naltrindole and bremazocine

Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [(3)H]bremazocine or with [(3)H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist.

Topics: Animals; Autoradiography; Benzomorphans; Brain; Gene Knockout Techniques; Male; Mice; Mice, Knockout; Naltrexone; Protein Multimerization; Protein Structure, Quaternary; Receptors, Opioid, delta; Receptors, Opioid, kappa; Subtraction Technique

Agonists at delta, mu, and kappa opioid receptors produce interacting effects in rodents and nonhuman primates. To further evaluate the determinants of these interactions, this study examined the effects of mixtures of delta + mu and delta + kappa agonists in rhesus monkeys (n = 4-5) using two behavioral procedures, an assay of schedule-controlled responding for food reinforcement and an assay of thermal nociception. Results were analyzed using dose-addition analysis. In the assay of schedule-controlled responding, the delta agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide (SNC80); the mu agonists methadone, fentanyl, morphine, and nalbuphine; and the kappa agonists (5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) and bremazocine all dose dependently decreased rates of food-maintained responding when administered alone. Fixed ratio mixtures of SNC80 + mu agonists produced additive or subadditive effects, whereas SNC80 + kappa agonist mixtures produced only additive effects. In the assay of thermal nociception, SNC80 produced no measurable effects when administered alone, whereas mu and kappa agonists produced dose-dependent antinociception. SNC80 + mu agonist mixtures produced superadditive effects manifested as leftward shifts in mu agonist dose-effect curves. This synergism was antagonized by the delta-selective antagonist naltrindole, suggesting that SNC80-induced enhancement of mu agonist antinociception was delta receptor-mediated. SNC80 did not enhance the antinociceptive effects of the highly selective kappa agonist U69,593, and it produced only a marginal enhancement of antinociception produced by the less selective kappa agonist bremazocine. These results suggest that delta agonists may selectively enhance the antinociceptive effects of mu agonists in rhesus monkeys. These results also confirm that opioid agonist interactions may depend on the receptor selectivity and relative doses of the agonists and on the experimental endpoint.

Topics: Analgesics, Opioid; Animals; Benzamides; Benzeneacetamides; Benzomorphans; Conditioning, Operant; Dose-Response Relationship, Drug; Drug Interactions; Hot Temperature; Macaca mulatta; Male; Naltrexone; Narcotic Antagonists; Pain; Piperazines; Pyrrolidines; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reinforcement Schedule

We hypothesized that the selectivity profile of the rat mu-opioid receptor for opioid receptor-selective ligands is determined by the nature of the amino acid residues at highly divergent sites in the ligand-binding pocket. To determine which characteristics of these residues contribute to opioid receptor ligand selectivity, we made various mutant receptors that replaced the Lys(303) and Trp(318) residues near the extracellular interface of transmembrane domains VI and VII, respectively. Ligand binding determinations using transiently transfected monkey kidney epithelial (COS-1) cells show that Lys(303) mutations cause little change in the receptor binding profile, whereas the Trp(318) mutant receptors have considerably lower affinity for micro-opioid receptor-selective ligands and greatly increased affinity for delta-opioid receptor-selective ligands. The nature of these mutations show that this effect is not due to sterics or charge alone. [35S]guanosine-5'-O-(3-thio)-triphosphate ([35S]GTPgammaS) activity assays show that these residues may influence functional, as well as binding selection. We conclude that a primary role for Trp(318) is to form a basis for ligand selectivity.

Topics: Amino Acid Substitution; Animals; Benzamides; Benzomorphans; Binding Sites; Binding, Competitive; COS Cells; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Fentanyl; Guanosine 5'-O-(3-Thiotriphosphate); Ligands; Morphine; Mutation; Naloxone; Naltrexone; Narcotic Antagonists; Peptides; Piperazines; Protein Conformation; Radioligand Assay; Rats; Receptors, Opioid, mu; Signal Transduction; Sulfur Radioisotopes

The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [(3)H]bremazocine by selective ligands for the mu (mu), delta (delta), and kappa (kappa) receptors revealed that only the delta -selective ligands could fully displace specific [(3)H]bremazocine binding, indicating the presence of only the delta -receptor in these cells. Saturation binding studies with the delta -antagonist naltrindole afforded a B(max)of 32 fmols/mg protein and a K(D)value for [(3)H]naltrindole of 0.46 n M. The binding affinities of various delta ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastomaxglioma cell line, NG108-15. Finally, various delta -agonists were found to stimulate the binding of [(35)S]GTP gamma S, confirming coupling of the cardiac delta -receptor to G-protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [(35)S]GTP gamma S to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled delta -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors.

Topics: Analgesics; Benzomorphans; Binding, Competitive; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Ligands; Myocardium; Naltrexone; Narcotic Antagonists; Receptors, Opioid, delta; Time Factors; Tumor Cells, Cultured

Modulation of depolarization-activated ionic conductances by opioid receptor agonists was investigated in isolated parasympathetic neurons from neonatal rat intracardiac ganglia by using the whole cell perforated patch clamp technique. Met-enkephalin (10 muM) altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and repolarization but the afterhyperpolarization was not appreciably altered. Under voltage clamp, 10 muM Met-enkephalin selectively and reversibly inhibited the peak amplitude of high-voltage-activated Ca2+ channel currents elicited at 0 mV by approximately 52% and increased three- to fourfold the time to peak. Met-enkephalin had no effect on the voltage dependence of steady-state inactivation but shifted the voltage dependence of activation to more positive membrane potentials whereby stronger depolarization was required to open Ca2+ channels. Half-maximal inhibition of Ba2+ current (IBa) amplitude was obtained with 270 nM Met-enkephalin or Leu-enkephalin. The opioid receptor subtype selective agonists, DAMGO and DADLE, but not DPDPE, inhibited IBa and were antagonized by the opioid receptor antagonists, naloxone and naltrindole with IC50s of 84 nM and 1 muM, respectively. The kappa-opioid receptor agonists, bremazocine and dynorphin A, did not affect Ca2+ channel current amplitude or kinetics. Taken together, these data suggest that enkephalin-induced inhibition of Ca2+ channels in rat intracardiac neurons is mediated primarily by the mu-opioid receptor type. Addition of Met-enkephalin after exposure to 300 nM omega-conotoxin GVIA, which blocked approximately 75% of the total Ca2+ channel current, failed to cause a further decrease of the residual current. Met-enkephalin inhibited the omega-conotoxin GVIA-sensitive but not the omega-conotoxin-insensitive IBa in rat intracardiac neurons. Dialysis of the cell with a GTP-free intracellular solution or preincubation of the neurons in Pertussis toxin (PTX) abolished the attenuation of IBa by Met-enkephalin, suggesting the involvement of a PTX-sensitive Gprotein in the signal transduction pathway. The activation of mu-opioid receptors and subsequent inhibition of N-type Ca2+ channels in the soma and terminals of postganglionic intracardiac neurons is likely to inhibit the release of ACh and thereby regulate vagal transmission to the mammalian heart.

Topics: Acetylcholine; Animals; Animals, Newborn; Anti-Arrhythmia Agents; Benzomorphans; Calcium; Calcium Channels; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Heart Conduction System; Ion Channel Gating; Ion Transport; Naloxone; Naltrexone; Narcotic Antagonists; Neurons; omega-Conotoxin GVIA; Parasympathetic Nervous System; Patch-Clamp Techniques; Peptides; Pertussis Toxin; Rats; Receptors, Opioid; Receptors, Opioid, kappa; Virulence Factors, Bordetella

The delta opioid agonist BW373U86 was examined alone and in combination with mu agonists in pigeons trained to discriminate the mu agonist fentanyl (0.056 mg/kg), the kappa agonist bremazocine (0.017 mg/kg), and distilled water in a three-choice drug discrimination procedure. BW373U86 (0.01-10 mg/kg) produced a dose-dependent increase in fentanyl-appropriate responding and complete generalization to fentanyl in four of five subjects. BW373U86 did not elicit bremazocine-appropriate responding in any of the subjects. Fentanyl-appropriate responding elicited by BW373U86 was antagonized by the delta selective antagonist naltrindole (0.1-10 mg/kg) but not by the mu selective antagonist naloxone (0.1-30.0 mg/kg). When BW373U86 was administered in combination with the mu agonists fentanyl, morphine and nalbuphine, a low dose of BW373U86 (0.01 mg/kg) that elicited primarily water-appropriate responding when administered alone did not produce a significant change in the ED50 values for fentanyl, morphine or nalbuphine. Higher doses of BW373U86 (0.1-1.0 mg/kg) increased levels of fentanyl-appropriate responding elicited by low doses of fentanyl, morphine and nalbuphine to levels similar to those produced by BW373U86 alone. These results indicate that BW373U86 shares discriminative stimulus properties with the mu agonist fentanyl in pigeons, possibly by acting at delta opioid receptors. However, BW373U86 does not potentiate the discriminative stimulus effects of mu agonists or share discriminative stimulus effects with the kappa agonist bremazocine.

Topics: Analgesics; Animals; Benzamides; Benzomorphans; Columbidae; Discrimination, Psychological; Female; Fentanyl; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Piperazines; Receptors, Opioid, delta

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Topics: Analgesics; Animals; Benzomorphans; Dynorphins; Models, Molecular; Naltrexone; Narcotic Antagonists; Nociceptin Receptor; Point Mutation; Rats; Receptors, Opioid

The actions of opioid receptor agonists and antagonists were studied in isolated rat aortic strips. Morphine (10(-7)-10(-6) M) had no contractile effect on resting strips but when added during the relaxation of the contractions induced by 10(-9) M noradrenaline, it induced a contractile response which was blocked by naloxone. The selective mu-opioid receptor agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, 10(-7)-10(-6) M), induced an increase in basal tension which remained after removal of endothelium or in Ca(2+)-free solution, but was inhibited by beta-flunaltrexamine. beta-Flunaltrexamine also inhibited the contractile response induced by DAMGO added during the relaxation of the contractions induced by noradrenaline. The delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin, had no effect on resting tension but potentiated the contractions induced by noradrenaline; these effects were abolished by naltrindol. The selective kappa-opioid receptor agonist, bremazocine, had no effect on resting tension and did not modify the amplitude of the contractions induced by noradrenaline. These results suggest that, at low concentrations, agonists of mu- and delta-opioid receptors may act as modulators of noradrenaline-induced responses, whereas at higher concentrations, mu-opioid receptor stimulation may have a direct contractile effect in isolated rat aorta.

Topics: Amino Acid Sequence; Analgesics; Analysis of Variance; Animals; Aorta; Benzomorphans; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Molecular Sequence Data; Morphine; Muscle Contraction; Muscle, Smooth, Vascular; Naltrexone; Norepinephrine; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, mu