enkephalin--ala(2)-mephe(4)-gly(5)- and naloxone-benzoylhydrazone

J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) is a recently developed antagonist of the opioid receptor-like 1 (ORL1) receptor. We compared the in vitro functional profile J-113397 on [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTPgammaS) binding to mouse brain with that of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 and naloxone benzoylhydrazone (NalBzoH). J-113397 antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding to mouse brain with an IC50 value of 7.6 nM, but had no effect on basal [35S]GTPgammaS binding by itself. [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 partially antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding but showed agonistic activity on ORL1 by itself. NalBzoH showed antagonistic activity on ORL1 receptor but had significant agonistic activity on other opioid receptors at lower doses. Schild plot analysis demonstrated competitive antagonism of J-113397 on ORL1 receptor in mouse brain. A [35S]GTPgammaS binding study using ORL1 receptor-deficient mice confirmed the selective antagonism of J-113397 on ORL1 receptor. These data indicate that J-113397 is the most potent and selective antagonist of ORL1 receptor in mouse brain that has yet been reported, and therefore will be a useful tool for characterization of ORL1 receptors in the brain.

Topics: Animals; Benzimidazoles; Binding, Competitive; Brain; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Naloxone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Opioid Peptides; Peptide Fragments; Piperidines; Receptors, Opioid; Sulfur Radioisotopes

A novel receptor, the opioid receptor-like orphan receptor (ORL1), is homologous to, but distinct from, classical opioid receptors. Although initially developed as an opioid receptor ligand, naloxone benzoylhydrazone (NalBzOH) is one of the few antagonists at ORL1. The present electrophysiological study of the effects of NalBzOH on the activation of ORL1 and mu-opioid receptors was performed in brain slices of the ventrolateral periaqueductal grey (PAG), a crucial site for opioid-induced supraspinal analgesia. Both orphanin FQ/nociceptin (OFQ/N), an ORL1 agonist, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, activated inwardly rectifying K+ (Kir) channels in the ventrolateral PAG. Of the neurons tested, 96% responded to OFQ/N, but only 65% to DAMGO. NalBzOH (3-30 microM) antagonized the effect of OFQ/N competitively with a pA2 of 5.67. NalBzOH also antagonized, but more potently and non-competitively, the effect of DAMGO. In contrast, NalBzOH did not affect baclofen-induced activation of Kir channels. NalBzOH alone, at concentrations up to 30 microM, had little effect on this inwardly rectifying channel. It is concluded that NalBzOH antagonizes the activation of Kir channels mediated by both ORL1 and mu-opioid receptors in the ventrolateral PAG. It acts not only as a competitive antagonist at ORL1, but also as a more potent and non-competitive antagonist at mu-opioid receptors.

Topics: Analgesics, Opioid; Animals; Brain; Drug Interactions; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Naloxone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Opioid Peptides; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, mu; Vasodilator Agents

Naloxone benzoylhydrazone (NalBzoH) is a potent mu antagonist in vivo. In a cell line stably transfected with MOR-1 (CHO/MOR-1), NalBzoH also was an antagonist when examined in adenylyl cyclase studies. In binding studies, it displayed high affinity for the mu receptor, confirming its earlier characterization in brain membranes. In competition studies under equilibrium conditions, NalBzoH and diprenorphine both retained their potency in the presence of the stable GTP analog 5'-guanylylimidophosphate, consistent with their mu antagonist properties, whereas the agonist DAMGO showed more than a 3-fold loss of affinity. The dissociation of 3H-diprenorphine was monophasic. However, kinetic studies revealed biphasic dissociations for both 3H-NalBzoH and 3H-DAMGO. The slow component of 3H-NalBzoH dissociation, corresponding to the higher affinity state, was dependent on coupling to G-proteins. It is selectively abolished by guanine nucleotides, leaving only the rapid dissociation phase. Furthermore, the slow dissociation component is eliminated by treatment of the cells with pertussis toxin, but not cholera toxin. In conclusion, NalBzoH is an unusual opioid. Functionally it is an antagonist, a classification consistent with its equilibrium binding in the presence of guanine nucleotides. Yet, kinetic studies reveal that it labels a G-protein coupled state of the receptor with high affinity.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; CHO Cells; Cholera Toxin; Cricetinae; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Naloxone; Narcotic Antagonists; Pertussis Toxin; Receptors, Opioid, mu; Transfection; Virulence Factors, Bordetella

Studies of various strains of mice revealed marked differences in their analgesic sensitivity towards morphine (mu), U50,488H (kappa 1) and naloxone benzoylhydrazone (NalBzoH; kappa 3). Sensitivity to mu and kappa analgesia varied independently of the other. Analgesic sensitivity to morphine remained relatively consistent among 3 different nociceptive assays for each strain. However, the sensitivity of an individual strain to NalBzoH remained highly dependent upon the assay used. CD-1 mice were sensitive to NalBzoH in all 3 assays, but in BALB/c mice NalBzoH produced analgesia only in the hot plate and cold water tail-flick assays. In Swiss-Webster mice, NalBzoH was active in the radiant heat and cold water tail-flicks but inactive in the hot plate. Although the levels of mu, kappa 1 and kappa 3 binding in whole brain homogenates did vary somewhat, they did not correlate with analgesic sensitivity. These results suggests that the genetic controls over mu and kappa analgesia operate independently and further illustrate the many difficulties in evaluating potential analgesics.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesia; Analgesics; Animals; Benzeneacetamides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Mice, Inbred Strains; Morphine; Naloxone; Pain; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Species Specificity