Eric J. Simon

Evidence from opiate binding studies that heroin acts through its metabolites.

The relative affinity to opiate receptors of heroin, 6-acetylmorphine and morphine was estimated by determining their ability to displace specifically bound 3H-naltrexone from rat brain opiate binding sites. In vitro hydrolysis of heroin to 6-acetylmorphine was monitored in the binding assay filtrate by use of a quantitative HPLC procedure. The rate of heroin hydrolysis was significantly slower at 0 degrees C than at 37 degrees C. The displacement of 1 nM 3H-naltrexone by unlabeled ligand at concentrations ranging from 7 to 500 nM was measured at 0 degrees C for 120 minutes, yielding IC50 values of heroin = 483 nM, 6-acetylmorphine = 73 nM and morphine = 53 nM. When the binding data for heroin were recalculated to include the displacement that could be attributed to the 6-acetylmorphine derived from heroin degradation during the incubation, all of the apparent heroin binding was accounted for by the 6-acetylmorphine. These results are consistent with previous reports of the low binding affinity of morphine congeners (e.g., codeine) that lack a free phenolic 3-hydroxyl group and support the view that heroin is a prodrug which serves to determine the distribution of its intrinsically active metabolites, 6-acetylmorphine and morphine.

Receptor affinity and pharmacological potency of a series of narcotic analgesic, anti-diarrheal and neuroleptic drugs

A series of 26 drugs was tested for in vitro binding to opiate receptors in the presence and absence of 0.1 M NaCl. The results were correlated with assays for in vivo pharmacological potency. Highly significant correlation was found between binding in the presence and absence of sodium ions and analgesic potency. For 10 drugs tested for anti-diarrheal potency significant correlation was observed with binding to brain opiate receptors when binding was carried out in sodium-containing medium. These data add support to the hypothesis that stereospecific opiate binding sites are pharmacological receptors which mediate analgesia and anti-diarrheal action. We found that neuroleptics can bind to opiate receptors with affinities in the micromolar range, in agreement with reports by others. The anti-diarrheal compound loperamide exhibits no significant central opiate effects but binds to opiate receptors from brain in vitro with high affinity. Evidency is presented suggesting that the lack of specific analgesic effect is the result of poor penetration through the blood--brain barrier. Our results lend further support to the similarity of opiate receptors in the brain and in the intestinal tract.

μ-, δ- and κ-opioid receptor populations are differentially altered in distinct areas of postmortem brains of Alzheimer’s disease patients

Abstract The putative role of the opioid system in cognitive and memory functions prompted us to search for possible changes in the cohort of the major opioid receptors, μ, δ and κ, in Alzheimer’s disease. The present study examines alterations in opioid receptor levels by quantitative autoradiography. These experiments were carried out on coronal sections of postmortem brains from Alzheimer’s disease patients and from aged-matched, dementia-free individuals. Brain sections were labeled with the tritiated forms of μ-, δ- and κ-opioid ligands; DAMGO ([ d -Ala2,N-Me-Phe4,Gly-ol5]-enkephalin), DPDPE ([ d -Pen2,5]-enkephalin) and bremazocine (in the presence of μ- and δ-ligands), respectively. Nonspecific binding was determined in the presence of naloxone (10 μM). Brain areas analyzed were caudate, putamen, amygdaloid complex, hippocampal formation and various cerebral and cerebellar cortices. Image analyses of autoradiographs show, that in comparison to the same areas in control brain, statistically significant reductions in μ-opioid receptor binding occur in the subiculum and hippocampus of Alzheimer’s disease brains. Binding of δ-opioid receptors is also decreased in the amygdaloid complex and ventral putamen of Alzheimer’s disease brains. In contrast, large increases of κ-opioid receptor binding are found in the dorsal and ventral putamen as well as in the cerebellar cortex of Alzheimer’s disease brains. Levels of μ-, δ- and κ-opioid receptor binding are unaltered in the caudate, parahippocampal gyrus and occipito–temporal gyrus. These results may suggest an involvement of the endogenous opioid system in some of the multitude of effects that accompany this dementia.

Selective changes in μ, δ and ϰ opioid receptor binding in certain limbic regions of the brain in Alzheimer's disease patients

Abstract 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 Alzheimers disease patients and age-matched control individuals. The most consistent finding in Alzheimers disease brains was an increase in ϰ 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 Alzheimers disease putamen showed a significantly higher level of total binding (85% increase). The amygdala of Alzheimers disease patients exhibited significantly lower levels of μ and δ binding (41% and 55% decrease, respectively). Total binding and binding to μ and δ receptors in frontal cortex, caudate and hippocampus of Alzheimers disease brains was indistinguishable from levels seen in these brain areas from control individuals.

Solubilization of a stereospecific opiate-macromolecular complex from rat brain

A [3H]etorphine-macromolecular complex has been solubilized from rat brain synaptosomal fraction by extraction with the nonionic detergent Brij 36T. Stereospecificity of binding to this solubilized complex was demonstrated by the finding that radioactivity in the complex was virtually eliminated when binding had occurred in the presence of excess levorphanol, an active narcotic analgesic, while it was unaffected by its inactive enantiomorph dextrorphan. Bound radioactivity was dissociated by proteolytic enzymes, sulfhydryl reagents, and heat, suggesting the presence of protein. The bound solubilized macromolecular moiety may be the opiate receptor.

Lectin binding of solubilized opiate receptors: Evidence for their glycoprotein nature

Abstract Lectin affinity chromatography was used to demonstrate that digitonin-solubilized opiate receptors contain a carbohydrate moiety. Receptors solubilized from toad, rat, chicken, bovine and human brains were retained on columns of wheat germ agglutinin (WGA)-agarose and eluted specifically with N-acetylglucosamine. The fraction retained and subsequently eluted ranged from 40–60% of the applied receptors. The eluted receptor was enriched approx. 30-fold. Evidence is presented which shows that the site of lectin interaction is functionally independent of the opiate binding site.

μ and δ-opioid receptor agonists induce mitogen-activated protein kinase (MAPK) activation in the absence of receptor internalization

Agonist-promoted internalization (endocytosis) of G-protein-coupled receptors (GPCRs), including all three opioid receptor types (μ, δ and κ), has been shown to occur via the clathrin endosomal pathway in response to receptor phosphorylation and the actions of the proteins, β-arrestin and dynamin. Many members of the GPCR family stimulate mitogen-activated protein kinases (MAPK or ERK) activity and, in several cases, it appears that MAPK activation is dependent on receptor internalization. We have reinvestigated the question of whether internalization is obligatory for MAPK activation by opioid receptors, using cell lines expressing the cloned μ or δ receptor. Morphine, which is known to activate both μ and δ receptors, does not induce their rapid internalization into clathrin-coated endosomes. However, morphine produced a robust stimulation of MAPK in both cell lines, as demonstrated by the appearance of phosphorylated MAPK. Moreover, pre-exposure of cells to the internalization inhibitors, concanavalin A or hypertonic sucrose, totally blocked DAMGO (μ-selective agonist) and DTLET (δ-selective agonist)-mediated receptor internalization, yet neither treatment affected MAPK phosphorylation induced by these peptides. Our results provide evidence that receptor internalization is not an obligatory requirement for MAPK activation by μ and δ opioid receptors. Hypotheses are presented to explain the seemingly contradictory results obtained from different laboratories.

Turnover of muscle and liver proteins in mice with hereditary muscular dystrophy

Abstract Male mice with hereditary muscular dystrophy and their normal litter mates were injected intravenously with dl -leucine-1-C 14 . The specific radioactivity of skeletal muscle and liver proteins was determined at various intervals following injection. No significant differences were observed between turnovers of liver proteins of dystrophic and normal mice. In marked contrast, turnover of muscle proteins was accelerated in dystrophic mice, with the increase in catabolism apparently exceeding that in synthesis. This increase occurred in myofibrillar as well as sarcoplasmic proteins. These results indicate that loss of muscle proteins in myopathic mice is not the result of defective protein synthesis but of increased rate of protein turnover in which the more rapid rate of synthesis is exceeded by an even faster rate of catabolism.

Role of Protein Kinase C (PKC) in Agonist‐Induced α‐Opioid Receptor Down‐Regulation

Abstract : Phosphorylation of specific amino acid residues is believed to be crucial for the agonist‐induced regulation of several G protein‐coupled receptors. This is especially true for the three types of opioid receptors (μ, δ, and α), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of μ‐ and δ‐opioid receptors and to potentiate agonist‐induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid‐activated PKC contributes to homologous receptor down‐regulation and then expand our focus to include the exploration of the mechanism(s) by which μ‐opioids produce PKC translocation in SH‐SY5Y neuroblastoma cells. [dAla2,N‐Me‐Phe4,Gly‐ol]enkephalin (DAMGO)‐induced PKC translocation follows a time‐dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control‐treated cells in a process we termed “reverse translocation.” The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L‐type calcium channel antagonist nimodipine attenuated opioid‐mediated effects on PKC and μ‐receptor down‐regulation, suggesting that this is a process partially regulated by Ca2+‐dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+‐sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down‐regulation. In addition to expressing conventional (PKC‐α) and novel (PKC‐ε) isoforms, SH‐SY5Y cells also contain a DAG‐and Ca2+‐independent, atypical PKC isozyme (PKC‐ξ), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC‐ξ is similarly sensitive to activation by μ‐opioids. PKC‐ξ translocates from the cytosol to the membrane with kinetics similar to those of PKC‐α and ε in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by μ‐opioid agonists is involved in the processes that result in μ‐receptor down‐regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat

Abstract Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.