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E J Simon

Publications and source records attributed to E J Simon.

At least 55 records · Page 3Linked to original sources
PubMed1992

Sulfhydryl groups on opioid receptors revisited. Evidence for two sulfhydryl groups at or near the active site of the mu opioid receptor.

Sulfhydryl groups were studied in opioid receptors solubilized from bovine striatal membranes and reconstituted into liposomes. This system has the advantage of permitting the complete uncoupling of tightly coupled opioid binding sites from guanine nucleotide binding proteins. Sensitivity of opioid receptors to N-ethylmaleimide (NEM) inactivation, as measured by [3H]bremazocine binding, was similar whether coupled or uncoupled from the G protein. Moreover, the binding of uncoupled receptors could be protected from NEM inactivation by preincubation with a ligand, as previously observed in coupled, membrane-bound receptors. These findings provide strong support of earlier results suggesting the presence of sulfhydryl groups on opioid binding sites. An examination of the major receptor types provided the following decreasing order of sensitivity to NEM: mu > delta > kappa. Mu agonist binding was found to be much more sensitive to NEM than antagonist binding, especially in the presence of NaCl, which affects the binding of the two types of ligands in opposite directions, as previously reported for membrane-bound receptors. At 100 microM NEM in the presence of 100 mM NaCl, [3H] (D-Ala2,N-methyl-Phe4,Gly-ol)-enkephalin (DAGO) binding is essentially eliminated, whereas [3H]bremazocine or [3H]naloxone binding is virtually unaffected. These results are most readily explained by the hypothesis that there are two sulfhydryl groups at or near the mu binding site; one essential for agonist (but not antagonist) binding, the other essential for antagonist and perhaps, also agonist binding. The sodium effect on NEM inactivation of antagonist binding was maintained in the uncoupled state indicating that this effect occurs at the level of the receptor protein.

Animals↗
PubMed1991

Effects of parabrachial opioid antagonism on stimulation-induced feeding.

The pontine parabrachial nucleus (PBN) contains gustatory relay neurons and a high concentration of opioid receptors. To investigate the involvement of PBN opioid activity in feeding behavior, antagonists were infused into the PBN bilaterally and effects on stimulation-induced feeding were determined. Naloxone, a mu-preferring antagonist, increased the lateral hypothalamic stimulation threshold for eliciting feeding behavior while nor-binaltorphimine, a kappa-selective antagonist, did not. Neither antagonist increased threshold when infused into dorsal pontine sites outside of the PBN or the fourth ventricle. In as much as PBN contains mu and kappa but no detectable delta receptors, the present results suggest that mu opioid activity within the PBN is involved in the mediation of feeding behavior.

Animals↗
PubMed1990

Brain stimulation-induced feeding alters regional opioid receptor binding in the rat: an in vivo autoradiographic study.

Although opioid antagonists block feeding behavior in a variety of animal models, the number and identity of CNS regions in which the inferred endogenous opioid activity mediates feeding have yet to be established. Furthermore, it is not yet clear whether the opioid activity that sustains feeding is a concomitant of the appetitive motivational state or the consummatory response. In an effort to address these issues, an in vivo autoradiographic method was used to visualize CNS regional changes in opioid release during appetitively motivating electrical stimulation in the lateral hypothalamus (ESLH) and during consummatory behavior elicited by such stimulation. Regional decreases in [3H]diprenorphine [(3H]Dpr) binding, suggesting increased release of an endogenous opioid peptide, were observed in the medial prefrontal cortex, medial septum, gustatory cortex, zona incerta, mediodorsal thalamus, and hippocampus of rats receiving ESLH. Decreased binding in the latter 4 structures did not appear when animals were allowed to eat during ESLH, suggesting that the inferred opioid release is associated with appetitive behaviors elicited by ESLH which are suppressed when food is available and consummatory behavior predominates. When animals were allowed to eat during ESLH, [3H]Dpr binding in anterior cingulate cortex decreased substantially, suggesting that feeding behavior specifically triggers opioid release in this region. ESLH and feeding were found to increase [3H]Dpr binding in a number of CNS regions. Alternative explanations for increased binding, including inhibition of tonic opioid release, changes in cerebral blood flow, and opioid receptor up-regulation are discussed.

Animals↗
PubMed1990

Differential effects of cyanogen bromide on ligand binding by mu, delta and kappa opioid receptors.

Guinea pig brain membranes treated with cyanogen bromide (CNBr) demonstrate a loss in the number of mu opioid receptors and a lower binding affinity of delta opioid receptors. These receptor changes are irreversible. Results from ligand protection experiments support the hypothesis that the location of the methionine groups, the sites at which CNBr cleaves peptides, differs between these two types of opioid receptors. Kappa receptors are significantly less sensitive to the action of CNBr than mu or delta receptors.

Animals↗
PubMed1990

No-carrier-added (NCA) N-(3-[18F]fluoropropyl)-N-norbuprenorphine and N-(3-[18F]fluoropropyl)-N-nordiprenorphine--synthesis, anatomical distribution in mice and rats, and tomographic studies in a baboon.

N-(3-Fluoropropyl)-N-norbuprenorphine (3a) and N-(3-fluoropropyl)-N-nordiprenorphine (4a) were synthesized by N-alkylation of norbuprenorphine (1) and nordiprenorphine (2) with 1-bromo-3-fluoropropane. The corresponding no-carrier-added (NCA) N-(3-[18F]fluoropropyl)-N-norbuprenorphine (3b) and N-(3-[18F]fluoropropyl)-N-nordiprenorphine (4b) were synthesized by N-alkylation of 1 and 2 with NCA 1-[18F]fluoro-3-iodopropane in a synthesis time of approximately 100 min from end of bombardment (EOB) with an overall radiochemical yield of approximately 15% (EOB) and a mass of 2-3 nmol. In vitro studies indicate that in the absence of sodium chloride, compounds 3a, 4a, N-propyl-N-norbuprenorphine (5), buprenorphine and diprenorphine are reasonably comparable in binding affinity for opioid receptors. In the presence of 100 mM sodium chloride, however, compounds 3a, 4a and 5, are clearly less potent than buprenorphine and diprenorphine. The anatomical distribution study of compound 3b in mice shows radioactivity accumulating in bone, indicating that in vivo defluorination may have occurred. Rat studies of both compounds 3b and 4b indicate the specific distribution of these two radioligands within certain cortical and subcortical regions of rat brain. However, the absolute uptake of compound 4b in rat brain was only half that of compound 3b. PET studies of 3b in a baboon revealed specific binding of compound 3b in striatum and cerebellum. At 1 h after injection, ratios of specific/non-specific binding of 3b in striatum and cerebellum of a baboon were 1.9 and 1.7 respectively.

Animals↗
PubMed1990

Conjugate addition ligands of opioid antagonists. Methacrylate esters and ethers of 6 alpha- and 6 beta-naltrexol.

Alpha- and beta-naltrexol derived esters 9 and 10 and ethers 11 and 12, each containing the alpha, beta-unsaturated ester functionality, were prepared as conformationally more flexible analogues of spiro-alpha-methylene-gamma-lactones 5 and 6. All were active in the opioid radioreceptor binding assay against [3H]bremazocine and more active against [3H]DAGO, indicating mu-subtype selectivity, but only ether 12 showed significant irreversible activity. We conclude that small structural changes, made in very closely related electrophilic opioids, lead to changes in receptor binding. All four compounds were long-acting antagonists to morphine in mice, with ester 10 being approximately equipotent with naltrexone.

Animals↗
PubMed1990

Electrophilic alpha-methylene-gamma-lactone and isothiocyanate opioid ligands related to etorphine.

Isothiocyanate and alpha-methylene-gamma-lactone analogues of 6,14-endo-ethenotetrahydrothebaine and -oripavine were prepared with the electrophilic groups being located at C-19 in the C-7 alpha-side chain. Isothiocyanates were prepared in the N-Me and N-CPM (N-cyclopropylmethyl) series, both as the phenols and 3-O-methyl ethers from the diastereomeric amines formed from reductive amination of thevinone (2) and N-(cyclopropylmethyl)northevinone (13). Although addition of the organozinc reagent from methyl alpha-bromomethacrylate to 25 failed, addition to 3-O-protected aldehydes 27 and 35 produced, after subsequent deprotection, alpha-methylene-gamma-lactones 29 and 37, respectively. In the opioid receptor displacement assays against [3H]bremazocine as the radiolabeled ligand, the phenolic compounds were most potent with N-CPM isothiocyanates 20 and 21 showing IC50s of 0.32 and 0.76 nM, respectively, and N-CPM alpha-methylene-gamma-lactone 37 having an IC50 = 1.0 nM. Compound 37 showed irreversible effects in the binding assay which were mu-selective, as demonstrated by analogous experiments using [3H]DAGO, and naloxone was found to protect against the irreversible effects. This observation suggests that a receptor-bound nucleophile is located at a position where it can readily reach the alpha-methylene group of lactone 37.

Chemical Phenomena↗
PubMed1990

Cross-linking of human [125I]beta-endorphin to opioid receptors in rat striatal membranes: biochemical evidence for the existence of a mu/delta opioid receptor complex.

In a modified Krebs buffer at 37 degrees C, the selective mu agonist [3H] D-Ala2,MePhe4,Gly-ol5]enkephalin [( 3H]DAMGO) and the nonselective mu/delta agonist human [125I]beta-endorphin [( 125I]beta-endH) bound to rat striatal membranes with a Kd of about 7 and 5 nM and a Bmax of about 95 and 260 fmol/mg of protein, respectively, consistent with labeling of mu receptors by the former ligand and labeling of both mu and delta receptors by the latter. The binding of 2 nM [125I]beta-endH was displaced by unlabeled DAMGO (IC50 30 nM), [D-Ala2-D-Leu5]enkephalin (IC50 60 nM) as well as by the selective delta agonists [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET, IC50 500 nM) and Tyr-Ala-Phe-Asp-Val-Val-Gly-NH2 (IC50 700 nM) in a monophasic manner within 2 to 3 log concentration units, suggesting an allosteric interaction between mu and delta sites labeled by [125I]beta-endH under these conditions. Accordingly, 500 nM DSTBULET caused almost 40% inhibition of the apparent Bmax without changing the apparent Kd of [3H] DAMGO. The kappa agonist U 50,488 was ineffective as competing ligand even at a concentration of 10 microM. Upon affinity cross-linking of [125I]beta-endH (2 nM) to rat striatal mu- and delta-opioid receptors, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized tissue under reducing conditions followed by autoradiography of the dried gels revealed a major broad band of covalently labeled protein with an apparent molecular weight of 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation.

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.

Animals↗
PubMed1989

Effects of the selective kappa opioid antagonist, nor-binaltorphimine, on electrically-elicited feeding in the rat.

Lateral ventricular injections of the 'nonspecific' opioid antagonist naloxone (100 micrograms) and the kappa-selective opioid antagonist nor-binaltorphimine (50 micrograms) elevated the electrical brain stimulation frequency threshold for eliciting feeding behavior. Mesopontine aqueductal injections of nor-binaltorphimine, on the other hand, lowered the feeding threshold while naloxone still elevated threshold. These findings suggest the existence of forebrain kappa receptors at which endogenous opioid activity results in a facilitation of feeding while kappa receptors in the brainstem seem to mediate an inhibitory effect.

Animals↗
PubMed1989

Distribution of 3H-morphine following lumbar subarachnoid injection in unanesthetized rabbits.

Morphine sulfate (40-100 micrograms) and 3H-morphine (125-200 pmol) were injected into the lumbar subarachnoid space of 18 unanesthetized rabbits through a surgically implanted catheter. Radioactivity remaining in the spinal cord 2, 4, 6, and 12 h later revealed recovery (mean +/- SEM) of 45 +/- 5.6% (n = 3), 30.5 +/- 14.1% (n = 4), 11.23 +/- 4.4% (n = 3), and 3.7 +/- 1.1% (n = 3), respectively, of the injected radioactivity. Tritiated morphine was found to be predominantly centered around the injection site, with limited rostral and caudal spread in the cord. No significant radioactivity was detected in plasma or cerebrospinal fluid (CSF) samples from the cisterna magna taken at 5, 15, 30, min and 1, 2, 4, 6, 12, and 24 h after receiving radioactive labeled drug (with the exception of that in one rabbit). Of the injected radioactivity, 75% was recovered in the urine in 12 h. These results suggest that the persistence of morphine in the spinal cord could account for its prolonged analgesic effect following intrathecal administration.

Animals↗
PubMed1989

Pharmacokinetics of [3H]-buprenorphine in the rat.

The present study was undertaken to evaluate the potential usefulness of 11C-buprenorphine (bup) as a ligand for investigating opioid receptors in living primates, including humans, using positron-emission transaxial tomography (PETT). Because PETT studies of receptor function are best carried out under conditions of low receptor occupancy, the pharmacokinetics, uptake into brain, and specific binding to opioid receptors within brain of 3H-bup were examined in rats under conditions in which occupancy of opioid receptors by 3H-bup never exceeded 2% of sites in the brain at any time point examined. Male Sprague Dawley rats (weight range 140-220 grams) were injected s.c. with either naloxone (10 mg/kg) or saline. Five min later, a saline solution containing [15, 16-3H] bup (39 Ci/mmole) was injected into tail veins at a dose of 0.4 microgram/kg body weight. At least 90-95% of radioactivity was cleared from the blood in the first 5 min. In saline pretreated rats, total brain uptake 15 min after injection of 3H-bup was about 0.4% of the administered dose. Ligand specifically bound to receptors may be estimated by comparing the amount of radioactivity in the brain following injection of labeled ligand alone to that obtained when a high concentration of an unlabeled competitor is pre- or co-administered. In the present study, average levels obtained in brain (excluding cerebellum) were higher in saline pretreated rats than in naloxone pretreated rats at all time points and the difference increased with time indicating specific binding to opioid receptors. Specific binding may also be estimated by comparing radioactivity accumulated in brain areas rich in opioid receptors with "background" levels achieved in areas known to be low in opioid receptors, e.g., the cerebellum in rats. In the present study, ratios of the amount of radioactivity in brain (excluding cerebellum) to the amount in the cerebellum increased with time (to about 4 after 60 min) in saline pretreated rats, but remained close to 1 in naloxone pretreated rats. The effects of biological variation were less when specific binding was estimated by the latter method since each animal served as its own control. Tissue distribution of radioactivity to other tissues (blood, skin, muscle, fat, liver, kidney) was similar in naloxone and saline pretreated rats. The results presented here suggest that 11C-bup or an 18F-labeled fluorinated derivative would be a useful ligand for PETT studies.

Animals↗