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E J Simon

Publications and source records attributed to E J Simon.

At least 73 records · Page 4Linked to original sources
PubMed1988

Affinity crosslinking of 125I-labeled human beta-endorphin to cell lines possessing either mu- or delta-type opioid binding sites.

The specific labeling of opioid receptor-related polypeptides was compared in two cell lines which differ in their opioid receptor population: SK-N-SH which contains predominantly mu-type opioid receptors, and NG-108-15, which contains exclusively delta-type opioid receptors. Labeling of opioid receptors was achieved by affinity cross-linking of membranes, using 125I-labeled human beta-endorphin, followed by solubilization in sodium dodecyl sulphate (SDS), SDS-gel electrophoresis and autoradiography. Different labeling patterns were obtained from these two cell lines. In SK-N-SH cells, 3 major proteins were labeled, corresponding to molecular weights of 92, 65 and 25 kDa, while in the NG-108-15 cells, 53-kDa and 25-kDa polypeptides were the major ones labeled. The radioactivity incorporated into the 92- and 65-kDa peptide bands derived from SK-N-SH cells was displaced by the mu-selective ligand Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) but not by the delta-selective ligand [D-Pen2,D-Pen5]enkephalin (DPDPE). The radioactivity incorporated into the NG-108-15-derived peptide bands was displaced by the delta-selective ligand, but not by the mu-selective ligand. This confirms our previous finding in mammalian brain which demonstrated that mu- and delta-opioid binding sites can be identified as distinct proteins which differ in molecular size.

Binding Sites↗
PubMed1987

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats.

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.

Analgesia↗
PubMed1987

Antibodies to dynorphin A(1-13) but not beta-endorphin inhibit electrically elicited feeding in the rat.

Highly specific antibodies to dynorphin A(1-13), infused into the lateral ventricle, elevated brain stimulation threshold for eliciting feeding behavior. Antibodies to beta-endorphin had little or no effect. Temporal analysis of the anorectic action indicated a striking similarity to the effect of systemically administered naloxone. These findings suggest that central dynorphin is involved in the control of ingestive behavior and that the anorectic action of naloxone may result from antagonism of dynorphinergic transmission.

Animals↗
PubMed1987

Selective changes in mu, delta and kappa opioid receptor binding in certain limbic regions of the brain in Alzheimer's disease patients.

Total opioid binding and levels of the three major types of opioid binding sites were measured in homogenates of various limbic structures from post-mortem brains of Alzheimer's disease patients and age-matched control individuals. The most consistent finding in Alzheimer's disease brains was an increase in kappa binding in all 6 areas of the limbic system examined, with the putamen and caudate regions showing significant increases of 114% and 53%, respectively. In addition, the Alzheimer's disease putamen showed a significantly higher level of total binding (85% increase). The amygdala of Alzheimer's disease patients exhibited significantly lower levels of mu and delta binding (41% and 55% decrease, respectively). Total binding and binding to mu and delta receptors in frontal cortex, caudate and hippocampus of Alzheimer's disease brains was indistinguishable from levels seen in these brain areas from control individuals.

Alzheimer Disease↗
PubMed1987

Interaction with lectin of kappa opioid binding sites solubilized from human placenta.

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.

Acetylglucosamine↗
PubMed1987

No-carrier-added (NCA) (+/-)-N-(3-[18F]fluoropropyl)-N-normetazocine-synthesis and PET studies in a baboon.

No-carrier-added (NCA) (+/-)-N-(3-[18F]Fluoropropyl)-N-normetazocine (2) was synthesized by N-alkylation of (+/-)-N-normetazocine (1) with NCA 1-[18F]fluoro-3-iodopropane in an overall radiochemical yield of 10% (EOB) with a mass of 3.5 nmol in a synthesis time of 90 min from end of bombardment (EOB). PET studies of 2 in a baboon did not indicate specificity for opiate receptor sites alone: The activity declined rapidly in the striatum, the frontal cortex and the cerebellum. The baboon total arterial plasma radioactivity clearance was very rapid and the metabolism of compound 2 in plasma was also very rapid. These results suggest that compound 2 is not a suitable radioligand for imaging opiate receptors in the human brain by positron tomography.

Animals↗
PubMed1987

Subunit structure and purification of opioid receptors.

Considerable evidence indicates the existence of multiple types of opioid receptors. The three major types have been named mu, delta and kappa. The earlier evidence was based on pharmacological as well as membrane binding experiments. This paper will emphasize more recent studies using solubilized opioid binding sites. Several laboratories, including our own, have succeeded in separating kappa receptors from other types. A similar separation of mu from delta receptors has not yet been achieved. By crosslinking experiments with 125I- human beta-endorphin we have been able to provide strong evidence for differences in molecular size between the major binding components of mu (65K) and delta (53K) receptors. It is not yet established whether the difference resides in the protein or carbohydrate portion of these glycoproteins. These results suggest that the three major types of opioid receptors represent distinct molecular entities. An active opioid binding protein solubilized from bovine striatal membranes has been purified to apparent homogeneity. The major purification steps involve affinity chromatography and lectin chromatography on immobilized wheat germ agglutinin. The purified material gave a single band of molecular weight 65K Da on SDS-PAGE. Its specific activity for opioid binding was ca. 13,000 pmol/mg protein and its properties are those of a component of the mu receptor.

Animals↗
PubMed1986

Identification of distinct binding site subunits of mu and delta opioid receptors.

Iodinated human beta-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65- and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108-15 cell line suggested that the 65-kDa peptide is a binding component of mu receptors while the 53-kDa peptide is a binding subunit of delta receptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly mu-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly mu-selective ligand [D-Pen2, D-Pen5]enkephalin (DPDPE). The labeling of the 41- and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to mu and delta receptors is not understood. Several possible explanations are presented.

Animals↗
PubMed1986

Antagonist-induced opiate receptor upregulation in cultures of fetal mouse spinal cord-ganglion explants.

Chronic exposure of fetal mouse spinal cord-ganglion explants to the opioid antagonist naloxone (10 microM, 7 days) produced a pronounced upregulation of mu opioid receptors. The antagonist action was stereospecific, as it was produced by (-)-, but not by (+)-naloxone, and was dose-dependent. Half-maximal naloxone-induced receptor upregulation occurred after two days; receptor density was maximal at 5 days. Exposure of the explant cultures to naloxone (10 microM) in the presence of the protein synthesis inhibitor, cycloheximide (1 microM; a concentration which blocks greater than 90% protein synthesis) resulted in receptor density changes that were similar to those observed in cultures exposed to naloxone alone. This finding suggests that antagonist-induced opiate receptor upregulation does not require the synthesis of new receptor molecules.

Animals↗
PubMed1986

Limbic regions of the brain of Alzheimer's disease patients show selective changes in mu, delta and kappa opioid receptor binding.

Total opioid binding and levels of the three major types of opioid binding sites were measured in homogenates of various limbic structures from post-mortem brains of Alzheimer's Disease (AD) patients and age-matched control individuals. AD brains showed an increase in kappa binding in all six areas of the limbic system examined, with the putamen and caudate regions showing significant increases of 114% and 53%, respectively. In addition, the AD putamen showed a significantly higher level of total binding (85% increase). The amygdala of AD patients exhibited significantly lower levels of mu and delta binding (41% and 55% decrease, respectively).

Alzheimer Disease↗
PubMed1986

Affinity crosslinking of 125I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites.

Affinity crosslinking of human 125I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

Cell Line↗
PubMed1986

The direct demonstration of binding of mu, delta and kappa agonists to digitonin-solubilized opioid receptors from bovine striatum.

Active opioid binding sites, that retain the ability to bind tritiated agonists have been obtained in good yield in digitonin/NaCl/Mg2+ extracts of morphine protected bovine striatal membranes. Ligand protection of binding sites and the presence of Mg ions were found to be absolute requirements for agonist binding in this solubilized opioid receptor preparation. Soluble preparations contained a ratio of mu: delta:kappa similar to that in the membranes.

Analgesics↗
PubMed1985

Purification of an active opioid-binding protein from bovine striatum.

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.

Affinity Labels↗
PubMed1985

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit.

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.

Animals↗