Predicted structures for kappa opioid G-protein coupled receptor bound to selective agonists.

Human kappa opioid receptor (κ-OR), a G protein-coupled receptor (GPCR), has been identified as a drug target for treatment of such human disorders as pain perception, neuroendocrine physiology, affective behavior, and cognition. In order to find more selective and active agonists, one would like to do structure based drug design. Indeed, there is an X-ray structure for an antagonist bound to κ-OR, but structures for activated GPCRs are quite different from those for the inactive GPCRs. Here we predict the ensemble of 24 low-energy structures of human kappa opioid receptor (κ-OR), obtained by application of the GEnSeMBLE (GPCR Ensemble of Structures in Membrane Bilayer Environment) complete sampling method, which evaluates 13 trillion combinations of tilt and rotation angles for κ-OR to select the best 24. To validate these structures, we used the DarwinDock complete sampling method to predict the binding sites for five known agonists (ethylketocyclazocine, bremazocine, pentazocine, nalorphine, and morphine) bound to all 24 κ-OR conformations. We find that some agonists bind selectively to receptor conformations that lack the salt bridge between transmembrane domains 3 and 6 as expected for active conformations. These 3D structures for κ-OR provide a structural basis for understanding ligand binding and activation of κ-OR, which should be useful for guiding subtype specific drug design.

[3H]ethylketocyclazocine binding to brain opioid receptor subtypes in alcohol-preferring AA and alcohol-avoiding ANA rats.

We measured brain regional patterns of [3H]ethylketocyclazocine binding to brain opioid receptors in ethanol-naive alcohol-preferring Alko, Alcohol (AA) and alcohol-avoiding Alko, Non-Alcohol (ANA) rats, by using quantitative autoradiography. This agonist ligand labels all opioid receptor subtypes. The proportions of mu- and delta-opioid receptor binding were evaluated by displacing the mu- and delta-opioid receptor components by the peptides Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol (DAMGO, 100 nM) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE, 100nM), respectively, the K-component being the naltrexone-sensitive binding left after removal of the above two components. The labeling patterns in the brains of the AA and ANA rats were consistent with the well-known distributions of the opioid receptor subtypes in nonselected rat strains and there was no major difference between the lines. The mu-opioid receptor binding was greater in the AA than ANA rats in several brain regions, most interestingly in the substantia nigra pars reticulata and striatal clusters with elevated shell/core ratios in the nucleus accumbens. The delta-opioid receptor binding did not differ between the lines, whereas the AA rats had more K-opioid receptors than the ANA rats in several brain regions, including limbic areas and basal ganglia. The observed results might indicate altered action of the opioidergic system on dopaminergic pathways in rats with differential alcohol preference.

Kappa1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

Taxol inhibits opioid binding on T47D human breast cancer cells.

In the T47D human breast cancer cell line, Taxol was found to compete for ethylketocyclazocine opioid binding (IC50 3.3 pM). In contrast, no interaction of the drug with [3H]diprenorphine binding occurred. Binding was multiphasic, in the absence of colchicine (10[-6] M), but monophasic in its presence, indicating an involvement of the cytoskeleton in this process. Alignment of Taxol binding domains on alpha and beta tubulin with the kappa opioid site revealed homology of these sites with the first extracellular loop of the receptor. These results indicate a possible new action of Taxol, indicating for the first time a membrane action of the agent.

Key residues defining the mu-opioid receptor binding pocket: a site-directed mutagenesis study.

Structural elements of the rat mu-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for mu/delta selectivity (Ile196 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-labeled D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin1-31, JOM-13, deltorphin II, dynorphin1-13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of mu, delta, and kappa agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile196 to Val resulted in a four- to fivefold decreased affinity for the mu agonists morphine and DAMGO, with no change in the binding affinities of kappa and delta ligands.

Effect of low pH treatment on opioid peptides binding to their receptors and functional coupling of G-proteins to adenylyl cyclase in the rat spinal cord.

Because low pH treatment is known to alter the coupling of G-proteins to brain receptors, and little is known about such an effect in the spinal cord, the present study was undertaken to examine whether preincubation of rat spinal cord membranes at low pH (pH 4.5) alters opioid receptor binding characteristics and sodium fluoride (NaF)-stimulated adenylyl cyclase (AC) activity (as a function of G, mediated). [3H][D-Ala2,MePhe4,Gly-ol]enkephalin (DAMGO) and [3H]ethylketocyclazosine (EKC) were used to label mu- and kappa-opioid receptors, respectively. AC activity was determined using ATP as substrate and cAMP formed was quantified. Low pH treatment of membranes did not affect the mu- and kappa-opioid binding characteristics in rat spinal cord. However, the low pH treatment significantly reduced the NaF-stimulated AC activity in rat spinal cord. It is concluded that low pH treatment causes selective changes in the functional coupling of Gs-proteins to AC without affecting the opioid receptor binding characteristics in the spinal cord.

Opioid receptor types for endogenous enkephalin in the thoracic ganglion of the crab, Carcinus maenas.

In crustaceans, the endogenous opioid peptides, enkephalins, are known to be concentrated in the thoracic ganglion, although they have been demonstrated in all parts of the nervous system. Bmax and Kd measurements have been obtained for the binding of ligands used to characterize delta- and kappa-type opioid receptors in vertebrates. High- and low affinity binding of [3H] [2-D-Pen5-D Pen] enkephalin ([3H]DPDPE) has been measured with a Kd = 9.2 +/- 2.4 nM, Bmax = 153 fmol/mg, and Kd = 243 +/- 27 nM, Bmax = 1.785 pmol/mg, respectively. In addition a kappa-type receptor with Kd 85.5 +/- 12.6 nM and Bmax = 21.138 pmol/mg protein has been recorded. Binding characteristics of several ligands were monitored. Electrophoretic studies of affinity chromatographically purified receptor fractions revealed a molecular mass of 60 kDa. Isoelectric focusing showed a specific binding of [3H]DPDPE to thoracic ganglion membranes at a pl of 5.5.

Thermodynamic parameters of frog brain kappa-opioid receptors.

The thermodynamic parameters for [3H]-ethylketocyclazocine binding in frog (Rana esculenta) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. KA values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18 degrees C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.

Location of regions of the opioid receptor involved in selective agonist binding.

To elucidate which portions of the opioid receptor molecules are involved in the ligand selectivity, we have expressed chimeric receptors between the rat delta- and mu-opioid receptors from cDNAs and analysed their ligand binding properties. We demonstrate that the major binding determinant for the delta-selective enkephalin-related peptide, [D-Pen2,D-Pen5]enkephalin, resides within the region comprising the transmembrane segments V-VII and the intervening loop regions. On the other hand, the region spanning from the intracellular loop I to the amino-terminal half of the transmembrane segment III is shown to be involved in determining high-affinity binding of the mu-selective enkephalin-related peptides, [D-Ala2, MePhe4,Gly-ol5]enkephalin and [D-Ala2,MePhe4,Met-ol5]enkephalin, whereas the major determinant for binding of the mu-selective alkaloids, morphine and codeine, is demonstrated to exist in the region spanning the transmembrane segments V-VII. These results indicate that distinct regions of the opioid receptor determine the selectivity for the delta- and the mu-selective enkephalin-related peptides and that the binding determinant for the mu-selective alkaloids is distinct from that for the mu-selective enkephalin-related peptides.

Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.

[3H]dynorphin1-8 binding sites in frog (Rana esculenta) brain membranes.

Opioid binding sites specific for [3H]dynorphin1-8 were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphin1-8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin1-13, bremazocine, levorphanol and naloxone. The specific binding of [3H]dynorphin1-8 can be significantly inhibited by Na+ ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a Kd approximately 7.5 nM is present in the membranes. The maximum density of binding sites (Bmax approximately 1.1 pmol [3H]dynorphin1-8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the mu- and delta-types of opioid receptor showed that in the frog brain most of the sites labelled by [3H]dynorphin1-8 are kappa-sites and that this is a rich source of such sites.

Identification, partial characterization, and hypothalamic distribution of kappa, mu, and delta opioid receptors in a passerine songbird (Junco hyemalis).

Brain tissues obtained from a passerine songbird (dark-eyed junco, Junco hyemalis) were used to identify and partially characterize central opioid receptors. We found that [3H]EKC (putative kappa ligand), [3H]DAMGO (putative mu ligand), and [3H]DPDPE and [3H]pCl-DPDPE (two putative delta ligands) bind to brain tissue preparations specifically, in a time-dependent fashion, and with a high affinity (Kd's < or = 5 nM). Binding sites are present at low concentrations (Bmax < 120 fmol/mg protein), and they are pharmacologically selective. In vitro autoradiography studies revealed a high density of delta receptors in hypothalamic regions (ventromedial and lateral hypothalamus) that regulate feeding behavior. Together with previous studies, these observations suggest that the central influence of opioids on avian food consumption depends on mechanisms located in hypothalamic regions.

Properties of the putative epsilon opioid receptor: identification in rat, guinea pig, cow, pig and chicken brain.

[3H]Ethylketocyclazocine, in the presence of mu, delta and kappa-1 blocking agents, labels a high-affinity, saturable binding site in rat brain which we previously concluded might be the beta-endorphin-selective epsilon opioid receptor. However, studies failing to establish clearly the existence of the site in species other than rat raised the possibility that it might be an artifact or a unique constituent of rat tissues. Neither appears to be the case. We report here that, like other types of receptors, the site is proteinaceous with a sulfhydryl group within the binding site itself. Furthermore, the site appears to exist in two interchangeable, GTP-gamma-S-sensitive states which are readily distinguished by beta-endorphin, but not (-)-[3H]ethylketocyclazocine, and which are likely to be the site coupled to and uncoupled from a G protein. The putative epsilon receptor is not a peculiarity of rat brain but is readily measurable in forebrain of guinea pig, cow, chicken and pig brain as well. In fact, in all of the species examined, it is more abundant than mu, delta or kappa-1 receptors, representing 38 to 55% of the total opioid receptor population. The binding selectivity profile for drugs and the structure-activity relationship for beta-endorphin analogs indicated that the pharmacological properties of the putative epsilon receptor in brain are remarkably correlated with those of the epsilon receptor that was first hypothesized to exist in rat vas deferens and of the epsilon receptor that has been hypothesized to mediate the supraspinal analgesic effects of beta-endorphin.

Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine.

Stereoselectivity of the binding sites for the specific kappa-opioid agonist [3H]U-69593, a benzeneacetamido based ligand was investigated in membrane suspension prepared from frog and rat brain, as well as guinea pig cerebellum, using the pure chiral forms of different unlabelled opiates. The ligand binding sites showed stereospecificity with at least three orders of magnitude differences in the affinities (measured as Ki values) of the opioid stereoisomer pairs both in rat and guinea pig membrane fractions. However, in frog brain membranes there was no substantial difference in potencies of the (-) and (+) isomers competing for the [3H]U-69593 binding sites. Another type of the kappa-site preferring opioid ligand, [3H]ethylketocyclazocine, a benzomorphan derivative was able to discriminate between (-) and (+) forms of the same compounds even in frog brain membrane preparation. Our data concerning binding profile of [3H]U-69593 in frog brain membranes are consistent with the observation that kappa opioid binding sites in frog (Rana esculenta) brain differ from those kappa-sites found in mammalian brains.

Neuroanatomical localization of kappa 1 and kappa 2 opioid receptors in rat and guinea pig brain.

The neuroanatomical localization of kappa opioid receptors in rat and guinea pig brain was determined by quantitative in vitro receptor autoradiography. Our study shows striking differences in kappa 1 and kappa 2 receptor distributions both between species and within each species. In the rat brain, kappa 1 sites (labeled by [3H]U-69,593) are of low density and confined to a small number of structures. These include the claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, midline nuclear group of the thalamus, superficial grey layer of the superior colliculus, and central grey. kappa 2 sites (labeled by [3H]ethylketocyclazocine or [3H]bremazocine under conditions in which mu, delta, and kappa 1 binding was suppressed) are more widely distributed throughout all levels of rat brain. kappa 2 sites occur at high density in the caudate putamen, nucleus accumbens, amygdala, thalamus, and interpeduncular nuclei. In guinea pig brain, kappa 1 sites predominate and are of high density in layers I and VI of the neocortex, claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, and molecular layer of the cerebellum. As in rat brain, kappa 2 sites in guinea pig are more uniformly and widely distributed throughout the brain than are kappa 1 sites. The highest density of kappa 2 sites is in the dorsal parabrachial nucleus, interpeduncular nuclei, mammillary nuclei, and posterior thalamic nuclei. Results from this study demonstrate important interspecies differences in the distribution of kappa 1 and kappa 2 opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.

A monoclonal antibody recognizing kappa- but not mu- and delta-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)

Modulation of mu, kappa and delta opioid receptors in the rat brain by isoflurane and enflurane.

This study was done to investigate whether inhalational anesthetics modulated the binding of specific ligands to opioid receptors in the brain of the rat. The effect of isoflurane and enflurane on the binding of specific ligands to various subtypes of opioid receptors in vitro was studied. Isoflurane inhibited the binding of [3H]naloxone to opioid receptors by 50% in the spinal cord, midbrain and cortex at 22, 49 and 50 mM, respectively. Enflurane was more potent than isoflurane in inhibiting the binding of [3H]naloxone. Scatchard analysis of the binding of [3H]naloxone, done in the presence of therapeutic level (5 mM) of isoflurane, suggested that it did not affect the KD (1.3 nM) but decreased the Bmax by 41% in the cortex. Isoflurane and enflurane, at large doses (30-50 mM), inhibited the binding of [3H]ethylketo-cyclazocine (EKC) to kappa receptors in midbrain, cortex and spinal cord. At a smaller dose (5 mM), they increased the binding of EKC in spinal cord. The binding of the analogs of enkephalin [3H]DSTLE(Tyr-D-Ser-Gly-Phe-Leu-Thr-enkephalin) to delta receptors and [3H]DAGO (Tyr-D-Ala-Gly-Methyl-Phe-Glyol-enkephalin) to mu receptors in the midbrain and cortex was inhibited by isoflurane at a significantly smaller concentration than the binding of [3H]naloxone, indicating that the binding of peptides was more susceptible to the inhibition by inhalational anesthetics than the binding of alkaloids, such as naloxone or EKC. These results suggest that the modulation of opioid receptors by inhalational anesthetics is a function of both the nature of the ligand and the tissue used for the receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for kappa 1 opioid receptor multiplicity in the guinea pig cerebellum.

Both [3H]U69,593 alone and [3H]ethylketocyclazocine (EKC) in the presence of mu and delta blockers ([D-Ala2,MePhe4,Glyol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE)) label kappa receptors in the guinea pig cerebellum. Dynorphin A(1-17) and nor-binaltorphimine (nor-BNI) potently competed the binding of both radioligands with Hill coefficients of approximately unity, strongly supporting a kappa classification of binding. However, saturation studies revealed that the Bmax for [3H]EKC binding was 45% greater than that for [3H]U69593, suggesting that [3H]EKC might be labeling more than one site. Although nonlinear regression analysis of dynorphin A(1-17) and nor-BNI competition of [3H]EKC binding best fit the curves with a one site model, competitions by dynorphin B, dynorphin A(1-9) and alpha-neoendorphin revealed Hill coefficients less than unity and were best fit to a two site model. Kinetic analysis also supported [3H]EKC binding heterogeneity. Together, these studies imply that under these conditions [3H]EKC labels more than one site in the guinea pig cerebellum. The sensitivity of all specific [3H]EKC binding to the selective kappa ligands dynorphin A(1-17) and nor-BNI indicates that both component are kappa while the differing sensitivities of dynorphin B, alpha-neoendorphin and dynorphin A(1-9) for these components support our previous hypothesis of kappa 1a and kappa 1b binding subtypes.

Autoradiographic distribution of mu, delta and kappa opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord.

The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of mu, delta and kappa opioid binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8), thoracic (T5-T7), lumbar (L3-L5) and sacral (S2-S3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for mu, delta, kappa sites, respectively. The physiological relevance of these data is discussed.