Interactive Fly, Drosophila

Adenylate cyclase and neurons

Olfactory signal transduction in the olfactory epithelium (OE) is mediated by second messenger cascades. Odorant receptors exhibit structural characteristics of the seven transmembrane G protein-coupled receptor superfamily. The application of rapid kinetics methodology has shown that odorants elicit a rapid elevation of either cAMP or IP3. The colocalization of a unique G protein, G_olf, the olfactory adenylyl cyclase (AC3), and cyclic nucleotide-gated (CNG) cation channels in the olfactory cilia suggests an important role of cAMP in olfactory signal transduction. Indeed, a variety of odorants stimulate adenylyl cyclase activity in membrane preparations from OE. Odorant receptor activation increases intracellular cAMP through interactions with G_olf. This leads to the opening of CNG cation channels, membrane depolarization, and the generation of action potentials. It was recently discovered that excitatory responses to both cAMP and IP3-producing odorants are undetectable in mice lacking functional olfactory CNG channels, further suggesting that cAMP may be a major second messenger mediating olfactory signaling. Thus, the coupling of olfactory receptors to adenylyl cyclase is a major mechanism for olfactory signal transduction (Wei, 1998 and references).

One of the characteristic features of odorant-induced second messenger signaling is the transient responsiveness to odorant. Rapid increases and subsequent decreases in cAMP or IP3 have been observed both in olfactory cilia and in primary olfactory neuron cultures. In vitro biochemical approaches and studies with transgenic mice have suggested several mechanisms for desensitization, including odorant receptor phosphorylation, activation of PDEs, and ion channel regulation. Although increases in intracellular Ca2+ are thought to terminate olfactory signaling, mechanisms for inhibition of olfactory signaling by Ca2+ are not well defined. Ca2+ influx through the CNG channels may trigger adaptation of odorant responses by targeting multiple steps in the cAMP signaling cascade. For example, olfactory receptor neurons express a Ca2+/calmodulin- (CaM) activated PDE, and PDE inhibitors prolong odor-induced cAMP formation in olfactory cilia. This suggests that a CaM-sensitive PDE may contribute to signal termination. Intracellular Ca2+ also decreases the sensitivity of the CNG channel to cAMP, suggesting that Ca2+-regulated channels may also participate in the adaptation process (Wei, 1998 and references).

AC3 and CaMKII are both expressed in the cilia of olfactory neurons. An immunohistochemical analysis of OE detects AC3 primarily in the apical ciliary layer. There is little or no AC3 detectable in olfactory receptor neuron cell bodies. CaMKII immunoreactivity is also evident in the apical ciliary layer as well as neuron cell bodies. The merged image indicates that AC3 and CaMKII are coexpressed in olfactory cilia, the structures that contain olfactory receptors and are the primary source of olfactory signaling. Therefore, CaMKII has the potential to regulate AC3 activity in olfactory neurons during olfactory signaling, since they are both expressed in the cilia of olfactory sensory neurons (Wei, 1998).

Odorant stimulation of increases in cAMP in the OE is mediated by AC3 (a Ca2+-inhibited adenylyl cyclase). Inhibition of AC3 by intracellular Ca2+ is mediated by CaM kinase II (CaMKII) phosphorylation of AC3 at Ser-1076; the mutation of Ser-1076 to alanine renders AC3 insensitive to Ca2+ inhibition. Since odorant stimulation of intracellular cAMP is accompanied by increased intracellular Ca2+, CaMKII phosphorylation of AC3 may contribute to the cAMP transients in olfactory cilia. To test this hypothesis, a polyclonal antibody specific for AC3 phosphorylated at Ser-1076 was generated. The phosphorylation of AC3 at Ser-1076 is significantly enhanced upon stimulation with an odorant, and this phosphorylation is mediated by CaMKII. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulates phosphorylation of AC3 at Ser-1076. To determine what fraction of olfactory neurons responds to odorants, cultured olfactory neurons were treated with citralva or isoamyl alcohol (IA) for 30 s and immunostained for phosphorylated AC3. Only 2% ± 0.4% of the cells show a detectable signal in the absence of odorant, whereas 11.0% ± 1.6%, 40% ± 7.0%, and 91% ± 9.2% of the cells are positive for phosphorylated AC3 when olfactory neurons are treated with IA, citralva, or forskolin, respectively. The high percentage of cells activated by citralva is not unexpected; citralva is a complex mixture of odorants that undoubtedly stimulates multiple receptors in olfactory cilia. Phosphorylation of AC3 is blocked by inhibitors of CaMKII, which also ablates cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses (Wei, 1998).

Molecular cloning of mammalian adenylyl cyclase has led to the discovery of nine different isotypes. These can be classified broadly into Ca2+-stimulated cyclases, Ca2+-inhibited cyclases, and protein kinase C-activated cyclases on the basis of their distinct regulation by intracellular Ca2+ and protein phosphorylation. Current evidence indicates that all three classes of adenylyl cyclase are expressed in the mammalian brain. Activation of cAMP synthesis by intracellular Ca2+ is thought to be the main mode of cAMP generation in the brain. The Ca2+-stimulated enzymes adenylyl cyclase I (AC1) and VIII (AC8) have been assigned important roles in synaptic plasticity. In terms of neurotransmitter function, AC1 and AC8 are both activated by Ca2+/calmodulin. Moreover, AC1 may generate cAMP as a coincidence detector for concerted signals from Gs-coupled receptors and ionotropic receptors that trigger changes in the membrane potential and an increase of intracellular free Ca2+. Adenylyl cyclases II (AC2) and VII (AC7) are activated by protein kinase C (Antoni, 1998).

The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca2+ and that this effect is blocked selectively by inhibitors of calcineurin (protein phosphatase 2B) such as FK506 and cyclosporin A. Adenylyl cyclase IX is inhibited by the same range of intracellular free Ca2+ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. A large number of neuronal perikarya in the anterior olfactory nucleus, the nucleus of the lateral olfactory tract, and the olfactory bulb are strongly positive for AC9 mRNA. The most intense mRNA signal is apparent in the limbic areas of the forebrain. In particular, neurons of the hippocampal complex, including the tenia tecta, the anterior hippocampus, indusium griseum, and hippocampus, are labeled very intensely for AC9 mRNA. In the hippocampus the CA1-CA3 pyramidal cell layers and the dentate gyrus granule cells show dense and specific labeling. Examination at higher magnification shows that close to 70% of the perikarya in the pyramidal layer of CA3 and at least 50% of the granule cells in the dentate gyrus express AC9 mRNA. Strong specific hybridization signal also is detected in neurons of the subiculum and the parasubiculum. Neurons in the piriform, the entorhinal, and the cingulate cortices are also highly positive for AC9 mRNA. Labeled cell bodies in the cingulate and the entorhinal cortices are most prominent in layers II and III. Other limbic areas showing distinct low-to-moderate levels of mRNA signal include the dorsal septal nucleus, the lateral, medial, and posterior amygdaloid nuclei, the medial habenular nucleus, and the interpeduncular nucleus. In the neocortex AC9 mRNA is observed mainly in neurons of layers II, III, and VI and the large pyramidal cells of layer V. There were no apparent topographical variations in this pattern. The caudate putamen contain a large number of cells expressing moderate levels of AC9 mRNA, whereas the globus pallidus is essentially devoid of specific signal. The anteroventral thalamic nucleus shows moderate mRNA signal; a lower level of signal is found in the paratenial and the centromedian thalamic nuclei. Both the supraoptic and the paraventricular nuclei contain low-to-moderate levels of mRNA. Low-level labeling also is detected in the median preoptic nucleus. In the cerebellar cortex Purkinje cells are weakly positive for AC9 mRNA. Other areas of the cerebellar system expressing AC9 mRNA include the cerebellar nuclei, the inferior olive, the pontine nuclei, and the red nucleus. Some reticular, serotonergic, and motor cells in the lower brainstem express moderate levels of AC9. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca2+/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades (Antoni, 1998).

Ca²⁺/calcineurin-inhibited adenylyl cyclase (AC9) is present in areas of the brain previously thought to contain only Ca²⁺-activated (AC1 and AC8) or protein kinase C-activated (AC2 and AC7) adenylyl cyclases. The distribution of AC9 mRNA in the brain suggests close proximity to or possible colocalization in AC1- and AC2-expressing neurons in limbic and neocortical areas. Functionally, comparative analysis of AC1- and AC9-derived cAMP production in HEK-293 cells shows that AC1 and AC9 are regulated by the same range of intracellular free Ca²⁺ concentrations. Taken together, these data indicate that AC9 is a major cAMP-synthesizing enzyme in brain and is modulated by Ca²⁺ by way of the protein phosphatase calcineurin (Antoni, 1998).

All mammalian adenylyl cyclases known so far conform to the "quasi-duplicated transporter" design consisting of two membrane-spanning domains each, followed by substantial cytoplasmic loops that have been designated C1 and C2, respectively. Current evidence indicates that the physical interaction of C1 and C2 underlies the catalytic activity of the enzyme. The interaction between C1 and C2 is facilitated by forskolin or G_salpha-GTP complexes that enhance the affinity of the interaction between C1 and C2 by ~50-fold. Therefore, overexpression of adenylyl cyclase in the region of 30-fold above normal, as in the present study, should produce substantial "basal" adenylyl cyclase activity, because the number of catalytically active C1-C2 complexes is increased by a similar factor as in the presence of forskolin or G_salpha-GTP. Taken together, the characteristics of cAMP production in cultured cells stably transfected with AC9 and treated with PDE blockers reflect the regulatory properties of the adenylyl cyclase catalytic moiety. The present experiments show that intracellular free Ca²⁺ inhibits cAMP synthesis by AC9. The inhibitory effect of Ca²⁺ on AC9 is blocked by FK506 and cyclosporin A, but not by L685,818, which conforms with the current knowledge on the pharmacology of calcineurin. The degree of inhibition of AC9 by Ca²⁺/calcineurin was modest, i.e., 20-40%, which could be attributable to a number of factors, including the high levels of adenylyl cyclase relative to calcineurin expressed in the cells or the inactivation of calcineurin by [Ca²⁺]_i. Furthermore, depending on the rate of cAMP hydrolysis of the system under study, a relatively small reduction (~30%) of the rate of cAMP synthesis may lead to a marked fall (~80%) of intracellular cAMP levels (Antoni, 1998).

A comparison of the patterns of expression of AC9 and other adenylyl cyclases clearly indicates the close proximity of neurons expressing AC9 to those expressing the Ca²⁺-stimulated cyclases AC1/AC8 as well as AC2, an adenylyl cyclase stimulated by protein kinase C. Indeed, the distribution of AC9 mRNA in the hippocampal formation is identical to that of AC1, except in the pyramidal layer of CA1-CA3 where AC1 mRNA is relatively low, whereas AC9 mRNA is highly abundant. Thus mRNAs for three classes of adenylyl cyclase are expressed in the pyramidal cells of CA1-CA3 and in the granule cells of the dentate gyrus. Whether or not this is also the case at the level of protein expression and to what extent these cyclases are colocalized in the same neurons remain to be clarified (Antoni, 1998).

Currently, any considerations of the physiological function of AC9 are speculative; however, two areas stand out as plausible and biologically significant. (1) In AtT20 cells, AC9 is the target of an intracellular Ca²⁺ feedback loop that also involves BK-type K⁺ channels and Ca²⁺/calmodulin-activated PDE. This system regulates the membrane potential and the levels of cAMP to maintain cellular excitability. AtT20 cells show the rhythmic firing of action potentials and intracellular free Ca²⁺ transients. Prominent expression of AC9, Ca²⁺-activated K⁺ channels, and Ca²⁺/calmodulin PDE, i.e., the components of Ca²⁺ negative feedback on cAMP levels and the membrane potential, is evident in hippocampal principal neurons, which also exhibit various modes of rhythmic firing. Thus AC9 may be involved in the generation of rhythmic electrical activity in the hippocampus and possibly in other parts of the forebrain. (2) Hippocampal neurons have been analyzed extensively with respect to the role of cAMP in synaptic plasticity. The activation of cAMP synthesis by [Ca²⁺]_i has been attributed as a key role in various types of long-term potentiation (LTP). However, the example of the Drosophila dunce mutant, which is deficient in a cyclic nucleotide PDE gene, indicates that the hydrolysis of cAMP is also a key process in memory formation and underscores the issue of a cAMP signal that is optimized in time as well as space. Calcineurin is a potent inhibitor of postsynaptic activity in hippocampal neurons and recently has been proposed as a "memory suppressor" gene. The overexpression of calcineurin in hippocampal neurons has been shown to impair a cAMP-dependent phase of tetanus-evoked LTP as well as hippocampus-dependent memory formation. The prominent expression of AC9 in the hippocampus and the pivotal role of the tuning of the cAMP Ca²⁺ signal in synaptic plasticity indicate that AC9 may be an important physiological target for protein kinase and phosphatase cascades that contribute to the long-term modulation of synaptic transmission (Antoni, 1998 and references).

Table of contents

rutabaga: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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