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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.

We have previously proposed that the reciprocal hindlimb scratching (RHS) of mice elicited by intrathecal injection of muscarinic agents is mediated by M1 muscarinic receptors. In this study, benzhexol potently inhibited pilocarpine-induced RHS (ID50 = 0.5 ng), methoctramine did not fully block RHS and RS 86 neither produced nor blocked RHS. These data (1) differentiate the three muscarinic agents using this in vivo endpoint, (2) substantiate binding studies characterizing benzhexol and methoctramine as putative M2 and M2 antagonists, respectively, and (3) further support using RHS for studying muscarinic agents.

Administration of p-octopamine by intracerebroventricular (i.c.v.) or intrathecal (i.t.) routes, but not orally, produced antinociception in the acetylcholine-induced abdominal constriction test (ED50 = 24.8 and 3.6 micrograms, respectively). Likewise, i.c.v. and i.t., but not peripheral (up to 200 mg/kg s.c.), administration increased latency in the 48 degrees C hot-plate test (ED50 = 11.5 micrograms i.c.v. and 0.2 micrograms i.t.). These actions were relatively long-lasting and not blocked by naloxone. Antinociception following i.c.v. administration was abolished in reserpinized mice or by pretreatment with i.t. phentolamine (2 micrograms). These results suggest a moderate antinociceptive action of p-octopamine involving non-opioid, reserpine-sensitive, central pathways.

Intrathecal (IT) administration of pilocarpine (0.25-2.0 micrograms) to mice produced a vigorous and dose-related reciprocal hindlimb scratching response that lasted for 10-15 minutes. Neither the intracerebroventricular administration of pilocarpine at up to 10 times the intrathecal ED90 dose nor the subcutaneous administration of 10 mg/kg pilocarpine caused as robust an effect as IT administration. The reciprocal hindlimb scratching produced by the ED90 dose of pilocarpine (2 micrograms, IT) was antagonized in a dose-related manner by simultaneous IT administration of atropine (ID50 = 0.002 micrograms), methysergide (ID50 = 1.89 micrograms), the substance P antagonist [D-Pro2,D-Trp7,9]-SP (ID50 = 4.94 micrograms), and the putative neurokinin B antagonist [D-Pro2,D-Trp6,8,Nle10]-NK (ID50 = 3.33 micrograms), but not by yohimbine (5 micrograms), phentolamine (2 micrograms), or naloxone (2.5 micrograms). These results suggest that pilocarpine-induced reciprocal hindlimb scratching is mediated spinally, that the effect is produced by an action of pilocarpine on muscarinic receptors in the spinal cord, and that neurokinin, and perhaps 5-HT, mechanisms might also be involved.

C57BL/6J-bg/bg (beige-J) mice have a blunted antinociceptive response to intracerebroventricularly (i.c.v.) injected morphine in the tail-flick test. Beige-J mice are also immunologically defective and exhibit the pathology of Chediak-Higashi syndrome (CHS). We transferred by i.v. injection 2 x 10(7) mononuclear spleen cells, devoid of PMNs, obtained from beige-J mice to normal C57BL/6J-bg/+ littermates that do not exhibit CHS or a blunted antinociceptive response to morphine. After 8 days, the normal littermates demonstrated significant (P less than 0.05) reduction in their analgesic responsiveness to morphine. This phenomenon was found to require B-cells and adherent cells in the adoptively transferred spleen cells. B-cells that had been purified by panning on anti-Ig-coated plates were sufficient to transfer the analgesic defect unless adherent cells were removed prior to immunocytoadherence. T-cells, in the presence or absence of adherent cells, failed to transfer the decreased sensitivity to morphine. These results demonstrate a novel neuroimmune interaction whereby B-lymphocytes and adherent cells, or a substance derived from them, are able to affect the antinociceptive action of morphine.

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.

The mu agonist, morphine, and the prototype kappa agonists, ketocyclazocine and ethylketocyclazocine (EK), were studied for their effects on gastrointestinal transit. Following s.c. administration, both morphine (0.3-3 mg/kg) and ketocyclazocine (0.3-10 mg/kg) antagonized transit of an opaque marker through the small intestines of mice. Morphine (0.1-1 microgram) was also effective after intracerebroventricular (icv) administration in mice whereas ketocyclazocine (0.3-30 micrograms) was not. Similarly, while both morphine (0.3-5 mg/kg) and EK (0.6-10 mg/kg) slowed transit after s.c. injection to rats, only morphine (1-10 micrograms), but not EK (0.3-300 micrograms), was active following icv administration. Icv infusion of the mu benzomorphan, phenazocine (10-100 micrograms), slowed transit in a dose-related manner. These results indicate that there may be an anatomically distinct distribution of receptors for benzomorphan kappa agonists in both the mouse and rat, with these opiate receptors not being located near the lateral cerebral ventricles. The difference in efficacy between morphine and ketazocines in slowing gastrointestinal transit after icv administration to rodents suggests that (a) inactivity in this endpoint is a characteristic of benzomorphan kappa compounds and (b) the model may serve as a useful screen when establishing in vivo profiles of kappa agonists in mice and rats.

Groups of rats were administered s.c. saline or morphine for 14 consecutive days and received morphine, saline or no treatment on day 15. Significant and consistent environmental cues were established through the treatment and test periods. Transit of a charcoal meal along the gastrointestinal tract, measured on day 15, was significantly greater in rats conditioned to daily morphine, followed by saline on day 15, than in any other group. This enhanced transit was approximately equal in magnitude to the amount of developed tolerance to morphine. These results support the hypothesis that tolerance to opiates may be partly explained by a compensatory conditioned physiological response opposing the acute effect.

Mice were exposed to whole-body irradiation (500 rads) from a 137Cs gamma-source and tested 2 h later for antinociception (tail-flick test) produced by intracerebroventricular administration of morphine or the more delta-selective opioid peptide, [D-Pen2,L-Pen5]enkephalin (DPLPE). Irradiation significantly attenuated the antinociception produced by morphine, but not by DPLPE. These results demonstrate a differential sensitivity of mu- and delta-opioid receptors to gamma-irradiation and, in addition, may be of clinical relevance for cancer patients receiving concurrent radiation therapy and opioid analgesics.

We recently discovered and reported that C57BL/6J-bgJ/bgJ (beige-J) mice have a deficiency in their analgesic response to intracerebroventricularly-administered morphine in the tail-flick test. Postulating a link between these findings and the known immunological defect of beige-J mice (Chediak-Higashi syndrome), we examined the effect of splenectomy on beige-J mice and the adoptive transfer of their mononuclear spleen cells to normal littermate controls (2 x 10(7) cells via tail vein). Eight days after these interventions, the splenectomized beige-J mice responded nearly as well as normal mice to centrally administered morphine in the tail-flick test. The adoptive transfer recipients, in contrast, nearly completely lost their response to the analgesic action of morphine in this test. From the combined results, the spleen appears to be a significant factor in the analgesic defect of beige-J mice and, furthermore, mononuclear splenocytes appear to be the source of a substance that can transfer this defect to otherwise normal animals.

Drugs are considered to produce their effects on biological tissues either by altering some physical property of cells or by interacting with specific cellular components, called receptors. Most drugs and endogenous neurotransmitters act on highly selective receptors located on the outer surface membrane of cells. These receptors were believed, until recently, to be stationary on the cell surface and to be present in unvarying numbers. Consequently, most early theorists modeled the drug-receptor interaction on the basis of stationary and static receptor molecules. The substantial advances in our understanding of drug action based on these models have partly justified this view. However, recent electron microscopic studies have revealed the presence of structures, including "coated" pits and vesicles, that appear to provide a mechanism by which cell surface receptors might be internalized in a process of endocytosis. The precise intracellular fate of these internalized receptors is unknown, but based on present understanding, it seems reasonable to believe that some are destroyed intracellularly whereas others are recycled to the cell surface. The importance of such processes to pharmacologic theory is a new awareness of a cellular pathway that is capable of internalizing drugs, receptors, or both. The implications of such a process to the theory of drug action extends to some unexplained drug phenomena such as down regulation, drug tolerance, tachyphyllaxis, and partial agonism. We present herein the theoretical framework for a model of drug action that incorporates the possibility of receptor internalization and subsequent degradation, recycling, or replacement.

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.

Intrathecal (IT) administration of pilocarpine to mice produces a vigorous and dose-related reciprocal hindlimb scratching (RHS) response (ED50 = 0.6 microgram) that is potently blocked by simultaneous IT administration of atropine (ID50 = 0.002 microgram). We now report that RHS is (1) also elicited by the more selective M1 agonist McN-A-343-11 (ED50 = 11.6 micrograms), (2) blocked by the selective M1 antagonist pirenzepine (ID50 = 0.001 microgram), and (3) is not blocked by the selective M2 antagonist AF-DX 116 BS at a dose up to 100 times the ID50 dose of pirenzepine. These results extend our earlier findings and suggest that the RHS elicited in mice by IT injection of muscarinic agonists is mediated through pirenzepine-sensitive (presumably M1) receptors and that RHS may be a convenient in vivo centrally mediated M1 endpoint.

A standard rabbit thoracic-aorta strip preparation was used to determine the effect of varying preload on KA(the dissociation constant), A50(the concentration that produces half maximal response) and the ratio KA/A50. Additionally, the stimulus-response relationships were obtained for the drug and preload conditions tested. Values of KA and A50 were calculated from dose-response curves obtained for three alpha-adrenergic agonists (phenylephrine, methoxamine and norepinephrine) in the presence and absence of partial irreversible blockade by phenoxybenzamine. For all three drugs, values of KA and A50 were found to be significantly greater at 1/4 gram preload than at 10 gram preload. However, the ratio KA/A50 was found to be independent of preload conditions. The stimulus-response relationships were all found to be non-linear, in contrast to the predictions of classical receptor theory. The significance of the constancy of KA/A50, rather than either KA or A50, and the non-linear relation between drug stimulus and response is discussed in terms of Stephenson's theory of drug action.

It is generally held that many biologically active compounds produce their effects through a sequence of events that are initiated when the substances combine with selective receptors located on the cell surface membrane. Activation of these receptors produces a stimulus that is somehow transmitted intracellularly. The transduction between the stimulus and the response is now known to be mediated, in many systems, by an intracellular intermediary or second messenger. A model describing the relation of agonist concentration, receptor occupation, and biological response in such a system is herein extended to include antagonist binding at two target sites the cell-surface receptor and the receptor for the second messenger. It is demonstrated that the shift of an agonist's dose-response curve is characteristic of the site of antagonism and that analysis of this shift can reveal the existence of a second messenger pathway or, if this is known, the site of action of the antagonist.

The antinociceptive effects of two mu-opioid receptor agonists, morphine and [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO), and a selective delta-receptor agonist, [D-Pen2, L-Pen5]enkephalin (DPLPE), were determined in C57BL/6J-bgJ (beige) and control mice (CRS-CDl and C57BL/6By) using a standard tail-flick assay. The antinociceptive response of C57BL/6J-bgJ mice to intracerebro-ventricularly administered morphine and DAGO was significantly reduced compared to controls, but there was no difference in the antinociceptive response to DPLPE. These results suggest that there is a genetic deficit of mu-opioid receptor number or a genetically-induced alteration in receptor function in regions of C57BL/6J-bgJ brains involved in antinociception, that delta-opioid receptors can mediate antinociception in mice, and that the C57BL/6J-bgJ strain may offer a practical new animal model for studying the function of opioid receptor subtypes.

C57BL/6J-bgJ (beige-J) mice respond poorly to the analgesic effects of centrally administered (intracerebroventricular; i.c.v.) morphine in the tail-flick test. In the present study C57BL/6J-bgJ mice, their littermate (heterozygous) controls, and normal Swiss mice were given carbachol (200 micrograms/ml) in their drinking water for three weeks. Carbachol had no effect on control animals. However, the carbachol-treated beige-J mice responded to i.c.v.-administered morphine (1 microgram) significantly better than untreated beige-J mice and nearly as well as the normal mice.