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FMRFamide (Phe-Met-Arg-Phe-NH2) and several analogs produce centrally-mediated, naloxone-reversible antinociception, but have minimal affinity for opioid receptor (sub)types. In the present study, the antinociception in mice (55 degrees C tail-flick test) produced by supraspinal (intracerebroventricular; i.c.v.) administration of [D-Met2]-FMRFamide (a stable analog of FMRFamide) was attenuated by pretreatment with i.c.v. oligodeoxyribonucleotide antisense to the opioid mu receptor or by antisense to the Gi2alpha G-protein subunit. These data suggest that [D-Met2]-FMRFamide produces its antinociception via an opioid interneuron.

In vitro studies have demonstrated that FMRFamide-related peptide receptors can be coupled to different G-proteins, mediating opposite stimulatory and inhibitory effects. The present study tested whether this duality might extend to effects in vivo. Antinociception in mice of ICV [D-Met2]FMRFamide, which produced agonist [ED50 = 36.3 micrograms (61.6 nmol)] and antagonist [ID50 = 0.72 microgram (1.22 nmol)] actions, was attenuated by 24-h pretreatment with ICV pertussis toxin (ID50 = 0.55 microgram) or cholera toxin (ID50 = 0.09 microgram), suggesting that [D-Met2]FMRFamide in vivo effects might also be explained by dual Gi/Gs, coupling.

The binding affinities at opioid receptor subtypes in rat or guinea pig brain membranes were determined for the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), the two mammalian-derived FMRFamide-related peptides F-8-Famide (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe- NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), and the two other FMRFamide-related peptides Tyr-Phe-Met-Arg-Phe-NH2 (Tyr-FMRFamide) and Pro-Gln-Arg-Phe-NH2 (Pro-Gln-RFamide). The mu and delta sites were labeled in rat brain membranes using tritiated [D-Ala2, N-MePhe4,Gly-ol5] enkephalin ([3H]DAMGO) and [D-Ala2,D-Leu5]enkephalin ([3H]DADLE), respectively. The kappa sites were labeled in guinea pig brain using [3H]U-69,593 after treatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 2. The kappa 2a binding sites were assayed using [Leu5]enkephalin to block kappa 2b sites and the kappa 2b sites were assayed using (-)-(1S,2S)-U50,488 to block kappa 2a sites. Neither FMRFamide nor any of the FMRFamide-related peptides (up to 61.0 microM) displayed significant affinity at any of the subtypes of opioid receptor. Hence, the known ability of FMRFamide and FaRPs to interact with the opioid system does not appear to be related to direct binding to these opioid receptors.

Morphine and the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) were administered to mice alone or in combination intracerebroventricularly (ICV) and the effect on locomotor activity was measured. Morphine given alone (0.5 micrograms) significantly increased horizontal locomotor activity compared to vehicle-treated controls. FMRFamide at low doses (0.01-10 micrograms) had no effect of its own, but blocked the morphine-induced increase in horizontal locomotor activity. Unlike the opiate antagonist naloxone (1.0 micrograms), FMRFamide (up to 10 micrograms) had no effect on morphine-induced decrease in vertical activity. These data further support a role for FMRFamide as a modulator of opiate action, but comparison to naloxone suggests that FMRFamide might not act as a pure competitive antagonist of this opiate effect.

Tritiated [D-Ala2,NMePhe4,Gly-ol5]-enkephalin ([3H]DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on [3H]DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.

First purified 11 years ago from clam ganglia, FMRFamide (Phe-Met-Arg-Phe-NH2) was quickly demonstrated to be cardioactive in several molluscan species. Subsequent discovery that FMRFamide, or FMRFamide-related peptides (FaRPs), were present in mammalian central nervous system and gastrointestinal tract prompted investigations into the effect of FMRFamide on mammals. FMRFamide has now been shown to be cardioexcitatory in mammals, to inhibit morphine-induced antinociception, and to block morphine-, defeat-, and deprivation-induced feeding. It also inhibits colonic propulsive motility, induces behavioral effects when administered intrathecally, and has been reported to have amnesic effects in rodents. A proposal has arisen that a FMRFamide-like substance is an endogenous opioid antagonist and has stimulated a search for such a substance. However, FMRFamide has only weak affinity for opioid receptors and not all the actions of FMRFamide appear to be explained by actions at opioid receptors. Alternative mechanisms have been proposed which suggest that FMRFamide acts as a neuromodulator.

Morphine and the two endogenous mammalian FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides known as morphine-modulating neuropeptides, F-8-Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), were administered intracerebroventricularly (ICV) to mice and the effect of each on colonic bead expulsion time was measured. Each of the three compounds delayed expulsion of a 3 mm glass bead placed in the distal colon. A-18-Famide was more potent than F-8-Famide [ED 50 = 2.3 micrograms (1.2 nmole) and 13.9 micrograms (13.0 nmole), respectively]. A-18-Famide: 1) did not block morphine-induced delay of bead expulsion time, and 2) was blocked by simultaneous administration (ICV) of 1.0 microgram of the competitive opiate antagonist naloxone. These data demonstrate apparent opioid modulatory or agonist-like, rather than antagonist-like, properties of A-18-Famide and F-8-Famide.

Isolated guinea pig ilea were contracted with acetylcholine (ACh) in the absence and presence of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2). FMRFamide (0.17-17 microM) enhanced ACh-induced contractions (observed as a leftward shift of the dose-response curve and increase in Emax) with maximal effect at 1.7 microM. FMRFamide had no effect when administered alone. These results extend the demonstration of a FMRFamide/ACh interaction to mammalian tissue and support the concept that FMRFamide, or mammalian equivalents, could play a modulatory role in mammals.