FMFRamide

EVOLUTIONARY HOMOLOGS (part 2/3)

Physiological effect of FMRFamides in arthropods

In the oviduct of Locusta migratoria, an FMRFamide-related peptide termed PDVDHVFLRFamide(SchistoFLRFamide) acts as a neuromodulator, inhibiting spontaneous and induced muscle contraction. N-terminal truncated peptides were used to show that PDVDHVFLRFamide hasseparated binding and activation regions. VFLRFamide is the minimum sequence required for binding,whereas HVFLRFamide is the minimum sequence for inhibitory biological activity. Thus the Hisresidue, which does not contribute to binding, is a critical amino acid for the activation of the receptor.The N-terminal PDVD appears to play little or no role in either binding or activation. VFLRFamide,which binds to the receptor but yields no inhibitory biological activity, is a strong antagonist ofPDVDHVFLRFamide (Wang, 1995a).

Benzethonium chloride(Bztc) serves as a nonpeptide mimetic analog agonist of the SchistoFLRFamide (PDVDHVFLRFamide) receptors found on locustoviducts. Bztc competitively displaces SchistoFLRFamide binding to both high- andlow-affinity receptors of membrane preparations. Bztc mimics the physiological effects ofSchistoFLRFamide on locust oviduct, by inhibiting myogenic and induced contractions in adose-dependent manner. Bztc is therefore recognized by the binding and activation regions of theSchistoFLRFamide receptors. This discovery provides a unique opportunity within insects to finallytarget a peptide receptor for the development of future pest management strategies (Lange, 1995).

The ventral nerve cord of the locust, Locusta migratoria, was examined for the presence ofFMRFamide-related peptides (FaRPs). Several FaRPs are present. Two sequences are identical to the two peptides previouslysequenced from brain and retrocerebral complex of Locusta. These two peptides(PDVDHVFLRFamide and ADVGHVFLRFamide) are inhibitory when tested on locust oviductcontractions. The other peptides are novel with sequences of GQERNFLRFamide,AXXRNFIRFamide, and AFIRFamide. The synthesized peptides are stimulatory when tested onlocust oviduct contractions, increasing the frequency and amplitude of spontaneous contractions andresulting in a basal contraction (Lange, 1994).

The FMRFamide-related peptides F1 and F2, originally isolated from lobster pericardial organs, havebeen shown to exert cardioexcitatory effects on isolated or semi-isolated crustacean hearts. In general the effects of F1 and F2were similar; however, F1 is more potent and its effects are of longer duration than those exertedby F2. Infusion of either F1 or F2 causes a decrease in heart rate and subsequent periods of acardia.These decreases in rate occur concurrently with a short-term increase in stroke volume of theheart, followed by a longer-term decrease in stroke volume. Hemolymph flow rates through theanterior aorta, anterolateral arteries, sternal artery, and posterior aorta also show the same trend,with an initial short-term increase in flow rate followed by a longer-term decrease with periods ofischemia. Hemolymph flow through the paired hepatic arteries simply decreases, but recovery topretreatment levels is faster than in the other arterial systems (McGaw, 1995).

The physiological actions of lobster peptide F1 (TNRNFLRFamide) have been examined on threedifferent lobster nerve-muscle preparations (exoskeletal, cardiac and visceral). The peptide, which isfound at high concentrations in a lobster neurosecretory gland, causes a long-lasting enhancement ofcontractility in each target tissue. On exoskeletal nerve-muscle preparations, peptide F1 has thefollowing actions: (1) it potentiates transmitter release from nerve terminals innervating exoskeletalmuscle, leading to an increase in both spontaneous and nerve-evoked release of transmitter; (2) it actsdirectly on the muscle, in the absence of nerve activity, to induce tonic contractions; and (3) it shows apotent desensitization that does not reverse with prolonged washing of the tissue. On each of the typesof muscle examined, peptide F1 is active at nanomolar concentrations and is 3-4 orders of magnitudemore potent than FMRFamide. These findings suggest that peptide F1 is a neurohormone withwidespread myogenic actions throughout lobster peripheral tissues. The molecular mechanism(s) bywhich the peptide acts are not yet known, but do not appear to involve cyclic AMP or cyclic GMP (Worden, 1995).

The effects of two recently identified neuropeptides were examined on crayfish hearts andon neuromuscular junctions of the crayfish deep abdominal extensor muscles. The two peptides,referred to as NF1 (Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2) and DF2(Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH2), increase the rate and amplitude of spontaneous cardiaccontractions and increased the amplitude of excitatory junctional potentials (EJPs) in the deepextensors. Both effects were dose-dependent, but threshold and EC50 values for the cardiac effects are at least 10 times lower than for the deep extensor effects. The heart responds equally well tothree sequential applications of peptide in any given preparation, but the responses of the deepextensors appear to decline with successive peptide applications. The results support the hypothesisthat these two neuropeptides act as neurohormones to modulate the cardiac and neuromuscularsystems in crayfish. Quantal synaptic current recordings from the deep extensor muscles indicate thatboth peptides increase the number of quanta of transmitter released from synaptic terminals. Neitherpeptide elicits a measurable change in the size of quantal synaptic currents. NF1 causes a smallincrease in muscle cell input resistance, while DF2 does not alter input resistance. These data suggestthat DF2 increases EJP amplitudes primarily by increasing transmitter release, while the increaseelicited by NF1 appears to involve presynaptic and postsynaptic mechanisms (Skerrett, 1995).

Physiological effect of FMRFamides in non-arthropod invertebrates

The physiological effects of two FMRFamide-related neuropeptides (PF1 and PF2)isolated from the free-living nematode Panagrellus redivivus were examined using neuromuscular preparations from the parasiticnematode Ascaris suum. PF1 and PF2 hyperpolarize muscle membrane and induce sustainedflaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. PF1reverses spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatorynematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. PF1 reversal oflevamisole contraction is blocked by pretreatment with agents that interfere with nitric oxide (NO)synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution,mimics PF1 and PF2. NO synthase activity is twice as abundant in A. suum hypodermis as in muscle, but was not present inreproductive tissue. The relative abundance of NO synthase activity in these tissues is similar to theobserved specific binding of [3H]PF1. These results suggest that the inhibitory effects of PF1 andPF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role inthis process analogous to that of the endothelium in vertebrate vasculature (Bowman, 1995).

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum,exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-likepeptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system that measures pharyngealpressure changes and therefore pumping. The pharynx does not contract spontaneously; however, 5-HT(10-1000 microM) stimulate pumping at a frequency of 0.5 Hz. FMRFamide had no apparent effecton pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamideinhibits the pumping elicited by 5-HT. The duration of inhibition is dose-dependent. The inhibition of the pharyngeal muscle is preceded byan initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx isinvolved in various nematode processes, including feeding, regulation of hydrostatic pressure andexcretion (Brownlee, 1995).

The endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) can accelerate the oscillation of reciprocally inhibitory pairs ofinterneurons that pace heartbeat in the medicinal leech. A model based on all available biophysical data of a two-cell heart interneuronoscillator provides a theoretical basis for understanding this modulation. Previously observed modulation of K+ currents byFMRFamide cannot account for this acceleratory effect in the model. This observation prompted the reexamination of K+currents in heart interneurons. Better methods have been devised for separation of the various components of K+ current in order to moreaccurately measure their activation and deactivation kinetics. FMRFamide activates a previouslyundetected K+ current (IKF), which has very slow activation and deactivation kinetics. Addition of physiologically measured amountsof IKF to the model two-cell oscillator can account for the acceleratory effect of FMRFamide (Nadim, 1997).

In Aplysiasensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels ofprotein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides thecAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), andconcomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT.These findings indicate that a receptor-mediated decrease in protein phosphorylation may play animportant part in the modulation of neurotransmitter release (Sweatt, 1989).

Modulatory effects of the four molluscan neuroactive peptides. FMRFamide(Phe-Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and (SER2)-Mytilusinhibitory peptide (SER2)-MIP (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inwardcurrent (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatinaganglia. Two Achatina giant neuron types were used: v-RCDN (ventral-right cerebral distinct neuron) andPON (periodically oscillating neuron). Achatin-I was applied locally to the neuron testedby brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfusedaround the ganglia. FMRFamide suppresses markedly the Iin elicited by theachatin-I of both v-RCDN and PON. APGW-amide also suppresses the Iin of v-RCDN. Oxytocin suppresses the Iin of PON (Liu, 1995).

FMRFamide immunoreactivity in the digestive tract of the bivalve mollusc Pecten maximus wasinvestigated by immunocytochemistry. Positive FMRFamide-like immunoreactivity is detected innerve fibers in close contact with exocrine alpha amylase secreting cells. Physiological studies onenzymatically dissociated cells of the stomach-digestive gland complex demonstrate the involvementof FMRFamide and analogs in the control of alpha amylase release from the cells. TheFMRFamide-induced secretion was shown to be time- and dose-dependent. In contrast to mostnaturally occurring vertebrate secretagogues that are hormones, FMRFamide appears to work as a paracrine factor (Favrel, 1994).

Physiological effect of FMRFamides in vertebrates

In the forced swimming induced immobility test, neuropeptide FMRFamideadministered via the intracerebroventricular (icv) route, prolongs the immobilization period in rats. Intraperitoneal administration of amitriptyline, imipramine, fluoxetine or amphetamine attenuatesFMRFamide-induced prolongation of immobility. Biochemical studies indicated that FMRFamidetreatment has significant effects on rat brain monoamines. FMRFamide significantly lowers the brainlevels of 5-hydroxytryptamine and norepinephrine at dosages that prolong immobility. FMRFamide prolongs the duration of immobility, perhaps by modulating the release ofneurotransmitters like 5-hydroxytryptamine and/or norepinephrine (Muthal, 1995).

Central administration of FMRFamide in rats dose dependently increases the duration of time spent inthe open arm of an elevated plus maze and enhance the number of drink contacts in the thirsty ratconflict test. Similarly, in the social interaction test, animals pretreated with FMRFamide spend sufficienttime in active social interaction as compared to controls. Neuropeptide FMRFamide antagonizes theanxiogenic effect of yohimbine and enhances the antianxiety effect of diazepam in rats. The resultsindicate anxiolytic action of FMRFamide; the mechanism of such an action may involveserotonergic transmission (Muthal, 1994).

Specific receptors for the octapeptide FLFQPQRFamide (NPFF), a mammalian FMRFamide-likeneuropeptide with anti-opiate properties have been identified in rat central nervous system. However,exploration of the biological role of this peptide requires a peptidase-resistant agonist. In this study, thestability and binding characteristics of [125I]DYLMeFQPQRFamide, a radioiodinated analogue ofNPFF, on rat spinal cord tissue were determined and compared with those of[125I]YLFQPQRFamide, the reference ligand that previously permitted characterization of NPFFbinding sites. In a binding assay, [125I]DYLMeFQPQRFamide remains intact in the presence ofmembranes without peptidase inhibitors, whereas [125I]YLFQPQRFamide is completelyhydrolysed. The specific binding is time-dependent, dose-dependent, saturable and reversible.[125I]DYLMeFQPQRFamide shares the same binding characteristics as [125I]YLFQPQRFamide(Kd = 0.07 nM; Bmax = 14.7 fmol/mg protein). Binding is not affected by various spinal cord opioidsor peptides. Autoradiographic studies indicate that binding sites are mainly located in the mostexternal layers of dorsal horn where high densities of NPFF binding sites have previously beendescribed. [125I]YLFQPQRFamide and [125I]DYLMeFQPQRFamide binding sites are bothGTP-regulated. These findings indicate that DYLMeFQPQRFamide should be of value in studies onNPFF-mediated actions in vivo (Devillers, 1994).

Neuropeptide FF (NPFF), a rat FMRFamide-like peptide with antiopioid properties, inhibitsmorphine-induced analgesia but also produces hyperalgesia. The mechanisms ofNPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. Theopening of voltage-sensitive Na+ channels with veratridine (20 microM) induces calcium-dependentNPFF release, which is abolished by tetrodotoxin (1 microM), suggesting that NPFF release dependson nerve impulse activity. NPFF release is a function of the extent ofdepolarization and is calcium dependent. The 30 mM K(+)-induced release is blocked by Co2+ orNi2+ but is unaffected by Ca2+ channel blockers of the L type or the N-type. In contrast, omega-agatoxin IVA leads to a 65%reduction in NPFF release, suggesting that P-type Ca2+ channels play a prominent role. The 35% NPFFremaining results from activation of an unknown subtype. The NPFF-like material insuperfusates recognizes spinal NPFF receptors, suggesting that NPFF release in the spinal cord has aphysiological role (Devillers, 1995).

The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and FMRF-amide (Phe-Met-Arg-Phe-NH2), known as anti-opioid agents, wereinvestigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Bothpeptides produced significant long-lasting analgesia in both tests without causing detectablemotor dysfunction. Pretreatment with systemic naloxone attenuates the initialantinociceptive effects (first hour) induced by both peptides in the tail flick test and only byFMRFamide in the paw pressure test. A subeffective dose of F8Famide enhances both theintensity and the duration of spinal morphine analgesia in both tests. In contrast, asubanalgesic dose of FMRFamide decreases the intensity and enhances the duration ofthe effect of morphine. Besides acting as antinociceptive agents in the spinalcord, F8Famide and FMRFamide can differentially modulate spinal opioid functions (Gouarderes, 1993).

The role of neuropeptide FF (NPFF) in the modulation of spinal nociception was studied in rats with carrageenan inflammation in the hind paw. Normally no NPFF-ir (NPFF immunoreactive) neuronal cell bodies are found in the spinal cord. During inflammation NPFF-neurons are seen in an area receiving innervation from the inflamed hind limb, but in rats pretreated with morphine no NPFF-ir neurons were found. NPFF or IgG from NPFF immunoserum administered intrathecally have no effect in thermal and mechanical nociceptive tests. Morphine produces significant antinociception in both tests in the inflamed paw, but the effect is not modified by NPFF. These findings differ from the effects of intrathecal administration of NPFF and opioids in acute thermal tests when no inflammation is present. The role of NPFF in the modulation of nociception in the spinal cord may be markedly changed during acute inflammation (Kontinen, 1997).

Neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) was evaluated in animal models of psychosis. FMRFamide produces blockade of conditioned avoidance response in rats and antagonizes apomorphine-induced climbing behaviour in mice. Similarly, FMRFamide at lower doses inhibits 5-hydroxy-L-tryptophan (5-HTP)-induced head twitches in rats. These effects of the peptide were similar to haloperidol. However, unlike haloperidol, FMRFamide per se does not induce any catalepsy in rats at the doses employed in the above paradigms. These results indicate antipsychotic-like activity for the neuropeptide FMRFamide with possible involvement of the dopaminergic/5-HT2 systems (Muthal, 1997).

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. The NPFF gene has been cloned from human, bovine, rat, and mouse, and the precursor mRNA is shown to encode for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). The brain and spinal cord mRNA distribution matches the distribution of NPFF and NPSF immunoreactivity. The mRNA levels in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). These results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. These results support a model for the role of these peptides in pain regulation at the level of the spinal cord (Vilim, 1999).

FMRFamide, a cardioexcitatory neuropeptide, directly activates a newly cloned amiloride-sensitive sodium channel that is expressed specifically in the brain and blocked by benzamil hydrochloride. The effects of short- and long-term intracerebroventricular infusion of FMRFamide on arterial pressure, sympathetic activity, vasopressin release, and brain renin-angiotensin system genes were examined in rats and the role of FMRFamide-activated brain sodium channels in salt-sensitive hypertension was studied. The intracerebroventricular preinjection of FMRFamide and subsequent intracerebroventricular infusion of 0.15 mol/L NaCl increases mean arterial pressure, heart rate, abdominal sympathetic activity, and plasma vasopressin concentration. The intracerebroventricular copreinjection with either benzamil or CV-11974 abolishes these increases. In rats administered a high-salt diet (8% NaCl), the continuous intracerebroventricular infusion of FMRFamide for 5 days increases mean arterial pressure, heart rate, urinary excretion of vasopressin and norepinephrine, and mRNAs of renin, angiotensin I-converting enzyme, and angiotensin II type 1 receptor in hypothalamus and brain stem. These increases are abolished by intracerebroventricular coinfusion of benzamil. In rats administered a low-salt diet (0.3% NaCl), however, increases in these variables were smaller than those in rats receiving a high-salt diet. Together, these findings suggest that brain FMRFamide-activated sodium channels may be involved in the mechanism of salt-sensitive hypertension through regulation of the brain renin-angiotensin system (Nishimura, 2000).

Functional specifity of FMRFamides

Continued: FMRFamide: Evolutionary homologs part 3/3

back to FMRFamide: Evolutionary homologs part 1/3

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