the EGL-4 cGMP-dependent kinase functions to reduce body size and decrease roaming. In wild-type animals, EGL-4 activity would be inhibited by sensory inputs. In che-2 mutants, EGL-4 would be inappropriately activated, resulting in a small body and decreased roaming. In egl-4;che-2 double mutants, EGL-4 function is eliminated, and body size and roaming are increased. Such a model of inhibitory sensory inputs is reminiscent of phototransduction in the vertebrate retina where light sensation causes a decrease in cGMP through activation of PDE. By analyzing other chb suppressors, it should be possible to identify additional components acting downstream of sensory inputs to regulate the growth and behavioral state of C. elegans (Fujiwara, 2002).

Synaptic vesicle traffic is supported by transient actin filaments and regulated by PKA and NO

Synaptic vesicles (SVs) can be pooled across multiple synapses, prompting questions about their dynamic allocation for neurotransmission and plasticity. This study found that the axonal traffic of recycling vesicles in primary cultures of hippocampal neurons is not supported by ubiquitous microtubule-based motility but relies on actin instead. Vesicles freed from synaptic clusters undergo ~1 microm bouts of active transport, initiated by nearby elongation of actin filaments. Long distance translocation arises when successive bouts of active transport were linked by periods of free diffusion. The availability of SVs for active transport can be promptly increased by protein kinase A, a key player in neuromodulation. Vesicle motion is in turn impeded by shutting off axonal actin polymerization, mediated by nitric oxide-cyclic GMP signaling (see Nitric oxide synthase) leading to inhibition of RhoA. These findings provide a potential framework for coordinating post-and pre-synaptic strength, using retrograde regulation of axonal actin dynamics to mobilize and recruit presynaptic SV resources (Chenouard, 2020).

Focusing on presynaptic vesicles that undergo activity-dependent fusion/retrieval, this study revealed that their axonal movements are longitudinally bidirectional, depend on actin, and do not require nocodazole-sensitive microtubules. SV AT was not detectably affected by nocodazole or colchicine, a microtubule-disrupting agent with a distinct mechanism. In contrast, the motion of vesicular transport packets, labeled via VAMP2/synaptobrevin but not endocytosed markers, was confirmed to be strongly directionally-biased and microtubule-dependent. These distinctions extend current views of axonal membrane traffic that emphasize microtubules and recall photobleaching experiments that first implicated actin in interbouton SV motion (Chenouard, 2020).

To clarify the structural determinants of SV dynamics, two-color imaging was used to monitor the motion of recycling vesicles while simultaneously tracking actin elongation. The initiation of vesicle movement corresponded spatiotemporally with the arrival of growing actin structures. Rather than actin-driven propulsion, the experiments support repeated binding and unbinding of SV to the newly laid actin track together with motor-mediated processive motion, in line with myosin V inhibition experiments. F-actin nucleation and elongation preceded SV transport, rather than being triggered by it as in Rab11a transport in oocytes. The formation of fresh actin tracks dictated the rate of SV AT occurrence and the speed of filament elongation put a ceiling on vesicle velocity. Actin filament polymerization and SV AT were slowed to a similar degree by exposure to latrunculin A, or by manipulating NO-cGMP-RhoA signaling. Unlike actin-based transport of axonal organelles in invertebrate axons, which persists after phalloidin-based actin stabilization, actin stabilization abolished SV traffic. Beyond differences in how actin polymerization is controlled or exploited, a common feature is that vesicle motion depends on actomyosin interaction. Continuously tracking QD-containing SVs, it was affirmed that they can travel for long distances along axons, doing so by combining episodes of directed AT, interspersed with diffusive motion. AT frequently started and ended at the periphery of synaptic clusters, but never in their core, confirming that AT reflected extra- rather than intra-synaptic mobility. Others have implicated SV-actin interactions in sub-diffusive motion, oscillating states, and mobility within synaptic clusters, possibly via acto-myosin2 complexes. In contrast, a major role for myosin II in extrasynaptic SV mobility was not apparent from a pharmacological intervention. Myosin V, a processive motor, seems better suited to carry out repetitive AT along axons. Interspersed diffusive pauses began when vesicles ceased to associate with nearby actin structures; directed motion resumed both without and with the arrival of a new growing actin structure as if temporarily disengaged vesicles can either re-engage with the original track or with a fresh one (Chenouard, 2020).

SVs moved bi-directionally, without an obviously preferred direction under basal conditions, enabling axonal re-distribution of SVs among synaptic sites. Dynamic actin filaments are suited for this function in growing in both axonal directions without strong bias. In contrast, axonal microtubules offer greater stability and singular polarization, befitting efficient and unidirectional fast transport between distant, pre-determined sites (soma to remote synaptic sites or vice-versa) (Chenouard, 2020).

By revealing checkpoints for SV trafficking, this work suggested scenarios by which neuromodulation could regulate interbouton exchange in association with learning. Intracellular cAMP can be elevated by dopamine-, serotonin- or activity-dependent activation of adenylate cyclase. cAMP elevation with forskolin enriched vesicles in the periphery of synaptic clusters, likely mediated by PKA phosphorylation of vesicular synapsin, liberating vesicles from synaptic confinement. Alternatives were considered such as PKA-mediated modulation of myosin V or myosin light chain kinase. Freeing vesicles from cytoskeletal constraints with PKA would make more vesicles eligible for interbouton trafficking, enlarging the vesicular 'superpool'. Indeed, it was observed that PKA stimulation increased the abundance of AT events without changing actin elongation or AT unitary properties (Chenouard, 2020).

Postsynaptic NMDA receptor activation locally enhances post- and presynaptic function in certain forms of LTP. Presynaptic enhancement is reflected by elevation of mini frequency, of uptake of presynaptic vesicular tags, of the number of synaptophysin-immunopositive puncta and of interbouton vesicle traffic. It has been shown that SV proteins accumulate at potentiated synapses by means of signaling via NO, cGMP, PKG, and RhoA. The observations of this study​ provide a way to link plasticity-inducing signals to the recruitment of presynaptic vesicular resources. It is propose dthat retrograde signaling by NO drives a local cascade of NO > ↑cGMP > ↑PKG > ↑RhoA to inhibit actin elongation and stall SV transport, enabling recruitment of stalled vesicles into synaptic pools at presynaptic sites destined for potentiation. This study found that raising NO or cGMP, key players in retrograde signaling, dampened actin filament elongation, and vesicle transport, involving both mDia1- and ROCK-dependent branches of signaling between RhoA and actin dynamics (Chenouard, 2020).

Such local recruitment of SVs could operate in tandem with a generalized PKA-dependent mobilization of SVs to concentrate SVs at potentiated postsynaptic sites, thus coordinating pre- and postsynaptic capabilities. By supporting short-haul SV traffic, actin-based SV movement facilitates local retrograde control, serving an altogether different function than microtubule-based, long-range, movements of transport vesicles (Chenouard, 2020).

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