Bremazocine Binding Research Articles

Effects of rat brain kappa 1- and kappa 2-opioid receptors after chronic treatment with non-peptide kappa-agonists.

Injection of kappa-agonist dynorphins and non-peptide kappa-agonists into the hippocampus induces a reduction in blood pressure. It has been postulated that kappa-opioid agonists and kappa-receptors are important in one mechanism of antihypertension and might have clinical potential for the treatment of hypertension. We have investigated whether chronic treatment with U-50488H and U-62066E, two non-peptide kappa-agonists, effects brain kappa 1- or kappa 2-receptor numbers or affinities in areas that might correlate with changes in blood pressure. kappa 1- and kappa 2-Opioid receptor affinities and densities were determined in cortex, hippocampus, hypothalamus, midbrain and pons after 14 days subcutaneous infusion of two non-peptide kappa-agonists, U-50488H and U-62066E, 9.6 mg kg day-1, by means of osmotic minipumps, to spontaneously hypertensive rats (SHR) and to Wistar-Kyoto (WKY) rats. This infusion significantly reduced blood pressure. Brains were removed within 48 h of the end of drug infusion and kappa-receptor binding studies were performed on homogenates from each brain area using [3H]U-69593 to assay kappa 1-receptors and [3H]bremazocine to assay kappa 2-receptors. U-62066E treatment seemed to cause a slight decrease in the number of [3H]bremazocine binding sites (kappa 2-receptors) from 98.2 +/- 9 to 74.9 +/- 8 fmol (mg protein)-1 in the hippocampus when compared with SHR controls. A small decrease in kappa 2-receptor density in the pons of WKY rats was also observed after U-50488H treatment (control, 51.2 +/- 5; U-50488H-treated, 24.3 +/- 9 fmol (mg protein)-1. Although SHR blood pressure values were consistently reduced by treatment with kappa-agonists, there was little if any significant change in apparent numbers of kappa 1- or kappa 2-receptors or their affinities in any of the brain regions examined. These data indicate that although chronic treatment with kappa-agonists reduces blood pressure in SHR, the treatment does not elicit major changes in brain kappa-receptors either in SHR or in WKY rats. The potential use of kappa-agonists for treating hypertension might not cause receptor changes in the brain and might, therefore, result in fewer side effects or negligible rebound hypertension.

Oxycodone and morphine have distinctly different pharmacological profiles: Radioligand binding and behavioural studies in two rat models of neuropathic pain

Previously, we reported that oxycodone is a putative κ-opioid agonist based on studies where intracerebroventricular (i.c.v.) pre-treatment of rats with the κ-selective opioid antagonist, nor-binaltorphimine (nor-BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (μ-selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor-BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)-diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post-STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an ∼3- to 4-fold decrease in potency. In rat brain membranes irreversibly depleted of μ- and δ-opioid binding sites, oxycodone displaced [ 3H]bremazocine (κ 2-selective in depleted membranes) binding with relatively high affinity whereas the selective μ- and δ-opioid ligands, CTOP ( d-Phe-Cys-Tyr- d-Trp-Orn-Thr-Pen-Thr- NH 2) and DPDPE ([ d-Pen 2,5]-enkephalin), respectively, did not. In depleted brain membranes, the κ 2b-ligand, leu-enkephalin, prevented oxycodone’s displacement of high-affinity [ 3H]bremazocine binding, suggesting the notion that oxycodone is a κ 2b-opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a κ 2b-opioid agonist with a relatively low affinity for μ-opioid receptors.

Purification and mass spectrometric analysis of the δ opioid receptor

A mouse δ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10.5 pmol/mg protein. [ 3H]Bremazocine exhibited high affinity binding to the epitope-tagged δ opioid receptor with a K D of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged δ receptor with K I's of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged δ receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The δ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of Sephacryl S300 gel filtration chromatography, wheat germ agglutinin (WGA)-agarose chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and SDS/PAGE permitted purification of the receptor. The purified δ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 65 kDa. MALDI-TOF mass spectrometry was used to identify and characterize peptides derived from the δ opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the δ opioid receptor.

κ-Opioid receptors are differentially labeled by arylacetamides and benzomorphans

Using Chinese Hamster Ovary cell membranes that stably expressed the human κ-opioid receptor, we investigated the hypothesis that κ 1- and κ 2-opioid receptors, historically defined by their phrmacological selectivity for either arylacetamides or benzomorphans are, in fact, different affinity states or binding sites on the same κ-opioid receptors. Receptor binding studies showed that GTPγS potently inhibited [ 3H](5α,7α,8β)-(+)- N-methyl- N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)-benzeneacetamide (U69,593) binding, compared to virtually no inhibition of [ 3H]bremazocine binding. Saturation binding experiments showed a three-fold decrease in [ 3H]U69,593 affinity in the presence of GTPγS, but GTPγS had no effect on [ 3H]bremazocine affinity. The κ-opioid receptor antagonist nor-binaltorphimine had a four-fold higher affinity for [ 3H]U69,593-labeled receptors than for [ 3H]bremazocine-labeled receptors. Functional selectivity studies, measuring the stimulation of [ 35S]GTPγS agonist-induced binding, showed a significantly higher U69,593-induced G protein-receptor activation in comparison to the stimulation observed with bremazocine. These results suggest that pharmacologically defined 1κ-opioid receptor subtypes may be different affinity states of the same receptor.

Purification and mass spectrometric analysis of the μ opioid receptor

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K D of 1.0 nM. The agonists [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO), morphine and [ d-Ala 2, d-Leu 5]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with K I's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)–agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.

Quantitative autoradiographic mapping of opioid receptors in the brain of δ-opioid receptor gene knockout mice

Using quantitative receptor autoradiography we have determined if deletion of the δ-opioid receptor gene ( Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse δ-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. δ-Opioid receptors were labeled with [ 3H]deltorphin I (7 nM), μ- with [ 3H]DAMGO (4 nM), and κ- with [ 3H]CI-977 (2.5 nM) or [ 3H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [ 3H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [ 3H]deltorphin I binding correlated significantly with those demonstrating high levels of the μ-receptor and were abolished in the presence of the μ-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [ 3H]DAMGO and [ 3H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [ 3H]bremazocine binding were higher in brains from −/− than +/+ mice. Our findings suggest that residual [ 3H]deltorphin I binding in the brain of δ-receptor gene knockout mice is the result of cross-reactivity with μ-sites and that there are no δ-receptor subtypes derived from a different gene. Changes in μ- and κ-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the δ-receptor. The differences in [ 3H]CI-977 and [ 3H]bremazocine binding indicate these ligands show differential recognition of the κ-receptor.

Inhibition of μ and δ opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

We reported recently that the ubiquitin–proteasome pathway is involved in agonist-induced down regulation of μ and δ opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl- l-Leucyl- l-Leucyl- l-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected μ, δ, or κ opioid receptors. The rank order of potency for leupeptin inhibition of [ 3H]bremazocine binding to opioid receptors was μ>δ>κ. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy- l-Leucyl- l-Leucyl- l-Leucinal), had significantly less effect on bremazocine binding to μ, δ, or κ opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.

Analysis of [ 3H] bremazocine binding in single and combinatorial opioid receptor knockout mice

Despite ample pharmacological evidence for the existence of multiple μ-, δ- and κ-opioid receptor subtypes, only three genes encoding μ-(MOR), δ-(DOR) and κ-(KOR) opioid receptor have been cloned. The KOR gene encodes κ 1-sites, which specifically bind arylacetamide compounds, and the possible existence of κ-opioid receptor subtypes derived from another κ-opioid-receptor gene, yet to be characterized, remains a very contentious issue. κ 2-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [ 3H]bremazocine in the presence of μ-, δ- and κ 1-opioid receptor blocking ligands. To investigate the genetic origin of κ 2-opioid receptors, we have carried out homogenate binding experiments with [ 3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68±12% of the binding sites arise from the MOR gene, 27±1% from the DOR gene and 14.5±0.2% from the KOR gene, indicating that the three known genes account for total [ 3H]bremazocine binding. Experiments in the presence of μ-, δ- and κ 1-opioid receptor suppressor ligands further showed that non-κ 1-opioid receptor labelling can be accounted for by binding to both the μ- and δ-opioid receptors. Finally, [ 3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [ 3H]bremazocine binding sites. Altogether the data show that the putative κ 2-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

An Immortalized Myocyte Cell Line, HL-1, Expresses a Functional δ -Opioid Receptor

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

Pharmacological profiles of selective non-peptidic δ opioid receptor ligands

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective δ receptor agonists. In this study, the affinities of a set of compounds for cloned δ and μ 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 δ opioid receptor and 240–1165 nM for the μ opioid receptor. We next sought to determine which domain of the δ receptor was critical for mediating the highly selective binding by analysis of ligand affinities for μ/δ 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 δ receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the μ 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 δ receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing δ 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 δ 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.

κ Opioid Receptor Stimulation of [ 35S] GTPγS Binding in Guinea Pig Brain

Although only one gene for κ opioid receptors has been cloned to date, κ 1 and κ 2 receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative κ receptor subtypes. To examine whether κ receptor subtypes can be distinguished at the level of the G-protein, the ability of the κ 1 agonist ( trans-(dl)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [ 35S]guanosine-5′-O-(γ-thio)-triphosphate ([ 35S]GTPγS) binding in guinea pig brain was compared with that of bremazocine and dynorphin. In membranes prepared from guinea pig striatum, both bremazocine and U-50488H stimulated [ 35S]GTPγS binding with the same relative efficacy, while dynorphin produced at least two-fold greater efficacy than the other two agonists. In vitro autoradiography of agonist-stimulated [ 35S]GTPγS binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the κ antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [ 35S]GTPγS binding with similar K e values. In agonist additivity experiments, the stimulation of [ 35S]GTPγS binding by the δ agonist [ d-pen 2,5, p-Cl-Phe 4]enkephalin (p-Cl-DPDPE) was approximately additive with the two κ agonists. Stimulation of [ 35S]GTPγS binding by the μ agonist [ d-Ala 2, N-Me 4, Gly 5-ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the μ antagonist properties of this compound. The combination of bremazocine and U-50488H together produced no greater stimulation of binding than either agonist alone, indicating that they were binding to the same site. These results demonstrate that bremazocine and U-50488H activate G-proteins in guinea pig brain through the same receptor, and suggest that κ 2 receptors are not coupled through the same signal transduction mechanisms as κ 1 receptors.

Sensitivity of opioid receptor binding to N-substituted maleimides and methanethiosulfonate derivatives.

A series of N-substituted maleimides was shown to effectively inactivate bremazocine binding to delta opioid receptors. Apparent second order rate constants for inactivation increased with increasing size of the N-substituent: N-methyl < N-ethyl < N-butyl < N-phenylmaleimide. It is suggested that the positive chain length effect is attributed to nonpolar interactions with the receptor in the vicinity of the reactive group. Binding to mu and delta opioid receptors was equally sensitive to inactivation by (2-aminoethyl)methanethiosulfonate; the [2-(trimethylammonium)ethyl] and (2-sulfonatoethyl) derivatives were less active. Site-directed mutagenesis of the mu opioid receptor indicated that Cys159, Cys190, Cys235, Cys292, or Cys321, residing in transmembrane domain 3, 4, 5, 6, and 7, respectively, were not the site of modification.

Opioid receptor ligand binding in the human striatum: II. Heterogeneous distribution of kappa opioid receptor labeled with [3H]bremazocine.

Selective kappa opioid receptor autoradiography with [3H]bremazocine (BRM) was used to examine regional and subregional kappa receptor distribution patterns at five rostrocaudal levels through the human striatum. [3H]BRM binding densities were measured in the individual striatal nuclei and in subregions therein. The distribution of [3H]BRM binding sites was found to have a strongly heterogeneous character. At the regional level a rostral-to-caudal decrease in [3H]BRM binding densities was observed. Also, a dorsal-to-ventral differentiation was seen, with higher values in the ventral striatum, especially in the nucleus accumbens, and lower values in the dorsal parts of the caudate nucleus and putamen. These findings suggest an association of kappa receptor function with limbic-related processes in the ventral striatum. Along the ventral edge of the nucleus accumbens and putamen, specific domains with extremely high [3H]BRM binding values were identified.

Studies on Inhibition of μ and δ Opioid Receptor Binding by Dithiothreitol and N-Ethylmaleimide: His223 IS CRITICAL FOR μ OPIOID RECEPTOR BINDING AND INACTIVATION BY N-ETHYLMALEIMIDE

The sensitivity of mu and delta receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor.

Kappa opioid receptors expressed on three related thymoma cell lines: Differences in receptor-effector coupling

The mouse thymoma R1.1 cell line was shown previously to express a single high-affinity K 1 opioid receptor that is negatively coupled through a pertussis toxin-sensitive G-protein to adenylyl cyclase. This study compared opioid receptor binding and inhibition of adenylyl cyclase activity in three unique derivatives of the R1.1 cell line. Membranes from the R1.G1 and R1E/TL8x.1.G1.OUA r.1 (R1EGO) cell lines bound both [ 3H]U69,593 and [ 3H](−)-bremazocine with similar affinities compared with R1.1 membranes, whereas membranes from the R1E/TL8x.1 (R1E) cell line did not posses any opioid binding sites, detected by radioreceptor binding. The B max values for [ 3H]U69,593 and [ 3H](−)-bremazocine binding to R1.G1 and R1EGO cell membranes were, respectively, 3- and 6-fold greater than those obtained with the parent R1.1 cell line. GTP and its nonhydrolyzable analog, Gpp(NH)p, inhibited [ 3H]U69,593 binding to all three cell lines. Stimulation of low- K m GTPase activity by the k-selective agonist (−)U50,488 was greatest in R1.G1 membranes, followed by R1EGO and R1.1. The maximal inhibition of forskolin-stimulated adenylyl cyclase activity by (−)U50,488 was 66 ± 2% in R1.G1 and 49 ± 2% in R1EGO, compared with 37 ± 1% in R1.1 membranes. Whereas maximal inhibition of adenylyl cyclase activity did not correlate with receptor number among cell lines, the inhibition of cyclic AMP production did correlate with stimulation of low- K m GTPase activity. The R1.1 cell line and its derivatives, R1.G1 and R1EGO, express a similar type of k opioid receptor, which exhibits differences in coupling to G-proteins and to adenylyl cyclase among cell lines. These cell lines provide an excellent model system for studying the regulation of opioid receptor-adenylyl cyclase coupling efficiency.

The κ-opioid receptor expressed on the mouse lymphoma cell line R1.1 contains a sulfhydryl group at the binding site

Studies were directed at determining whether the κ-opioid receptor expressed on the mouse R1.1 thymoma cell line contained either a disulfide bond or a sulfhydryl group at the opioid binding site. The binding of the κ-opioid receptor agonist [ 3H](−)-bremazocine to R1.1 cell membranes was unchanged following treatment with the disulfide bond-reducing reagent dithiothreitol at concentrations up to 130 mM. However, treatment of membranes with the sulfhydryl-alkylating reagent N-ethylmaleimide, followed by extensive washing, reduced [ 3H](−)-bremazocine binding by as much as 90%. Inhibition of [ 3H](−)-bremazocine binding by N-ethylmaleimide was concentration- and time-dependent. When R1.1 cell membranes were treated with 1 mM N-ethylmaleimide for 10 min at 24°C, the B max value for [ 3H](−)-bremazocine binding decreased by 50%, with no change in receptor affinity. N-Ethylmaleimide-induced reduction of [ 3H](−)-bremazocine binding was attenuated by pretreatment of membranes with the κ-selective opioids U50,488 and U69,593. The results indicate that a sulfhydryl group is present at or near the binding site on the κ-opioid receptor expressed by the R1.1 thymoma cell line.

Characterization of opioid binding sites in the neural and intermediate lobe of the rat pituitary gland by quantitative receptor autoradiography.

Previous studies have suggested an involvement of enkephalins in regulation of oxytocin (OXT) and vasopressin (AVP) release, which seems to disagree with the very low affinities of Met- and Leu-enkephalin for the kappa opioid receptor. As opioid receptors in the neural lobe exclusively exist of kappa receptors, we studied the binding characteristics of larger pro-enkephalin derived peptides for opioid binding sites in the neural lobe by means of light microscopic receptor autoradiography. In addition, the pharmacological characteristics of opioid binding sites in the neural lobe were compared with those in other parts of the pituitary. In the neural as well as the intermediate lobe both high and low affinity 3H-bremazocine binding sites were present. Binding to these sites was completely displaceable by both naloxone and nor-binaltorphimine suggesting that these sites represent kappa opioid receptors. Also with regard to selectivity and affinity characteristics to other ligands, opioid binding sites in the neural and intermediate lobe were quite similar. In the anterior lobe a very low level of bremazocine binding was present, which could not be displaced by nor-binaltorphimine. Displacement studies with pro-enkephalin and pro-dynorphin derived peptides showed that both groups of peptides could bind to opioid binding sites in the neural and intermediate lobe. Especially the relatively large pro-dynorphin and pro-enkephalin derived peptides, such as dynorphin 1-17 and BAM22, appeared to be very potent ligands for these opioid binding sites and were much more potent than smaller fragments, such as dynorphin 1-8, and Met- and Leu-enkephalin. These results contradict the existence of a mismatch in the neural (and intermediate) lobe with regard to the local type of opioid peptides and receptors present.

Kappa opioid binding sites on the R1.1 murine lymphoma cell line: sensitivity to cations and guanine nucleotides

The present study describes the characterization of an opioid binding site on membranes prepared from the R1.1 cell line, a murine thymoma. Specific (−)[ 3H]bremazocine binding was saturable, stereoselective, and limited to a single high affinity binding site with a K d value of 15.2±1.6 pM and a B max value of 54.8±6.0 fmol/mg of protein. The κ-selective alkaloids and dynorphin peptides inhibited (−)[ 3H]bremazocine binding with K i values of less than 1 nM, in contrast to μ- and σ-selective ligands. The high affinity of this site or α-neo-endorphin and U50,488 suggests that this κ opioid binding site resembles the κ 1 b subtype. NaCl, as well as other mono- and divalent cations, inhibited (−)[ 3H]bremazocine binding. In the presence of NaCl, the nucleotides GTP, GDP, and the nonhydrolyzable analog guanylyl-5′-imidodiphosphate (Gpp(NH)p) also decreased (−)[ 3H]bremazocine binding, suggesting that this κ opioid binding site is coupled to a G-protein. In summary, R1.1 cells posses a single high affinity κ opioid receptor that shares many properties with brain κ 1 b opioid receptors.

Guanine nucleotide-binding protein-coupled and -uncoupled states of opioid receptors and their relevance to the determination of subtypes.

Opioid receptors are currently classified as mu, delta, and kappa types, but various subtypes have also been proposed. We have investigated whether subtypes exist by using [3H]bremazocine. [3H]Bremazocine binds to twice as many naloxone-sensitive sites as other nonselective opioid agonists, as shown in four membrane types that have very different ratios of mu, delta, and kappa receptor types. [3H]Bremazocine binding is completely inhibited by an excess (in unlabeled form) of other opioid ligands, with Hill coefficients of 0.8-0.95. These paradoxes can be explained if there are high- and low-affinity states of the mu, delta, and kappa receptors and bremazocine binds with similar affinities to both states. We propose that these states are the guanine nucleotide-binding protein (G-protein)-coupled form and the uncoupled form of each receptor. As evidence for this proposal, the [3H]bremazocine binding suffered little or no loss with G-protein-uncoupling treatments, whereas binding of other opioid agonists was fully sensitive. We conclude that [3H]bremazocine offers a tool for the measurement of the total pools of coupled and uncoupled opioid receptors and that much of the previous characterization of opioid receptor subtypes reflects, instead, a significant pool of G-protein-uncoupled opioid receptors.

Bremazocine agonistic and antagonistic activity on withdrawal contraction in two isolated preparations

Previously, we reported that oxycodone is a putative κ-opioid agonist based on studies where intracerebroventricular (i.c.v.) pre-treatment of rats with the κ-selective opioid antagonist, nor-binaltorphimine (nor-BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (μ-selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor-BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)-diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post-STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an ∼3- to 4-fold decrease in potency. In rat brain membranes irreversibly depleted of μ- and δ-opioid binding sites, oxycodone displaced [3H]bremazocine (κ2-selective in depleted membranes) binding with relatively high affinity whereas the selective μ- and δ-opioid ligands, CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2) and DPDPE ([d-Pen2,5]-enkephalin), respectively, did not. In depleted brain membranes, the κ2b-ligand, leu-enkephalin, prevented oxycodone’s displacement of high-affinity [3H]bremazocine binding, suggesting the notion that oxycodone is a κ2b-opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a κ2b-opioid agonist with a relatively low affinity for μ-opioid receptors.