Desensitization by histamine of H2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H2 receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man.     

Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1

The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.     

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes.

The A1 adenosine receptor was purified approximately 13,000-fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity-chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16,545-16,551; Nakata, H. (1990) J. Biol. Chem. 265, 671-677]. The purified human brain receptor showed the ligand-binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.     

Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats.

Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.     

Purification of A1 adenosine receptor from rat brain membranes

The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.     

Histamine and VIP interactions with receptor-cyclic AMP systems in the human gastric cancer cell line HGT-1

In HGT-1 cells incubated at 20 degrees C for 15 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX), histamine (10(-4)M) increased basal cAMP levels from 2.12 +/- 0.14 to 22.9 +/- 2 pmol per 10(6) cells, with a potency of 6.4 X 10(-6)M. IBMX was added in order to inhibit cAMP degradation by low and high Km cAMP-phosphodiesterases (cAMP-PDE). The use of specific H1, H2 agonists or antagonists indicated that the histamine effect was due to an interaction with typical H2 -receptors that are involved in gastric acid secretion. Cyclic AMP levels were also increased (10-fold) by vasoactive intestinal peptide VIP (3 X 10(-11) - 10(-8)M). Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) and secretin were respectively 80 and 3600 times less potent than VIP and did not produce additive effect when tested in combinations with VIP. This observation indicates that these two peptides, structurally related to VIP, are acting through the recognition sites for VIP. Combination of VIP and histamine results in additive stimulation on intact cells as well as on membrane-bound adenylate cyclase, suggesting the existence of two cell populations bearing respectively the two sets of receptors. Two other human cancer cell lines originating from nongastric tumors (HT-29 and HL-60) possess only VIP or histamine receptors, respectively, indicating the gastric cellular originality of the HGT-1 cells. It is concluded that HGT-1 cells possess both VIP and histamine H2 receptors with similar pharmacological properties to those characterized in normal human fundic glands (1,2). Therefore, this cell line can be a good model to study drugs used therapeutically during the treatment of patients for gastric ulcer or cancer.     

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.     

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.     

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.     

Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.     

N-methyl-D-aspartate/phencyclidine receptor complex of rat forebrain: purification and biochemical characterization

The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.     

Muscarinic acetylcholine receptor from rat brain. Partial purification and characterization.

A protein capable of binding atropine and (3H)propylbenzilylcholine mustard was solubilized and purified (200-fold) from rat brain. Pronase and trypsin, but not phospholipases, diminished the binding capacity of the solubilized receptor. The molecular weight of the salt-solubilized receptor as determined by gel filtration in the absence of detergents is 30,000. The purified protein showed specificity of binding toward muscarinic ligands. the high and low affinity dissociation constants of the receptor.atropine complex are 0.3 nM and 0.15 muM. Binding of atropine is pH-dependent with an optimum at 7.1. Ca2+ influences the binding of atropine and maximal binding occurs at 0.5 mM Ca2+. The subcellular distribution of the receptor was also examined.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

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)