enkephalin--leucine-2-alanine and bremazocine

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.

Topics: Amino Acid Sequence; Analgesics; Analgesics, Opioid; Benzomorphans; Binding, Competitive; Cells, Cultured; Cloning, Molecular; Down-Regulation; Enkephalin, Leucine-2-Alanine; GTP-Binding Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Indoles; Isoquinolines; Kidney; Ligands; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Morphine; Mutagenesis, Site-Directed; Naloxone; Narcotic Antagonists; Quinolines; Radioligand Assay; Receptors, Opioid, delta; Receptors, Opioid, mu; Tritium

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 changes in the latency for tail withdrawal in response to noxious heating of the skin induced by microinjection of opioid or serotonergic agonists into the anterior pretectal nucleus (APtN) was studied in rats. The mu-opioid agonist DAMGO (78 and 156 picomol), but not the delta-opioid agonist DADLE (70 and 140 pmol), the kappa-opioid agonist bremazocine (0.24 and 0.48 nanomol) or the sigma-opioid agonist N-allylnormetazocine (0.54 nanomol), produced a dose-dependent antinociceptive effect. The 5-HT1 agonist 5-carboxamidotryptamine (19 and 38 nanomol) and the 5-HT1B agonist, CGS 12066B (1.12 and 2.24 nanomol), but not the non-selective 5-HT agonist m-CPP (41 to 164 nanomol), 5-HT2 agonist alpha-methylserotonin (36 and 72 nanomol) and 5-HT3 agonist 2-methylserotonin (36 and 72 nanomol), produced a dose-dependent antinociceptive effect. These results indicate that the antinociceptive effects of opioid or serotonergic agonists microinjected into the APtN depend on drug interaction with local mu or 5-HT1B receptors, respectively.

Topics: Analgesics; Animals; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Microinjections; Pain; Phenazocine; Piperazines; Quinoxalines; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Serotonin; Serotonin Receptor Agonists

Binding sites were characterized in rat whole spinal cord crude membrane preparations using selective labelling techniques with multiple methods of mathematical analysis of experimental curves. Mathematical analysis of single [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (DAGO) saturation curves suggested binding of the [3H]-ligand at one site, while displacement curves of low concentrations of [3H]-DAGO with selective mu-ligands indicated the presence of high- and low-affinity sites. All the [3H]-DAGO curves processed simultaneously by LIGAND analysis showed the presence of high (27%) and low (73%) affinity components, with a total Bmax of 3.19 pmol/g tissue. Eighty percent of [3H]-[D-Ala2,D-Leu5] enkephalin (DADLE) binding was displaced by DAGO with high affinity, indicating that a high percentage of [3H]-DADLE binding was at mu-sites. Saturation curves of [3H]-DADLE after inhibition of mu-sites by unlabelled DAGO (delta-sites) were monophasic with non-linear fitting analysis and the Bmax was 0.90 pmol/g tissue. Most mathematical analysis of single saturation curves of [3H]-(-)-bremazocine indicated binding at more than one site. DAGO, DADLE, U-69,593 and PD 117302 displaced 0.15 nM of [3H]-(-)-bremazocine biphasically: the percentages of displacement calculated with the non-linear fitting program were respectively 50 (mu-sites), 64 ((mu + delta)-sites), 18 and 25 (kappa-sites). Haloperidol displaced [3H]-(-)-bremazocine only at microM concentrations. suggesting no binding at sigma-sites. In the presence of 225 nM of DAGO, DADLE displaced only 21% of [3H]-(-)-bremazocine 0.15 nM binding (delta-sites). Most mathematical analysis of saturation curves of [3H]-(-)-bremazocine, after inhibition of binding at mu- and delta-sites by DAGO and DADLE, still indicated binding at more than one site and the selective kappa-ligands U-69,593 and PD 117302 displaced [3H]-(-)-bremazocine in these experimental conditions. LIGAND analysis of saturation and inhibition curves of [3H]-(-)-bremazocine by U-69,593 and PD 117302 showed the presence of high (43%) and low (57%) affinity components, with a total Bmax of 2,73 pmol/g tissue. Thus in rat spinal cord there are at least two mu-sites bound by [3H]-DAGO which amount together to approximately 47% of total opioid sites, delta-sites bound by [3H]-DADLE amounting to approximately 13%, kappa-sites and other unknown sites (possibly a kappa-subtype) bound by [3H]-(-)-bremazocine, which together are approximately 40% of total opioid s

Topics: Animals; Benzomorphans; Binding Sites; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Male; Rats; Receptors, Opioid; Spinal Cord

Opioid receptors in membranes prepared from guinea-pig cerebellum were modified irreversibly by treatment with a water soluble carbodiimide, 1-ethyl,3-(3-dimethylaminoethyl)carbodiimide (EDAC). This decreased the number of [3H]bremazocine binding sites (Bmax reduced from 140 to 100 fmol/mg by 1 mM EDAC) without changing their affinity. When the EDAC concentration used was sufficient (500 mM) to inactivate almost all of the opioid receptors, the modification was partly prevented by inclusion of high concentrations (100 microM) of opioid agonists ([D-Ala2, MePhe4, Glyol5]-enkephalin, [D-Ala2, D-Leu5]-enkephalin,(+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzo(b)thiophene-4-acetamide hydrochloride), although they exhibited equal efficacy irrespective of their mu, delta or kappa type selectivity. However, almost all of the opioid binding sites were protected when a guanine nucleotide analogue (GppNHP, 100 microM) was also included with the agonists during carbodiimide treatment.

Topics: Animals; Benzeneacetamides; Benzomorphans; Binding Sites; Brain Chemistry; Carbodiimides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Guanylyl Imidodiphosphate; Guinea Pigs; Pyrrolidines; Receptors, Opioid

Binding capacities and affinities (KD) of ligands at mu-, delta- and kappa-opioid binding sites were determined by selective labelling techniques together with analysis of saturation curves in seven regions of the guinea-pig brain. The kappa-sites predominated over the other sites in most regions and were 90% of the total in the cerebellum; binding capacities at kappa-sites were highest in the cortex, intermediate in the cerebellum, striatum and mesencephalon and lowest in the diencephalon, hippocampus and pons-medulla. At the mu-sites, binding capacities were highest in the diencephalon and mesencephalon, with intermediate levels in the pons-medulla, cortex and striatum, and low levels in the hippocampus and cerebellum. The highest binding capacity at the delta-sites was in the striatum, intermediate in the cortex, diencephalon and hippocampus, low in the mesencephalon and pons-medulla and not detectable in the cerebellum. No regional differences in binding affinities were found at mu-, delta- and kappa-sites with [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin (KD = 1.10-2,61 nM), [3H]-[D-Ala2,D-Leu5]enkephalin (KD = 0.81-1.94 nM) and [3H]-(-)-bremazocine (KD = 0.083-0.185 nM). Thus in guinea-pig brain there are regional differences in opioid binding capacity and in the distribution of mu-, delta- and kappa-sites, but not in binding affinities.

Topics: Animals; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; In Vitro Techniques; Male; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

1. It has recently been suggested that opioid antagonists may be divided into those possessing negative intrinsic activity (e.g. naloxone) and those with neutral intrinsic activity (e.g. MR2266). 2. MR2266 was chronically administered to rats by subcutaneous infusion at a dose of 0.3 mg kg-1 h-1 for 1 week. 3. This dose reduced ingestive behaviour and blocked the antinociceptive effects of a kappa-agonist, indicating occupation of opioid receptors in vivo. 4. No supersensitivity could be detected to the antinociceptive actions of mu or kappa agonists, either one or two days after cessation of treatment. 5. No up-regulation of mu, delta or kappa binding sites was observed. 6. Since naloxone induces both supersensitivity and receptor up-regulation under equivalent conditions, the results suggest that negative intrinsic activity may be required for these phenomena to occur.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Benzomorphans; Binding, Competitive; Drinking; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Narcotic Antagonists; Pain; Pain Measurement; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Sensory Thresholds; Up-Regulation

Equilibrium binding saturation studies with [3H]bremazocine, under mu- and delta-suppressed conditions and [3H]U69593 have demonstrated that both radioligands bind with high affinity to an apparently homogeneous population of binding sites in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. In competition studies, the absolute affinities and slopes of the inhibition curves for several unlabelled ligands against [3H]bremazocine were not significantly different to those against [3H]U69593 and were consistent with binding to the kappa-opioid binding site. In the intestinal layers underlying the longitudinal muscle-myenteric plexus [3H]bremazocine, under kappa-selective conditions, recognized both a high and low affinity site. In contrast, [3H]U69593 bound to a homogeneous population of binding sites. The [3H]U69593 binding site and the [3H]bremazocine high affinity site demonstrated comparable characteristics to the single, kappa site identified in the longitudinal muscle layer. The nature of the low affinity site was not investigated due to difficulties associated with low specific binding, and its significance therefore remains to be investigated.

Topics: Animals; Benzeneacetamides; Benzomorphans; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Ileum; In Vitro Techniques; Kinetics; Muscle Contraction; Muscle, Smooth; Naloxone; Pyrroles; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa; Thiophenes

The kappa-agonists bremazocine and dynorphin(1-13), the sigma-agonist SKF 10.047 as well as the delta-agonists [D-Ala2,D-Leu5]-enkephalin (DADL) and Met-enkephalin, but not the mu-agonist morphine, applied subarachnoidally to the caudal portion of the transected spinal cord (at the T3-T4 level) induced postural asymmetry of the hind limbs in rats. Asymmetry was registered visually. The leg was regarded as flexed if its projection on the longitudinal axis of the animal was smaller than that of its counterpart. The side of the flexed leg depended upon the type of drug: bremazocine, dynorphin(1-13) and Met-enkephalin predominantly induced flexion of the right leg, SKF 10.047 induced flexion of the left leg (at some doses there is no side preference), while in the case of DADL the side of the flexed leg depended upon the dose of the drug. Comparison of electromyographic activity of the symmetric biceps and quadriceps femoris revealed that bremazocine considerably facilitates the flexion reflex of the right hind limb without affecting the left limb reflex. As a rule, a flexed leg determined visually exhibited higher EMG activity of the biceps femoris as compared with a symmetric one. The opiate antagonist naloxone significantly reduced the percentage of animals with postural asymmetry. The magnitude of asymmetry and the side of flexion were not constant in some animals, but changed with time. However, the mean magnitude of asymmetry, the percentage of animals with asymmetry and the left/right flexion ratio in each group of animals remained constant. The side of flexion also depended upon the level of spinal cord transection: bremazocine and Met-enkephalin injected subarachnoidally following transection at the T1-T4 and T5-T6 levels predominantly induced flexion of the right and the left leg, respectively. Asymmetry did not develop in physiologically intact animals given bremazocine, even if the spinal cord was cut later, i.e. transection of the spinal cord was necessary for the development of asymmetry. These data indicate that the neurons which maintain muscular tone of the hind limbs and which are located symmetrically to the sagittal plane, have different sensitivities to kappa-, sigma-, and delta-agonists. In most animals, neurons with a higher specificity to some agonist are localized on one side of the sagittal plane.

Topics: Animals; Benzomorphans; Decerebrate State; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Functional Laterality; Male; Muscles; Naloxone; Narcotics; Phenazocine; Posture; Rats; Rats, Inbred Strains; Receptors, Opioid; Spinal Cord; Time Factors

The opioid mu-agonist morphine, the delta-agonist [D-Ala2,D-Leu5]enkephalin (DADL) and the kappa-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N1). After application of the sigma-agonist SKF 10.047 the inhibition was followed by facilitation of EP. The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Bremazocine and morphine predominantly inhibited the left cortical EP, whereas DADL was a potent inhibitor of the right cortical EP. Thus, the opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to the kappa- and mu-type, while delta-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Besides, we have studied in vivo binding of the kappa-agonist [3H]ethylketocyclazocine ([3H]EKC) and the delta-agonist [3H]DADL to LVC and RVC. The binding was specific and could be accounted for by the interaction with membrane opioid receptors and/or specific uptake of these drugs by cortex cells. [3H]EKC and [3H]DADL exhibited a more effective binding to LVC and RVC membranes, respectively. The results obtained in vitro apparently indicate that LVC and RVC differ in the number of kappa- and delta-binding sites. Thus, turtle brain may have a side-specific mechanism for selective neurochemical regulation of neuron activity in LVC and RVC realized predominantly via kappa- and delta-receptors, respectively.

Topics: Animals; Benzomorphans; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Evoked Potentials, Visual; Functional Laterality; Morphine; Naloxone; Receptors, Opioid; Turtles; Visual Cortex

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.

Topics: Analgesics; Animals; Benzomorphans; Binding, Competitive; Body Weight; Brain Chemistry; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Male; Membranes; Nerve Tissue Proteins; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

The kappa-opioid compound U-69,593 was studied in rats in vitro in binding assays to assess its selectivity at the single types of opioid sites and in vivo to assess its analgesic activity and effect on intestinal propulsion. In vitro the U-69,593 inhibition curve of [3H]-(-)-bremazocine binding suppressed at mu- and alpha-sites was biphasic and the inhibition constant (Kl) at the high-affinity site (10-18 nM) was two orders of magnitude smaller than the Kl at the low-affinity site. The Kl at mu- and alpha-sites were respectively 3.3 and 8.5 microM. Thus [3H]-(-)-bremazocine, suppressed at mu- and alpha-sites, may still bind more than one site, which U-69,593 might distinguish. In vivo U-69,593 i.p. prolonged the reaction time of rats on a 55 degrees C hot-plate and the dose of naloxone required to antagonize this effect was 40 times the dose that antagonized morphine-induced antinociception, suggesting the involvement of the kappa-receptor. In the intestinal transit test U-69,593 at doses between 0.5 and 15 mg/kg i.p. only slightly slowed intestinal transit of a charcoal meal in rats with no dose-relation; it partly but significantly antagonized morphine-induced constipation. These results suggest that the kappa-type of opioid receptor, with which U-69,593 interacts may induce analgesia, but has no appreciable role in the mechanisms of opioid-induced inhibition of intestinal transit in rats.

Topics: Animals; Benzeneacetamides; Benzomorphans; Binding Sites; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Gastrointestinal Motility; In Vitro Techniques; Male; Naloxone; Nociceptors; Pyrrolidines; Rats; Receptors, Opioid

The neurochemical basis for the supersensitivity to mu or kappa opiate agonists observed after chronic naloxone infusion (reported in the preceding paper) has been investigated using the technique of quantitative in vitro autoradiography. The binding of [3H]-D-Ala2MePhe4Glyol enkephalin (3H-DAGO) to mu opioid sites was increased in many brain regions after chronic administration (7 days) of a low dose of naloxone (0.5 mg/kg/hr). The greatest increases were seen in the hippocampal strata moleculare and lucidum. The binding of [3H]-D-Ala2-D-Leu5 enkephalin (3H-DADL) to delta sites and of [3H]-bremazocine to kappa sites were not significantly affected in any region. In contrast, after administration of a higher dose of naloxone (3.0 mg/kg/hr), the binding to mu, delta and kappa sites was increased in many brain areas, suggesting that up-regulation is observed only when a sufficient proportion of the receptors in vivo are occupied by the antagonist. However, a different regional pattern of up-regulation was seen for each class of opiate binding site. The greatest increases in kappa binding in the brain were seen in the periaqueductal gray and the hippocampal molecular and pyramidal cell layers. The substantia nigra showed up-regulation of kappa but not of mu sites. Differences in the pattern of mu and kappa binding site up-regulation were also noted in the spinal cord. This regionally specific modulation of opiate binding sites suggests that, for each receptor type, the level of tonic activation varies between individual brain areas. In addition, the data demonstrate that all three types of opiate receptor can undergo up-regulation "in vivo."

Topics: Animals; Autoradiography; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Kinetics; Naloxone; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord; Substantia Nigra

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1-13). Specific [3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1-13). Delta opioid sites, labelled with [3H][D-Ala2,D-Leu5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [3H][D-Ala2, NMePhe4,Gly-ol5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

Topics: Adrenal Medulla; Animals; Benzomorphans; Cattle; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse, but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vivo, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol.

Topics: Animals; Benzomorphans; Binding, Competitive; Brain Chemistry; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethanol; In Vitro Techniques; Limbic System; Male; Mice; Mice, Inbred C57BL; Narcotics; Oligopeptides; Receptors, Opioid

Intrathecal injection of pertussis toxin (1 microgram) in rats produced a marked decrease in the antinociceptive effect of the intrathecally administered opioid agonists [D-Ala2,D-Leu5]enkephalin, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and bremazocine. The effect of the toxin was time-dependent, since it was more pronounced at 6 than at 2 days after its injection. The pertussis toxin-catalyzed ADP ribosylation of a 40 KDa substrate in membranes prepared from the spinal cord of toxin-injected rats was strongly reduced as compared to controls. The data indicate that the antinociceptive effect produced by opioid agonists with different receptor preference is initiated at receptor sites which interact with G-protein substrates of pertussis toxin.

Topics: Adenosine Diphosphate; Analgesics; Animals; Benzomorphans; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Membranes; Nociceptors; Pertussis Toxin; Rats; Rats, Inbred Strains; Reaction Time; Receptors, Opioid; Ribose; Spinal Cord; Virulence Factors, Bordetella

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5'-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity mu-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris-HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+, 2.9 mM and Li+, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the mu-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris-HCl. In 50 mM Tris-HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris-HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited. [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris-HCl, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Batrachotoxins; Benzomorphans; Brain Chemistry; Cations, Monovalent; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Etorphine; Guanine Nucleotides; Guanylyl Imidodiphosphate; In Vitro Techniques; Male; Naloxone; Rats; Receptors, Opioid; Temperature

D-Ala2, D-Leu5-enkephalin (DADLE) and dynorphin1-13 (Dyn1-13) inhibited striatal adenylate cyclase activity, both basal and dopamine-stimulated (DA), in rats and guinea pigs. The kappa-agonists bremazocine (BRZ), U-50,488 (trans-3,4-dicloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide), and U-69,593 (5 alpha, 7 alpha 8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl-1-oxaspiro (4.5)dec-8yl) benzeneacetamide inhibited only the basal adenylate cyclase activity, and such an effect was restricted to guinea pig striatum, an area known to contain a high density of kappa-binding sites. Moreover, BRZ was found to antagonize the inhibitory effect of both DADLE and Dyn1-13 in rat striatum.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclases; Analgesics; Animals; Benzeneacetamides; Benzomorphans; Corpus Striatum; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Male; Morphinans; Peptide Fragments; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid

The experiments on alert rats have shown that dissociation in opioid regulation of behavioural and hemodynamic pain manifestations is determined at a spinal opiate receptor level. Opiates and opioids suppress blood pressure nociceptive reactions to mu-opiate receptors, while sigma-opiate receptors are involved into the generation of autonomic activating effect in opiate analgesia.

Topics: Animals; Benzomorphans; Blood Pressure; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Hemodynamics; Morphine; Nociceptors; Rats; Receptors, Opioid

The opiate antagonist naloxone modifies the electric activity of some identified neurons of the Helix lucorum which have not been preliminary exposed to the effect of exogenous opioids. Some neurons are excited while others are inhibited by naloxone, and in both cases the reaction may have both a short and long latent period. The reactions depend on naloxone dose and become less expressed or are blocked when naloxone is administered together with the agonists of opiate receptor (morphine, D-Ala2, D-Leu5-enkephalin, bremazocine and beta-endorphin). Opioids alone do not produce any effect on neurons. The effect of naloxone on neurons is assumed to be a result of the elimination by the opiate antagonist of the tonic effect of endogenous opioids by their replacing on opiate receptors which are constantly stimulated by endogenous ligands.

Topics: Animals; Benzomorphans; beta-Endorphin; Drug Interactions; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Helix, Snails; Membrane Potentials; Morphine; Naloxone; Neurons; Receptors, Opioid

We investigated the effects of dynorphin A (Dyn A), a heptadecapeptide, on the population spikes of the guinea pig hippocampal CA3 pyramidal neurons, in vitro, using paired-pulse stimulation of the mossy fibers. Dyn A produced facilitatory and inhibitory effects on the population spikes in the preparations with lower and higher degrees of paired-pulse facilitation, respectively. Morphine and D-Ala2, D-Leu5-en-kephalin, mu- and delta-agonist, respectively, predominantly potentiated the population spikes, while kappa-agonists such as U-50, 488H and bremazocine mainly caused an inhibition. These results suggest that Dyn A has two separate (excitatory and inhibitory) effects on the guinea pig hippocampal CA3 neurons through mu-(delta) and kappa-opioid receptors, respectively.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzomorphans; Drug Interactions; Dynorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Guinea Pigs; Hippocampus; In Vitro Techniques; Male; Morphine; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Synaptic Transmission

[3H]Bremazocine, in the presence of saturating concentrations of mu and delta receptor blocking agents, was used to label putative kappa opiate binding sites in rat brain. The binding of [3H]bremazocine under these conditions was completely displaced with high affinity by U-50488H and dynorphin1-17, and the potency of a series of opiate ligands was consistent with an action at kappa receptors. Therefore, [3H]bremazocine, in the presence of mu and delta blockers, was used to localize U-50488H-displaceable kappa binding sites by autoradiography. A distribution different from that of mu and delta receptors was seen, with levels highest in the claustrum, striatum, medial preoptic area, suprachiasmatic nucleus, medial amygdala and superior layer of the superior colliculus. The results show that the U-50488H-displaceable kappa sites have a distinct distribution which is discussed in terms of the possible functional roles of kappa receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Autoradiography; Benzomorphans; Binding Sites; Binding, Competitive; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Morphinans; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa

Receptor binding experiments with masking of mu- and delta-receptors in the presence of an excess of unlabelled dihydromorphine and [D-Ala2, D-Leu5]enkephalin have been carried out. They indicate that 12 to 17% of original high affinity binding of [3H]Mr 2034 [-)(1R,5R,9R,2"S)-5,9-dimethyl-2-tetrahydrofurfuryl-2'-hydroxy-6, 7-benzomorphan hydrochloride), an opioid kappa-agonist, could then be detected as kappa-receptor sites in brain membranes both from untreated rats and from rats pretreated with naloxazone, too. Because of the irreversible blockade of the high affinity binding sites by naloxazone (naloxone hydrazone) treatment, the masking effects of mu- and delta-selective ligands seem to be mediated by the low affinity binding sites of these opioid receptor types. As could be shown before with [3H]Mr 2034, another kappa-agonist, [3H]bremazocine does not seem to be affected in its binding properties by naloxazone treatment of the rat in vivo. Displacement studies with several opioid agonists and antagonists, [3H]Mr 2034 and [3H]ethylketocyclazocine as radioligands in brain membranes from naloxazone treated rats and untreated controls provided further support for the evidence of two different kappa-receptors.

Topics: Animals; Benzomorphans; Binding, Competitive; Cyclazocine; Dihydromorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; In Vitro Techniques; Kinetics; Male; Morphinans; Naloxone; Rats; Receptors, Opioid; Receptors, Opioid, kappa

In this study we examined the interaction of opiates with kappa binding sites in the bovine adrenal medulla. [3H]Ethylketocyclazocine (EKC), [3H]etorphine, and [3H]bremazocine stereoselective bindings were used to assay these interactions. The kappa sites were found to be heterogeneous: [3H]bremazocine identified with high affinity all subtypes of these sites. [3H]EKC, in the presence of saturating concentrations of [D-Ala2, D-Leu5]-enkephalin (DADLE) (5 microM), was used to identify kappa 1 sites, on which dynorphin A (1-13) bound with high affinity. Either [3H]EKC or [3H]etorphine in the presence of 5 microM DADLE identified the kappa 2 subtype. This subtype was found to interact with beta-endorphin and especially with the octapeptide Met5-enkephalyl-Arg6-Gly7-Leu8. Furthermore, [3H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 microM DADLE. This residual interaction was found to be equally stereoselective and presenting kappa selectivity. Met5-enkephalyl-Arg6-Phe7 interacted preferentially with this site. The three kappa subtypes interacted differentially with monovalent (Na+, K+, and Li+) and divalent (Ca2+, Mg2+, and Mn2+) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent KD for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A2-phospholipase) produced different effects on each subtype of the kappa site, suggesting a different protein (or protein-lipid?) composition.

Topics: Adrenal Medulla; Animals; Benzomorphans; Binding Sites; Calcium; Cattle; Cyclazocine; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Ethylketocyclazocine; Ethylmaleimide; Etorphine; Guinea Pigs; Kinetics; Lithium; Manganese; Narcotics; Oligopeptides; Phospholipases A; Potassium; Receptors, Opioid; Receptors, Opioid, kappa; Sodium; Stereoisomerism

The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu-selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314-316), and the binding of [3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [3H]bremazocine to BIT/FIT-treated membranes was greatly increased by conducting the assay at 0 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0 degrees C in the presence of 0.4 M NaCl using BIT/FIT-treated membranes and that rat brain is endowed with a high level of kappa binding sites.

Topics: Alkylating Agents; Animals; Autoradiography; Benzomorphans; Brain; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; In Vitro Techniques; Membranes; Rats; Receptors, Opioid; Receptors, Opioid, kappa; Sodium Chloride

A new potent analgesic drug, 1-(1-phenylcyclohexyl)-4-phenyl-4-piperidinol (PCP-4-Ph-4-OH), derived from phencyclidine was tested for its interactions with different types of opioid receptors. The antinociceptive effect of PCP-4-Ph-4-OH in the mouse writhing test (ED50 = 0.3 mg/kg) is reversed by low doses of naloxone (pA2 = 6.98). The potency of PCP-4-Ph-OH in the inhibition of the electrically induced contractions of the guinea-pig ileum (IC50 = 17 nM) is 8-fold higher than that in the mouse vas deferens preparation (IC50 = 130 nM). The concentration of naloxone required to double the IC50 (Ke) of PCP-4-Ph-4-OH is 1.5 to 1.9 nM in both preparations. In opioid radioreceptor assays, PCP-4-Ph-4-OH displays 60- to-300 fold higher affinity for the [3H] dihydromorphine (mu) and D-[3H]Ala2-MePhe-Gly-ol5-enkephalin (mu) binding sites than for D-[3H]Ala2-D-Leu5-enkephalin (delta) sites in rat brain and [3H]bremazocine (kappa) sites in guinea-pig cerebellar membrane preparations. These results suggest that PCP-4-Ph-4-OH interacts with high affinity and selectivity with mu opioid receptors.

Topics: Analgesia; Animals; Benzomorphans; Binding, Competitive; Cerebellum; Dihydromorphine; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Muscle Contraction; Naloxone; Phencyclidine; Rats; Receptors, Opioid; Receptors, Opioid, mu; Vas Deferens

The binding of labelled naloxone, morphine and (D-Ala2,D-Leu5)enkephalin (DADL) to oocyte membranes of the toad Bufo viridis was investigated. The opiate antagonist naloxone binds to the membranes much more effectively than morphine or DADL. The binding of [3H]naloxone is reversible and saturating. The bound [3H]naloxone is readily replaced by unlabelled naloxone or bremazocine (kappa-agonist), far less effectively by morphine (mu-agonist) and SKF 10.047 (sigma-agonist) and is not practically replaced by DADL (delta-agonist), beta-endorphin (epsilon-agonist) and other neuropeptides. Analysis of experimental results in Scatchard plots revealed two types of binding sites with a high (Kd = 15 nM) and low (Kd = 10(3) nM) affinity for naloxone. The number of sites responsible for the binding of naloxone possessing a high affinity is 16 pmol-/mg of oocyte homogenate protein, i.e., 20-50 times as great as in the toad or rat brain. Trypsin and p-chloromercurybenzoate decrease the binding of [3H]naloxone. The oocyte extract is capable of replacing the membrane-bound [3H]naloxone, on the one hand, and of inhibiting the smooth muscle contracture of the rabbit vas deferens, on the other. This inhibition is reversed by naloxone and can also be induced by bremazocine, but not by morphine, DADL and SKF 10.047. In all probability oocytes contain compounds that are similar to opiate kappa-agonists. It may also be possible that these compounds mediate their effects via specific receptors and are involved in the control over maturation of oocytes and early development of toad eggs.

Topics: Animals; Benzomorphans; Binding Sites; Bufonidae; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Female; In Vitro Techniques; Kinetics; Male; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Oocytes; Rabbits; Receptors, Opioid; Vas Deferens

The presence of binding sites for ligands of mu and delta, in addition to kappa selectivity, have been demonstrated in the lumbo-sacral spinal cord of the rat by direct binding of [3H]ligands. These findings have been confirmed by competition studies. This information helps explain the observed pharmacology of opioids after intrathecal administration.

Topics: Animals; Benzomorphans; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Male; Narcotics; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

3H-Tifluadom labels specifically recognition sites of opioid kappa receptors. Membranes of guinea-pig whole brain bind 3H-tifluadom with two affinities, in contrast to the cerebellum where almost all opioid sites are kappa.

Topics: Animals; Benzodiazepines; Benzomorphans; Binding Sites; Brain; Cyclazocine; Dihydromorphine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Guinea Pigs; In Vitro Techniques; Male; Morphine; Receptors, Opioid

In this paper we examine the binding of [3H]FOXY (tritiated-6-beta-fluoro-6-desoxy-oxymorphone) to membranes of rat brain. Using the site-directed alkylating agents BIT and FIT, evidence is presented that [3H]FOXY selectively labels mu opiate binding sites in vitro. Further, BIT and FIT did not significantly affect [3H]bremazocine binding to kappa receptors. Scatchard plots of [3H]FOXY binding were somewhat curvilinear, suggesting the presence of two classes of mu binding sites. At concentrations up to 19 nM, 90 percent of the total binding was specific. The combination of high mu-selectivity and low nonspecific binding suggests the [3H]FOXY may prove to be a powerful tool for studying the opiate mu receptor.

Topics: Animals; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Fentanyl; Hydromorphone; Isothiocyanates; Kinetics; Oxymorphone; Rats; Receptors, Opioid; Receptors, Opioid, mu; Thiocyanates; Tomography, Emission-Computed

Some in vivo agonist and antagonist properties of the putative k-compound bremazocine were characterized in rats. Bremazocine, at doses from 0.015-32 mg/kg i.p., delayed nociceptive reaction on a 55 degrees C hot-plate with a dose-response curve not readily fitting a single straight line; this effect was antagonized by high doses of naloxone. In the same rats bremazocine did not delay the intestinal transit of a charcoal meal fed 5 min earlier and prevented morphine-induced constipation. This antagonism appeared to be opioid-specific and competitive, with apparent pA2 value 8.56. Catatonia induced by etorphine (0.004 mg/kg s.c.) and constipation induced by etorphine (0.004 mg/kg s.c.) and D-Ala2-D-Leu5-enkephalin (0.1 mg/kg i.p.) were completely antagonized by bremazocine (0.03-8 mg/kg i.p.). Antinociception induced by morphine (10 mg/kg i.v.) and etorphine (0.004 mg/kg s.c.) was only partly prevented. Naloxone (1 mg/kg) and bremazocine (0.015-1 mg/kg i.p.) precipitated a withdrawal syndrome, evaluated as jumping frequency, in rats rendered dependent to morphine. These data suggest the involvement of more than one opioid receptor population in bremazocine action in vivo.

Topics: Analgesics; Animals; Benzomorphans; Catatonia; Constipation; Dose-Response Relationship, Drug; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Gastrointestinal Motility; Humans; Male; Morphinans; Morphine; Naloxone; Rats; Substance Withdrawal Syndrome; Substance-Related Disorders

Kappa opiate drugs differ from other opiates in their unique sedative actions and lack of cross-tolerance. We have visualized kappa opiate receptors by in vitro autoradiography using the kappa drugs [3H]ethylketazocine ([3H]EKC) and [3H]bremazocine. Though these ligands also label mu and delta opiate receptors, their binding is rendered kappa specific by coincubation with morphine and [D-Ala2, D-Leu5]enkephalin (DADL-Enk) to displace mu and delta interactions, respectively. Labeling patterns with [3H]EKC and [3H]bremazocine are the same and differ markedly from localizations of mu and delta opiate receptors visualized with [3H]dihydromorphine and [3H]DADL-Enk, respectively. The highest density and most selective localization of putative kappa receptors occurs in layers V and VI of the cerebral cortex. In these layers cells are localized which project to the thalamus regulating sensory input to the cortex. Receptors in these layers could account for the unique sedative and possibly analgesic effects of kappa opiates.

Topics: Analgesics; Analgesics, Opioid; Animals; Autoradiography; Benzomorphans; Brain Stem; Cerebral Cortex; Cyclazocine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; Guinea Pigs; Male; Morphine; Organ Specificity; Receptors, Opioid; Receptors, Opioid, kappa; Tritium

Opiate binding sites of the kappa-subtype were visualized in guinea-pig brain sections using in vitro autoradiography. kappa-Binding sites, defined as [3H](-)-bremazocine binding in the presence of high concentrations of [D-Ala2,MePhe4,Gly-ol5]enkephalin and [D-Ala2,D-Leu5]enkephalin, were found in the cortical laminae V and VI, hippocampal dentate gyrus, and lateral habenulae. The distribution of kappa-sites in the guinea-pig differs considerably from the distribution of mu- and delta-sites which others have found in the rat.

Topics: Animals; Autoradiography; Benzomorphans; Binding, Competitive; Brain; Cerebral Cortex; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Hypothalamus, Middle; Male; Receptors, Opioid; Receptors, Opioid, kappa

Cultured hippocampal pyramidal cells responded to field stimulation with a short latency excitation followed by a long-lasting inhibition. This sequence was transformed into a bursting response by bath application of 10(-8) M FK 33-824, 10(-6) M (D-Ala)2(D-Leu)5-enkephalin and 10(-5) M bremazocine. Bremazocine and ethylketocyclazocine stereospecifically blocked the effects of FK 33-824. The results indicate that the excitatory responses were predominantly mediated by mu-receptors.

Topics: Animals; Benzomorphans; Culture Techniques; Cyclazocine; D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; Dose-Response Relationship, Drug; Electrophysiology; Endorphins; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Hippocampus; Membrane Potentials; Morphinans; Rats; Receptors, Opioid; Receptors, Opioid, mu