Rat Brain Opiate Receptors Research Articles

Binding of strychnocarpine and related β-carbolines to brain receptors in vitro

The affinity of strychnocarpine and related β-carbolines for serotonin, benzodiazepine, tryptamine, opiate and GABA receptors in rat brain was studied. Strychnocarpine showed a low to very low affinity for all the receptors tested. The weak binding to tryptamine receptors might explain part of the tremorigenic effects found earlier in the in vivo studies.

Opioid receptors and endogenous opioid peptides.

Medicinal Research ReviewsVolume 11, Issue 4 p. 357-374 Article Opioid receptors and endogenous opioid peptides Dr. Eric J. Simon, Dr. Eric J. Simon New York University Medical Center, 550 First Avenue, New York, New York 10016 Dr. Eric J. Simon: is Professor of Psychiatry and Pharmacology at New York University Medical Center in New York. In 1973 his laboratory reported the biochemical demonstration of opiate receptors in rat brain. He has continued to work in the area of opioid receptors and endogenous opioid peptides and has made many important contributions. Dr. Simon received his B.S. degree in Chemistry from Case Institute of Technology, Cleveland, Ohio and his M.S. and Ph.D. degrees in Organic Chemistry from the University of Chicago. He received postdoctoral training in the Department of Biochemistry at Columbia University Medical School (PS). Before entering the opiate field, he did biochemical research in a number of areas ranging from porphyrin biosynthesis to muscular dystrophy and vitamin E metabolism. Among his contributions are a widely used synthesis for succinyl (and other acyl) coenzyme A thioesters and the isolation and characterization of two previously unknown metabolites of α-tocopherol from human and animal urine. For his pioneering research on opiate receptors Dr. Simon has received many honors, including the Research Pacesetter Award from the National Institute on Drug Abuse (1977), The Louis and Bert Freedman Foundation Prize from the New York Academy of Sciences (1980), the Nathan B. Eddy Award from the Committee on Problems of Drug Dependence (1983), and an Honorary Doctorate (1982) from the University of Paris (Sorbonne). He is a member of many professional societies including the American Chemical Society, the American Society of Pharmacology and Experimental Therapeutics, American Society of Biological Chemists, American Society for Neuroscience, and New York Academy of Sciences (member of Board of Governors 1985–88). For 4 years he served as chairman of the International Narcotic Research Conference. Dr. Simon has been on the Faculty of New York University since 1959 and has served on many governmental advisory committees and as a consultant for a number of pharmaceutical companies.Search for more papers by this author Dr. Eric J. Simon, Dr. Eric J. Simon New York University Medical Center, 550 First Avenue, New York, New York 10016 Dr. Eric J. Simon: is Professor of Psychiatry and Pharmacology at New York University Medical Center in New York. In 1973 his laboratory reported the biochemical demonstration of opiate receptors in rat brain. He has continued to work in the area of opioid receptors and endogenous opioid peptides and has made many important contributions. Dr. Simon received his B.S. degree in Chemistry from Case Institute of Technology, Cleveland, Ohio and his M.S. and Ph.D. degrees in Organic Chemistry from the University of Chicago. He received postdoctoral training in the Department of Biochemistry at Columbia University Medical School (PS). Before entering the opiate field, he did biochemical research in a number of areas ranging from porphyrin biosynthesis to muscular dystrophy and vitamin E metabolism. Among his contributions are a widely used synthesis for succinyl (and other acyl) coenzyme A thioesters and the isolation and characterization of two previously unknown metabolites of α-tocopherol from human and animal urine. For his pioneering research on opiate receptors Dr. Simon has received many honors, including the Research Pacesetter Award from the National Institute on Drug Abuse (1977), The Louis and Bert Freedman Foundation Prize from the New York Academy of Sciences (1980), the Nathan B. Eddy Award from the Committee on Problems of Drug Dependence (1983), and an Honorary Doctorate (1982) from the University of Paris (Sorbonne). He is a member of many professional societies including the American Chemical Society, the American Society of Pharmacology and Experimental Therapeutics, American Society of Biological Chemists, American Society for Neuroscience, and New York Academy of Sciences (member of Board of Governors 1985–88). For 4 years he served as chairman of the International Narcotic Research Conference. Dr. Simon has been on the Faculty of New York University since 1959 and has served on many governmental advisory committees and as a consultant for a number of pharmaceutical companies.Search for more papers by this author First published: July 1991 https://doi.org/10.1002/med.2610110402Citations: 78 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume11, Issue4July 1991Pages 357-374 RelatedInformation

Total synthesis of fully tritiated leu‐enkephalin by enzymatic coupling

AbstractThis paper describes the total enzymatic synthesis of Leu‐enkephalin (Tyr‐Gly‐Gly‐Phe‐Leu) in which all residues were labelled with tritium. Carboxypeptidase Y from Saccharomyces cerevisiae was the coupling enzyme. [3H]‐Tyr‐NH2, [3H]‐Gly‐Oet, [3H]‐Phe‐NH2 and [3H]‐Leu‐NH2 were prepared with specific radioactivities ranging between 20 and 60 Ci/mmol (740 to 2220 GBq/mmol). Using a microscale procedure, we obtained a fully tritiated hormone having a specific radioactivity equal to 139 Ci/mmol (5143 GBq/mmol), in agreement with the summation of the specific radioactivities of constituting residue. The radioactive hormone had antigenic properties identical to those of native Leu‐enkephalin. It also bound to rat brain opiate receptors like the parental hormone.

Binding characteristics of a series of dimeric tripeptide enkephalins for δ opiate receptors in rat brain and NG108–15 cells

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 μM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 μM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1μM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to −80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 μM. Unsulfated CCK-8 (100pM to 1 μM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

Autoradiographic localization of delta opiate receptors in rat and human brain

In vitro quantitative receptor autoradiography was performed on frozen sections of rat and human brain to visualize delta opiate receptors using the specific ligand [ 3H][ d-Pen 2, d-Pen 5]enkephalin. For comparison, rat brain sections were also labelled with [ 3H] d-Ala 2, d-Leu 5-enkephalin. Compounds which block mu and kappa binding were included to make the [ 3H] d-Ala 2, d-Leu 5-enkephalin binding more specific. The two ligands had similar, but not identical, distributions in rat forebrain sections. Sites labelled with [ 3H][ d-Pen 2, d-Pen 5]enkephalin were distributed heterogeneously within the layers of the frontal and parietal cerebral cortex, with high densities in the superficial and deep cortical layers. The claustrum and striatum had the most delta sites, whereas the globus pallidus had no delta binding. The distribution of [ 3H] d-Ala 2, d-Leu 5-enkephalin binding sites was similar to that of [ 3H][ d-Pen 2, d-Pen 5]enkephalin, except that there was less heterogeneity in the frontal cortex. In the human brain regions studied, the highest delta binding was in caudate, putamen, temporal cortex and amygdala. There was less heterogeneity in the binding of [ d-Pen 2, d-Pen 5]enkephalin in the human cortex than in the rat. No delta binding was seen in the medial and lateral segments of the globus pallidus. In both species, a discrepancy between the high enkephalin content of the globus pallidus and the absence of delta binding was apparent.

The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech, and rat

We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [ 125I]β-endorphin ([ 125I]βE), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for μ, δ, and κ opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [ 125I]β-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their μ, δ, or κ specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking. An evolutionary conservation of the opiate receptor is suggested by the presence of the same 3 major cross-linking products in Tetrahymena, leech, and rat.

Specific binding of ?-and ?-ligands by rat brain opiate receptors in the presence of reaferon

Specific binding of ?-and ?-ligands by rat brain opiate receptors in the presence of reaferon

Human antiidiotypic antibody against opiate receptors.

Sera containing antibodies to beta-endorphin from 2 patients with major depressive disorder were shown to have antidiotypic antibodies that specifically inhibited reactivity between anti-beta-endorphin IgG and beta-endorphin. Autologous and homologous antiidiotypic anti-anti-beta-endorphin IgG antibodies were isolated by affinity chromatography. The purified antiidiotypic antibody did not bind beta-endorphin but competed with [125I]beta-endorphin for rat brain opiate receptors. Normal IgG that was similarly treated had negligible competitive effects. The antibody bound to the membrane preparation; such binding was inhibited by opiate receptor ligands. Binding of the antiidiotype to a 60,000-dalton protein from rat brain was detected by Western immunoblot analysis. This protein corresponds in molecular weight to proteins proposed to be components of opiate receptors. These findings imply that immune reactivity to neuropeptides could contribute to psychiatric impairment.

Enzymatic reconstitution of brain membrane and membrane opiate receptors.

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.

Visualization of mu1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique.

We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of mu1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the mu1 binding of [3H]dihydromorphine, leaving mu2 sites, while low morphine concentrations eliminate the mu1 binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving delta sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent mu1 binding sites. The regional distributions of mu1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5]enkephalin. High levels of mu1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of mu1 sites in analgesia. Other regions with high levels of mu1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to mu1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D-Leu5]enkephalin and 20% to 52% for [3H]dihydromorphine.

Rabbit anti-idiotypic antibodies raised against monoclonal anti-morphine IgG block μ & δ opiate receptor sites

Rabbit antibodies have been raised against murine monoclonal anti-morphine Fab fragments. Following affinity purification, these antibodies competitiveky inhibit morphine binding to anti-morphine monoclonal antibodies, yet do not recognize normal mouse IgGs, suggesting that the antibodies produced are anti-idiotypic with respect to the anti-morphine IgG. More importantly, the purified antibodies competitively inhibit binding of morphine, naloxone, and D-ala-2-D-leu-5-enkephalin to rat brain opiate receptors.

Sulfonic acid enkephalin: Binding specificity to rat brain opiate receptors

We tested sulfonic-acid enkephalin, a SO 3H-Tyr derivative of the Leu-enkephalin, for its binding capacity to rat brain opiate receptors, by competition against several tritiated enkephalins. Using [ 3H]DADLE as the competitor, we demonstrated that sulfonic-acid enkephalin binds to rat brain opiate receptors, and using [ 3H] DSLET and [ 3H]DADLE as competitors, we demonstrated that sulfonic-acid enkephalin binds preferentially to δ-opiate receptors. The affinity ratio of sulfonic-acid enkephalin for δ-opiate receptors versus μ-opiate receptors, is considerably higher than that of the parent compound, Leu-enkephalin, and of DADLE and is similar to the affinity ratio of DSLET for the same receptors.

Mathematical modeling of opiate binding to mouse brain membrane.

The heterogeneity of rat brain opiate receptors was examined by analyzing competition data. The binding of three prototypical tritiated opioid agonists, [3H]-dihydromorphine ([3H]-DHM), [3H]-D-ala2-D-leu5-enkephalin ([3H]-DADLE), and [3H]-ethylketocyclazocine ([3H]-EKC) was determined in the presence of varying concentrations of each of these unlabeled ligands, generating nine displacement curves. A computer program was then used to find the best fit of a model system to these data, assuming two, three or four independent binding sites. The best fit was a four-site model. One of these sites is specific for DHM; two are relatively selective for DHM and DADLE respectively, but also bind EKC. The remaining site binds only EKC with high affinity. These results, together with displacement data using naloxone, FK33824, and D-ala2-met5-enkephalinamide, are discussed in terms of current opiate receptor models.

Effect of sodium chloride on opiate receptor binding in spontaneously hypertensive rats: dependence on age.

The effect of 100 mM sodium chloride on the binding of [3H]-naltrexone to rat brain opiate receptors in spontaneously hypertensive (SHR) and normotensive (WKY) rats was studied. The percentage increase in binding in the presence of sodium chloride did not vary with age in WKY rats. Brain homogenates from 4 week old SHR rats incubated with 100 mM sodium chloride exhibited a similar increase in binding compared to age matched WKY rats. In contrast, brain preparations from 6, 14 and 20 week old SHR rats were more sensitive to sodium chloride, and the increase in binding of [3H]-naltrexone was significantly greater in these animals than in corresponding normotensive ones. Since blood pressure is increased in SHR rats compared to WKY rats at these ages, these results suggest that elevated blood pressure may be correlated with an increase in opiate receptor sensitivity to sodium chloride. The effect of in vivo sodium chloride was examined by feeding the animals a diet containing 4% salt. This concentration of salt did not significantly alter the binding of [3H]-naltrexone to rat brain homogenates prepared from 8 week old SHR rats. These results suggest that higher levels of sodium chloride and longer exposure to the diet may be required to observe the salt sensitivity produced by 100 mM salt in the in vitro radioreceptor assay.

Castration affects male rat brain opiate receptor content.

We previously reported that saturable stereospecific binding of [3H]-naltrexone in rat brain homogenates prepared from castrated male rats was greater than the corresponding binding in intact animals. We now report that we have replicated these results and that the difficulty of other investigators in observing these differences is due to methodological factors. Specifically, when samples were filtered individually and rapidly, differences between castrated and intact rats were maintained. The increase in binding was also observed when tissues were washed to remove endogenous opioids prior to incubation, when [3H]-naloxone was used as the ligand, and when various antagonists were used as displacers in the radioreceptor assay.

Mathematical modeling of opiate binding to mouse brain membrane

The heterogeneity of rat brain opiate receptors was examined by analyzing competition data. The binding of three prototypical tritiated opioid agonists, [ 3H]-dihydromorphine ([ 3H]-DHM), [ 3H]-D-ala 2-D-leu 5-enkephalin ([ 3H]-DADLE), and [ 3H]-ethylketocyclazocine ([ 3H]-EKC) was determined in the presence of varying concentrations of each of these unlabeled ligands, generating nine displacement curves. A computer program was then used to find the best fit of a model system to these data, assuming two, three or four independent binding sites. The best fit was a four-site model. One of these sites is specific for DHM; two are relatively selective for DHM and DADLE respectively, but also bind EKC. The remaining site binds only EKC with high affinity. These results, together with displacement data using naloxone, FK33824, and D-ala 2-met 5-enkephalinamide, are discussed in terms of current opiate receptor models.

Prenatal and postnatal exposure to diazepam: Effects on opioid receptor binding in rat brain cortex

The effects of chronic perinatal or postnatal administrations of diazepam on opiate receptor development in cerebral cortex and striatum were studied at two different post-treatment ages, using tritiated ehtylketocyclazocine, the prototypic κ-opiate, for measurements of opiate binding site ontogeny. Prenatal plus postnatal exposure to diazepam markedly decreased the total number of binding sites for [ 3H]ethylketocyclazocine in the rat cerebral cortex and striatum at 14 days of age. Postnatal diazepam treatment alone did not alter [ 3H]ethylketocyclazocine binding site development in cortex or striatum. These results suggest that development of opiate receptors in rat brain can be altered by prenatal exposure to diazepam.

Autoradiographic distribution of mu and delta opiate receptors in rat brain using highly selective ligands

We have characterized the autoradiographic distribution of mu and delta opiate receptors in rat brain using [ 3H]-Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO) and [ 3H]D-Thr 2, Thr 6leu-enkephalin (DTLET), two highly selective ligands for mu and delta opiate receptors, respectively. Both ligands label an apparent single class of sites. Ligand selectivity shows that [ 3H]DAGO and [ 3H]DTLET are more selective ligands for their respective receptors than [ 3H]dihydromorphine (DHM) and [ 3H][D-Ala 2, D-Leu 5]enkephalin (DADLE). Autoradiographically, mu opiate receptors are highly discretely distributed in certain areas. High densities are found in “patches” in caudateputamen, nucleus accumbens, cingulate cortex, habenula, various thalamic nuclei, amygdala, superior colliculus, and interpeduncular nucleus. Delta opiate receptors are mostly concentrated in the external plexiform layers of the olfactory bulb, caudate-putamen, olfactory tubercule, claustrum, and deep layers (layers V and VI) of the cortex. Since [ 3H]DAGO and [ 3H]DTLET are more selective ligands for their respective opiate receptor subtypes, they should be better ligands to characterize the fine distribution of mu and delta opiate receptors.

Visualization and solubilization of rat brain opiate receptors with a "kappa" ligand selectivity pattern.

1. Specific binding of [3H]ethylketocyclazocine (EkappaC), a prototype kappa-opiate agonist, to slide-mounted rat striatal sections is increased in the presence of 100 mM NaCl at 4 degrees C. 2. Under similar incubation conditions, binding of mu and delta prototype opiates is reduced to almost undetectable levels. 3. Correlation (P less than 0.01) of the ligand selectivity pattern of [3H]EKC displacement with the potencies of various opiate drugs in inhibiting the contractions of the rabbit vas deferens, a kappa-opiate receptor bioassay, suggests that the binding site under study represents the pharmacologically relevant kappa-opiate receptor. 4. Visualization of these kappa-opiate receptors with tritium-sensitive film reveals a striking, highly discrete brain distribution pattern (e.g., striatal patches, habenular stripe) which is similar to that of [3H]dihydromorphine and [3H]naloxone. 5. Soluble [3H]EKC binding sites obtained from rat membranes also possess a kappa-like ligand selectivity pattern, with bremazocine being a potent displacer while mu and delta ligands are almost inactive. 6. A possible explanation of these data is that the "kappa"-opiate binding site in rat brain is one transitional state of an opiate receptor capable of assuming distinct conformations with characteristic ligand selectivity patterns. Other possibilities such as pre and post-synaptic locations should also be considered.