Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.     

Differences of binding characteristics of non-selective opiates towards mu and delta receptor types

[3H]ET (etorphine), which is considered either as an "universal" ligand or a mu agonist, interacts with identical affinities KD = 0.33-0.38 nM to hybrid cells and rabbit cerebellum, pure delta and mu-enriched opioid receptor preparations, respectively. In rat brain tissue, [3H]ET binding is inhibited by DAGO (Tyr-D-Ala-Gly-(Me)-Phe-Gly-ol), a mu selective agonist, in a competitive manner without apparent modification of the maximal number of sites. Furthermore, even at a DAGO concentration (300 nM) which should be sufficient to block [3H]ET interaction with mu sites, no variation in the total capacity of the tritiated ligand is observed. In contrast, DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), a delta-preferential agonist, blocks [3H]ET binding in rat brain at a concentration able to saturate delta-sites. At higher concentrations, where DTLET cross reacts with mu-sites, this ligand exhibits similar properties to those of DAGO. These data are very different from those obtained with [3H]EKC (ethylketocyclazocine), another "universal" ligand, the binding properties of which are easily explained by the occurrence in rat brain tissue of independent sites exhibiting pharmacological profiles of mu, delta and kappa sites. Our results underline the possible misinterpretation of binding data obtained by using [3H] etorphine as a non selective ligand.     

Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?

The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of distinct binding site subunits of mu and delta opioid receptors

Iodinated human beta-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65- and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108-15 cell line suggested that the 65-kDa peptide is a binding component of mu receptors while the 53-kDa peptide is a binding subunit of delta receptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly mu-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly mu-selective ligand [D-Pen2, D-Pen5]enkephalin (DPDPE). The labeling of the 41- and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to mu and delta receptors is not understood. Several possible explanations are presented.     

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing the kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20 nM) TENA did not significantly antagonize the delta agonist, [D-Ala2,D-Ala5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors from irreversible blockage by beta-CNA. The results of this study indicate that TENA is the most selective kappa antagonist yet reported.     

Modification by Cholecystokinin Octapeptide of the Binding ofμ-, δ-, and K-Opioid Receptors

Previous study has shown that cholecystokinin (CCK) octapeptide (CCK-8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium-labeled agonists for the mu-[(tryrosyl-3,5-3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAGO], delta- ([tyrosyl-3,5-3H][D-Pen2,5]enkephalin ([3H]DPDPE], and kappa- ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK-8. In the competition experiments, CCK-8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 mumol/L. In the saturation experiments, CCK-8 at concentrations of 0.1 nmol/L to 1 mumol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK-8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK-8 inhibits the binding of mu- and kappa-opioid receptors via the activation of CCK receptors.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Receptor autoradiography of mu and delta opioid peptide receptors in spontaneously hypertensive rats.

The receptor autoradiographic distribution of opioid peptide receptors in spontaneously hypertensive rats (SHR) was compared to that of Sprague-Dawley (SD) rats, using the highly selective mu and delta opioid receptor ligands, [3H]DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol) and [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin), respectively. Although the distribution of these binding sites was similar in both strains, SHR showed significantly higher binding densities of mu receptors in 16 of 27 areas examined. These included the patch and matrix components of the caudate-putamen (CPu), olfactory tubercle, endopiriform nucleus, anterior cingulate cortex, ventral tegmental area lateroposteral thalamic nucleus and the ventral part of the dentate gyrus. In contrast, SHR had lower [3H]DAGO binding sites in the CA1 of the hippocampus. Conversely, SHR showed higher binding densities of delta receptors in 7 of 20 areas examined, including the CPu, CA2 and CA3 areas of the hippocampus and the central grey. High-to-low lateromedial gradients of striatal delta receptors were observed in both strains. Because opioid peptides are known to participate in locomotive behavior in rodents and in the control of blood pressure, the present results support a role of opioid peptidergic systems in the manifestation of hyperactivity and hypertension observed in SHR.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

The occurrence of different kappa opioid receptors (K1 and K2) in frog (Rana esculenta) brain membranes

Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.     

Prolonged in vivo antagonism of central mu- and delta-opioid receptor activity by beta-funal trexamine

beta-Funaltrexamine (beta-FNA) was tested in the spinal cord and supraspinally against inhibition of reflex bladder contractions produced in the anesthetized rat by the opioid-receptor selective agonists [D-Ala2, MePhe4, Gly (ol)5]enkephalin (DAGO, mu-agonist) and [D-Pen2, D-Pen5] enkephalin (DPDPE, delta-agonist). All agents were microinjected either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). beta-FNA (1-8 micrograms) produced long-lasting antagonism of both DAGO and DPDPE. Complete recovery from its effects was only observed some 24-32 h later. Higher doses of beta-FNA (4 and 8 micrograms i.t.) produced short-lived agonistic activity though the selectivity of this was not determined. It was concluded that beta-FNA was a potent, long-lasting antagonist at central opioid receptors in vivo but was unselective for the mu and delta opioid receptor.     

Physiological analysis of delta opioid receptors in the guinea pig myenteric plexus

Ilea taken from guinea pigs that had been chronically exposed to morphine exhibit a greater tolerance to morphine and normorphine than to the opioid peptides D-ala2-D-leu5-enkephalin (DADLE) or D-met2-pro5-enkephalinamide (DMPE). This differential tolerance strongly implies the existence of at least two different types of opioid receptor in the guinea pig myenteric plexus or two different mechanisms of interaction between opioids and their receptor complex. Since DADLE is considered to be the prototypic ligand for the delta receptor, the above results imply the presence of delta receptors in the guinea pig myenteric plexus and furthermore, that this subtype of opioid receptor is associated with the modulation of release of enteric acetylcholine.     

Activation of central opioid receptors may induce gastric mucosal defence in the rat.

The effect of different opioid peptides on acidified ethanol- and indomethacin-induced gastric mucosal lesions was studied following intracerebroventricular (i.c.v.) administration. It was found that both the selective delta opioid receptor agonists--deltorphin II, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)-, mu-opioid receptor agonist--[D-Ala(2), Phe(4), GlyT-ol]-enkephalin (DAGO)--as well as beta-endorphin inhibited the mucosal damage induced by both ethanol and indomethacin in pmolar dose range. In contrast, the gastric acid secretion was not influenced by DADLE in the dose of 16 nmol/rat and only a slight reduction (40%) was induced by DAGO in the dose of 1.9 nmol/rat. The protective effect was abolished in both ulcer models by bilateral cervical vagotomy. N(G)-nitro-L-arginine, an inhibitor of NO synthase, reduced the protective action in ethanol-induced, but not in indomethacin-induced gastric damage. The results suggest that activation of supraspinal delta and mu-opioid receptors resulted in inhibition of gastric mucosal lesions elicited by ethanol or indomethacin. The gastroprotective action is independent from the effect of opioids on acid secretion. Vagal nerve is involved in conveying the central action to the periphery. The mechanism of the gastroprotective effect of opioids is different in ethanol- and indomethacin-ulcer models: prostaglandins and nitric oxide are likely to be involved in the protective action of opioid peptides in ethanol-, but not in the indomethacin-ulcer model.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.     

Opioid receptor selectivity reversal in deltorphin tetrapeptide analogues.

Deltorphin N-terminal tetrapeptides [DEL A: H-Tyr-D-Met-Phe-His-R, where R = -NH2, -NH-NH2, -OCH3, -OH, -NH-NH-CO-R' (R' = -CH3 or adamantane); DEL C: H-Tyr-D-Ala-Asp-R (R = -OH, -NHCH3)], were used in a receptor binding assay with [3H]DADLE and [3H]DPDPE for delta sites, and [3H]DAGO for mu sites; tetrapeptide Ki delta values were similar with either [3H]-delta ligand. DEL A tetrapeptides C-terminally substituted with -NH2, -NH-NH2, -OCH3, and -OH had 10 to greater than 1,000-fold decreased Ki delta values, while Ki mu increased 5 to 100-fold to yield mu selectivity. C-Terminal substitution with -NH-NH2 and -OCH3 conferred highest mu selectivities; adamantyl and acetyl hydrazide derivatives were non-selective. DEL-(1-4)-OH peptides had decreased delta and mu affinities: DEL A-[Asp4]-(1-4)-OH and DEL C-(1-4)-OH had low affinities (greater than 1 microM), however, the Ki delta of the former was 5-fold greater than the latter, and the Ki mu was less by 15-fold. The data suggest that the "message" domain of DEL exhibits receptor selectivity different from that of the heptapeptide.     

Characterization of kappa1 and kappa2 opioid binding sites in frog (rana esculenta) brain membrane preparation

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa₁ is the dominat receptor subtype, frog brain contains mainly the kappa₂ subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa₂ ligands (Met⁵)enkephalin-Arg⁶-Phe⁷ and etorphine also show high affinities to the frog brain. Kappa₁ binding sites measured in the presence of 5 M /D-Ala²-Leu⁵/enkephalin represent 25–30% of [³H]ethylketocyclazocine binding in frog brain membranes. The kappa₂ subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala²-(Me)Phe⁴-Gly⁵-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.Abbreviations used DAGO /D-Ala²-(Me)Phe⁴-Gly⁵-ol/enkephalin - DALE /D-Ala²-L-Leu⁵/-enkephalin - EKC ethylketocyclazocine - DHM dihydromorphine - CAM N-cyclopropylmethylnorazidomorphine - nor-BNI nor-binaltorphimine - MR2034 (-)-(1R,5R,9R)-5, 9-dimethyl-2 (L-tetrahydrofuryl-2'-hydroxy-6,7benzomorphan) - MR2035 (+)-(1R,5R,9R)-5,9-dimethyl-2 (L-tetrahydrofuryl-2'-hydroxy-6,7-benzomorphan), U50488H=3,4-dichloro-N-/2-(1-pyrrolidinyl) —cyclohexo/-benzene-acetamide - PD117302 trans-N-methyl-N-/2-(1-pyrrolidinyl) — cyclohexyl/-benzo (b) thiophene-4-acetamide     

Sigma and opioid receptors in human brain tumors

Human brain tumors (obtained as surgical specimens) and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [3H]1,3-di-o-tolylguanidine (DTG), whereas opoid receptor subtypes were measured with tritiated forms of the following: mu, [D-ala2,mePhe4,gly-ol5]enkephalin (DAMGE); kappa, ethylketocyclazocine (EKC) or U69,593; delta, [D-pen2,D-pen5]enkephalin (DPDPE) or [D-ala2,D-leu5]enkephalin (DADLE) with mu suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels (pmol/mg protein) found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. kappa opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.     

A Monoclonal Antibody Recognizing K- but Not γ- and δ-Opioid Receptors

A monoclonal antibody (mAb), KA8 that interacts with the kappa-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified kappa-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (kappa-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain kappa-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when mu- and delta-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when mu-, delta-, and kappa 2-sites were blocked by 5 microM DADLE. However, the mu-specific [3H]DAGO and the delta-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the kappa- but not the mu- and delta-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)