-
Proceedings of the National Academy of... Aug 1980The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i)...
The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i) 125I-labeled[D-Ala2, D-Leu5]enkephalin or 125I-labeled[D-Ala2,N-Me-Phe4,Met(O)5ol]-enkephalin to brain membranes; (ii) [3H]ethylketocyclazocine to brain membranes; and (iii) [3H]diprenorphine and [3H]naloxone to neuroblastoma cell and brain membranes, respectively. According to their relative binding potencies and the effects of Na+ and GTP on the binding to these two receptors, opiates and enkephalins can be classified into seven classes: (i) morphine-type mu agonists; (ii) enkephalin-type delta agonists; (iii) mixed agonists-antagonists; (iv) putative kappa agonists; (v) putative sigma agonists; (vi) nalorphine-type antagonists; and (vii) opiate antagonists. Studies with [3H]ethylketocyclazocine do not reveal specific kappa receptors distinct from those already described that bind morphine and enkephalins. The benzomorphan analogs ketocyclazocine and ethylketocyclazocine (putative kappa agonists) and N-allylnormetazocine (putative sigma agonist) bind to morphine (mu) and enkephalin (delta) receptors with similarly high affinities. The potency of putative kappa agonists, measured by competition with binding of the 3H-labeled antagonist, is greatly reduced by the presence of Na+ and GTP; the "Na+ and GTP ratios" are similar to those of morphine and enkephalins. However, Na+ and GTP greatly decrease the potency of binding of putative sigma agonists to enkephalin receptors but only slightly decrease the binding to morphine receptors. These data suggest that putative kappa agonists have agonistic activity toward both receptors, whereas putative sigma agonists behave as agonists for enkephalin receptors but have antagonist activity for morphine receptors. Mixed agonist-antagonists also show smaller difference in affinity to both receptors. These findings may have important implications for understanding the differences in the pharmacological effects of these drugs.
Topics: Animals; Benzomorphans; Brain; Cyclazocine; Diprenorphine; Endorphins; Enkephalins; Ethylketocyclazocine; Guanosine Triphosphate; Morphinans; Morphine; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Sodium; Synaptic Membranes
PubMed: 6254028
DOI: 10.1073/pnas.77.8.4469 -
Proceedings of the National Academy of... Sep 1982Kappa 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...
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
PubMed: 6127675
DOI: 10.1073/pnas.79.18.5703 -
Proceedings of the National Academy of... Jun 1980The receptor binding of the kappa agonist [3H]ethylketocyclazocine to brain homogenates in vitro and ketocyclazocine (kappa) analgesia in vivo has been investigated and... (Comparative Study)
Comparative Study
The receptor binding of the kappa agonist [3H]ethylketocyclazocine to brain homogenates in vitro and ketocyclazocine (kappa) analgesia in vivo has been investigated and compared to morphine, a mu agonist. Saturation analysis of [3H]ethylketocyclazocine binding in both mice and rats yielded biphasic Scatchard plots similar to those of opiate mu agonists, antagonists, enkephalins, and endorphins. Treatment of brain membranes with monovalent and divalent cation, chelating agents, protein-modifying reagents, and enzymes affected [3H]ethylketocyclazocine binding in a manner similar to that of [3H]morphine. Naloxazone, a long-acting antagonist that selectively abolished high-affinity [3H-DAla2,Met5]enkephalinamide binding in vivo, also selectively blocked high-affinity [3H]ethylketocyclazocine binding. Evaluation of analgesia with writhing and tail-flick assays in animals whose high-affinity binding sites were blocked by naloxazone demonstrated a 6- to 7-fold increase in median effective dose (ED50) values of ketocyclazocine. This decrease in analgesic potency was comparable to morphine's decreased potency in similarly treated mice. These biochemical and pharmacological results suggest that the analgesic properties of both kappa and mu agonists may be mediated through the same subpopulation of receptors, the high-affinity binding sites.
Topics: Analgesics; Animals; Brain; Cyclazocine; Dihydromorphine; Dose-Response Relationship, Drug; Enkephalin, Methionine; Enkephalins; Ethylketocyclazocine; In Vitro Techniques; Mice; Morphine; Motor Activity; Naloxone; Rats; Receptors, Opioid; Receptors, Opioid, kappa
PubMed: 6251477
DOI: 10.1073/pnas.77.6.3691 -
Neuropharmacology Apr 1986Scatchard analysis of the binding to opioid receptors of [3H]ethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting...
Scatchard analysis of the binding to opioid receptors of [3H]ethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting of the data resulted in a minimal model of two independent saturable binding sites. The KD values for the high- and low-affinity sites were 0.58 and 38 nM for [3H]EKC, and 0.13 and 22 nM for [3H]etorphine. The corresponding density of binding sites was 157 and 418 fmol/mg protein for [3H]EKC, and 220 and 289 fmol/mg protein for [3H]etorphine. The KD values calculated from the association and dissociation rate constants corresponded to those observed at equilibrium. In the course of equilibrium binding, various opioids competed with [3H]EKC and [3H]etorphine preferentially at the high-affinity opioid receptor sites. No difference between the competition patterns of putative mu and kappa ligands was observed. The kinetics of association and dissociation of [3H]EKC and [3H]etorphine revealed that the apparently homogeneous high-affinity binding site observed at equilibrium consisted of two components characterized by their fast and slow equilibrium times, respectively. While none of the mu and kappa opiates investigated altered the rate of dissociation of [3H]EKC or [3H]etorphine, in the presence of sodium ions the rapidly dissociating binding component of [3H]etorphine became refractory to inhibition by mu but not kappa agonists. The results underline the advantages of evaluating both equilibrium binding and the kinetics of ligand-receptor interactions.
Topics: Animals; Brain; Computers; Cyclazocine; Drug Interactions; Ethylketocyclazocine; Etorphine; Kinetics; Ligands; Membranes; Morphinans; Rats; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu
PubMed: 3012397
DOI: 10.1016/0028-3908(86)90229-7 -
Proceedings of the National Academy of... Oct 1986The excitatory amino acid receptors selectively activated by N-methyl-D-aspartate (N-Me-D-Asp) (also known as NMDA) are a major determinant of central nervous system...
The excitatory amino acid receptors selectively activated by N-methyl-D-aspartate (N-Me-D-Asp) (also known as NMDA) are a major determinant of central nervous system neuronal excitability. We report here that rat brain synaptic plasma membranes contain a distinct population of L-[3H]glutamate binding sites with pharmacological properties indicative of the N-Me-D-Asp receptor. The N-Me-D-Asp sites are readily distinguished from other L-[3H]glutamate binding and uptake sites by their sharp pH optimum, more rapid association rate, preferential localization in synaptic structures, and lack of dependence on temperature and inorganic ions. As with other receptor systems, ligand binding at the N-Me-D-Asp site is reduced by guanine nucleotides but not by adenosine nucleotides. Binding is insensitive to ketamine and cyclazocine, indicating that sigma opiates inhibit N-Me-D-Asp excitation at a site different from that of the N-Me-D-Asp binding site. The quantitative pharmacological properties of N-Me-D-Asp-sensitive L-[3H]glutamate binding sites determined in a well-defined dendritic field (stratum radiatum of CA1) by quantitative autoradiography closely correlate to those of both the electrophysiologically identified N-Me-D-Asp receptors in the same dendritic field and the N-Me-D-Asp sites studied in membrane preparations. Under conditions that selectively reveal N-Me-D-Asp receptors, these sites are found to exhibit considerable anatomical specificity as evidenced by variations within cortical, striatal, and thalamic regions. Autoradiography also showed that regions in rodent and primate brain that are especially sensitive to anoxic and excitotoxic neuronal damage (e.g., Sommer's sector or CA1) have a high level of N-Me-D-Asp sites. Since N-Me-D-Asp receptors are known to contribute to these causes of neuronal loss, their selective distribution partially accounts for the pattern of selective damage seen in these pathological conditions.
Topics: Animals; Aspartic Acid; Autoradiography; Binding, Competitive; Glutamates; Guanine Nucleotides; Hydrogen-Ion Concentration; Kinetics; N-Methylaspartate; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Subcellular Fractions; Synaptic Membranes; Temperature
PubMed: 3020547
DOI: 10.1073/pnas.83.19.7532 -
British Journal of Pharmacology Nov 1986In conscious saline loaded rats, the kappa-opioid agonists tifluadom, U50488, and ethylketocyclazocine, given subcutaneously, induced a characteristic diuresis which...
In conscious saline loaded rats, the kappa-opioid agonists tifluadom, U50488, and ethylketocyclazocine, given subcutaneously, induced a characteristic diuresis which could be antagonized by naloxone. Bilateral adrenal demedullation significantly reduced adrenal gland catecholamine content and plasma adrenaline levels, but did not significantly affect plasma corticosterone levels, indicating that the adrenal cortex remained both intact and functional. Seven days following bilateral adrenal demedullation, the subcutaneous administration of the kappa-agonists no longer induced diuresis. However, demedullation did not affect the diuretic response to frusemide or clonidine, nor did it affect the antidiuretic response induced by the mu-opioid agonists morphine and buprenorphine. Adrenal catecholamines do not appear to be involved in kappa-opioid-induced diuresis, since pretreatment with propranolol, prazosin and idazoxan did not affect the diuretic response in intact animals. The results indicate a link between the adrenal medulla and kappa-opioid-induced diuresis and suggest that a peripheral mechanism may also be involved in mediating this effect.
Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adrenal Medulla; Animals; Benzodiazepines; Catecholamines; Corticosterone; Cyclazocine; Diuresis; Ethylketocyclazocine; Male; Naloxone; Narcotics; Pyrrolidines; Rats; Sympatholytics
PubMed: 3542107
DOI: 10.1111/j.1476-5381.1986.tb11160.x -
The Journal of Pharmacology and... Nov 1979Rats learned drug discriminations in a shock-escape T-maze task. They were trained to turn right in the maze following injection of a drug (D) and left when no injection...
Rats learned drug discriminations in a shock-escape T-maze task. They were trained to turn right in the maze following injection of a drug (D) and left when no injection (N) was given. Number of training sessions before criterion performance (STC) was used to indicate degree of discriminability of the training drug. STC decreased monotonically as dosage increased, and reached a minimum of 3 to 26 with various agonists. Most agonists were not highly discriminable. Daily maintenance injections of morphine, 200 to 600 mg/kg, increased the STC of morphine, 15 mg/kg, significantly, but complete tolerance to discriminable drug actions was not observed. After rats discriminated D vs. N, they were tested with novel drugs to determine which would elicit D choices. Most morphine-like agonists substituted for one another during substitution tests; the tested agonists included alphaprodine, codeine, fentanyl, heroin, meperidine, methadone, morphine, piminodine and propoxyphene. In a few instances, one of these agonists failed to substitute for another. Naloxone and naltrexone antagonized the discriminable effects of morphine. Cyclazocine, levallorphan, naltrexone, dextromethorphan, ethoheptazine and the narcotic agonists did not substitute for one another, suggesting that six dissimilar discriminable effects were produced by these drugs.
Topics: Analgesics, Opioid; Animals; Antitussive Agents; Avoidance Learning; Discrimination Learning; Male; Rats; Structure-Activity Relationship
PubMed: 41087
DOI: No ID Found -
Proceedings of the National Academy of... Jul 1981The high-affinity binding of benzomorphan drugs (ethylketocyclazocine and N-allylnorcyclazocine) and [DAla2,DLeu5] enkephalin was examined in a mouse...
The high-affinity binding of benzomorphan drugs (ethylketocyclazocine and N-allylnorcyclazocine) and [DAla2,DLeu5] enkephalin was examined in a mouse neuroblastoma--Chinese hamster brain clonal hybrid cell line (NCB-20). Scatchard analysis of saturation binding isotherms indicated the presence of a single binding site for 3H-labeled [DAla2,DLeu5]enkephalin (Kd = 3 nM) and multiple binding sites for [3H]ethylketocyclazocine (Kd = 4 and 20 nM) and N-[3H]allylnorcyclazocine (Kd = 0.5 and 15 nM). Both ethylketocyclazocine and N-allylnorcyclazocine competed (Ki = 10 and 30 nM, respectively) with [3H][DAla2,DLeu5]enkephalin binding in NCB-20 cells but neither [DAla2,DLeu5]enkephalin nor morphine could completely inhibit the specific binding of [3H]ethylketocyclazocine (7 nM) or N-[3H]allylnorcyclazocine (3 nM). Furthermore, not all benzomorphan drugs (e.g., ethylketocyclazocine) were totally efficacious in displacing 3 nM N-[3H]allylnorcyclazocine binding in the presence or absence of high concentrations of [DAla2,DLeu5]enkephalin. The data presented suggest that benzomorphan drugs interact with three distinct high-affinity binding sites: (i) a site that binds enkephalin and morphine in addition to ethylketocyclazocine and N-allylnorcyclazocine; (ii) a site that binds both ethylketocyclazocine and N-allylnorcyclazocine but not enkephalin and morphine; and (iii) a site that binds N-allylnorcyclazocine but not enkephalin, morphine, or ethylketocyclazocine. The first of these sites was comparable to the delta opiate receptor expressed in NG108-15 and N4TG1 cell lines based on the potency series obtained for various opiates and benzomorphan drugs in competition studies with [3H][DAla2,DLeu5]-enkephalin. However, the specific high-affinity benzomorphan binding sites thus far are unique and may represent biochemical correlates of kappa and sigma opiate receptors which have been proposed to exist on the basis of physiological studies.
Topics: Analgesics, Opioid; Animals; Benzomorphans; Binding Sites; Binding, Competitive; Brain; Cell Line; Cyclazocine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Hybrid Cells; Kinetics; Morphinans; Neuroblastoma; Phenazocine; Rats; Receptors, Opioid
PubMed: 6117074
DOI: 10.1073/pnas.78.7.4309 -
British Journal of Pharmacology Oct 19971. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine...
1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.
Topics: Acetylcholine; Animals; Benzeneacetamides; Dopamine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Ethylketocyclazocine; In Vitro Techniques; Male; N-Methylaspartate; Naloxone; Neostriatum; Neural Inhibition; Peptide Fragments; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu
PubMed: 9351509
DOI: 10.1038/sj.bjp.0701394 -
Japanese Journal of Pharmacology Oct 1983Aversive (nociceptive) biting-like responses induced by micro-application of bradykinin solution onto rat tooth pulp were dose-dependently suppressed by non-narcotic...
Aversive (nociceptive) biting-like responses induced by micro-application of bradykinin solution onto rat tooth pulp were dose-dependently suppressed by non-narcotic drugs such as baclofen and lidocaine as well as carbamazepine and phenytoin, which are employed for clinical treatment of trigeminal neuralgia. The potency order of these drugs on a molar basis is baclofen (4.20) greater than carbamazepine (1.00) greater than lidocaine (0.94) greater than phenytoin (0.19). Such responses were also inhibited by morphine, pentazocine and cyclazocine (potency ratio of the three general analgesics, 1.00:0.46:8.11), indomethacin (a non-narcotic and anti-inflammatory analgesic) and alpha-chloralose (an anesthetic). The latter drug produced an analgesic effect at doses much lower than those used for anesthesia. These findings suggest that our method is feasible for evaluating the activities of general and particular analgesic drugs in the trigeminal regions.
Topics: Analgesics; Analgesics, Opioid; Anesthetics; Animals; Baclofen; Bradykinin; Chloralose; Dose-Response Relationship, Drug; Indomethacin; Lidocaine; Male; Morphine; Nociceptors; Rats; Rats, Inbred Strains
PubMed: 6139496
DOI: 10.1254/jjp.33.933