Eli Hazum

Multiple opiate receptors

Abstract The discovery of opiate receptors has been followed by the exciting discovery of endogenous opiate-like substances; the enkephalins and endorphins. These opioid peptides bind to opiate receptors with high affinity and mimic some of the effects of morphine itself. They have been postulated to act as neurotransmitters or neuromodulators in regulating pain perception, locomotor activity, body temperature, behaviour, and neuroendocrine functions. The discovery of enkephalin has also led to the discovery of multiple types of opiate receptor and in this article Chang, Hazum and Cuatrecasas discuss whether the complex effects of opioid peptides are mediated by single or multiple types of opiate receptors.

Competitive interaction between endothelin and sarafotoxin: Binding and phosphoinositides hydrolysis in rat atria and brain

Binding studies with the structurally similar vasoconstrictor peptides 125I-endothelin and 125I-sarafotoxin b, the former of mammalian origin and the latter derived from snake venom, reveal their mutually exclusive binding to rat atrium and various regions of the rat brain. In these tissues endothelin, like sarafotoxin, induces phosphoinositide hydrolysis which is in part Ca2+-independent. It is suggested that endothelins and sarafotoxins share common binding sites and mechanisms of action.

Increased biological activity of dimers of oxymorphone and enkephalin: Possible role of receptor crosslinking

Abstract Four analogs of oxymorphone, oxymorphaminoethylthiol, oxymorphamino-ethyldisulfide, oxymorphaminoethyl-nitrobenzoic acid disulfide and oxymorphone thiazolidine, as well as the enkephalin analogs, enkephalin-thiol, Tyr-D-Ala-Gly-Phe-Leu-Lys(ϵ-NH)COCH 2 CH 2 SH and the enkephalin-dimer, [Tyr-D-Ala-Gly-Phe-Leu-Lys(ϵ-NH)COCH 2 CH 2 S-] 2 , were examined for binding to enkephalin and morphine receptors. The analogs gained substantial affinity for enkephalin and lost affinity for morphine receptors. The affinity of the dimers of both opiates and enkephalins was slightly greater than that achieved by the corresponding thiol monomers. However, in the guinea pig ileum the dimeric analogs were much more active than the monomers. Receptor dimerization or cross-linking may be involved in the biological activity of opiates and opioid peptides.

Fluorescent and photo-affinity enkephalin derivatives: Preparation and interaction with opiate receptors

Abstract The fluorescent and photo-affinity derivatives of enkephalin, Tyr-D-Ala-Gly-Phe-Leu-Lys-Ne-Rhodamine (II) and Tyr-D-Ala-Gly-Phe-Leu-Lys-Ne-nitro-azidophenyl (III), were prepared by conventional methods followed by chemical modification. The two peptides inhibit the binding of 125I-labeled enkephalin to brain membrane preparations, with apparent IC50 values of 5.9 nM and 5.5 nM for peptides II and III, respectively. The iodinated derivative of peptide III binds specifically to brain membrane preparations with an apparent Kd of about 2.1 × 10−9M.

Naloxazone irreversibly inhibits the high affinity binding of [125I]D-ala2-D-leu5-enkephalin

Abstract Scatchard analyses of [125I]D-ala2-D-leu5-enkephalin binding in rat brain membranes are curvilinear, suggesting low and high affinity sites. Treating the membranes with naloxazone abolishes the high affinity binding with slight effect on low affinity binding. Displacement of [125I]-D-ala2-D-leu5-enkephalin binding by morphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no inhibition by low concentrations of morphine. Naloxazone treatment has little effect on displacements by unlabeled D-ala2-D-leu5-enkephalin. Binding in N4TG1 neuroblastoma cells, which demonstrates a linear Scatchard plot with single affinity constant similar to that of the low affinity binding in brain, is less sensitive to naloxazones actions. Naloxazone treatment in vivo inhibits D-ala2-D-leu5-enkephalin analgesia.

Digestion of insulin receptors with proteolytic and glycosidic enzymes-effects on purified and membrane-associated receptor subunits

Abstract Insulin receptors, photoaffinity-labeled in rat liver and human placenta membranes, and receptor purified from rat liver membranes, were treated with proteolytic and glycosidic enzymes and analyzed by SDS-polyacrylamide gel electrophoresis. The protease fragments from the purified receptor were similar to those from the photoaffinity-labeled receptors, if these were solubilized prior to digestion. However, the intact photoaffinity-labeled membranes were less sensitive to proteases, and different fragments were produced. Thus, solubilization exposes sites very sensitive to proteolysis. The labeled tryptic fragments of placenta membranes remained membrane-bound. Neuraminidase altered the mobility of the intact receptor and of several of the large tryptic fragments, indicating that these contain sialic acid and are probably exposed on the outer surface of the membrane.

Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells

The mitogenic activity of endothelin (ET) was studied in osteoblast-like cells, MC3T3-E1. [3H] Thymidine incorporation induced by ET was markedly lower than that of platelet-derived growth factor (PDGF). ET synergistically stimulated [3H] thymidine incorporation induced by PDGF with an apparent ED50 value of 2.5 nM. Treatment of MC3T3-E1 cells with ET and subsequent immunoblotting of the cell extracts with antiphosphotyrosine antibodies followed by labeling with [125I] protein A resulted in the identification of several phosphotyrosine-containing proteins. The intensity of these labeled phosphoproteins significantly increased when the cells were treated with a combination of ET and PDGF. Genistein, an inhibitor of tyrosine kinases, blocked [3H] thymidine incorporation as well as protein tyrosine phosphorylation stimulated by either ET, PDGF or the combination of ET and PDGF. These findings suggest that tyrosine phosphorylation could play a role in the comitogenic activity of ET in osteoblast-like cells.

Receptor redistribution induced by hormones and neurotransmitters: Possible relationships to biological functions

Abstract Studies on the patterns and mobilities of various receptors for polypeptide hormones have indicated that generally the occupied receptors appear to be initially distributed uniformly over the cell surface and quickly form clusters which are subsequently internalized. In contrast, clusters of opiate receptors appear to form only slowly and they do not become internalized. It is now recognized that receptor microaggregation or cross-linking, perhaps independent of internalization, may be important for hormonal action in some systems. On the other hand, in certain other systems of for certain special functions, internalization of receptor-bound proteins may serve as a selective transport system which directs specific ligands to specific subcellular organelles.

Rapid degradation of [3H] leucine-enkephalin by intact neuroblastoma cells

Abstract Enkephalin, a brain peptide with morphine-like activity, is rapidly degraded by N4TG1 neuroblastoma cells which contain opiate receptors. The enzymic activity is temperature dependent and it is maximal at 37°C with an apparent Km of 5 × 10 −5 M. The enzyme can be inhibited by bacitracin and puromycin with apparent Ki values of 3.2 × 10 −5 M and 2.3 × 10 −7 M, respectively. Digesting intact cells with trypsin greatly diminishes the ability of the cells to degrade enkephalin and suggests that the degradative enzyme is probably localized at the cell surface. Opiates such as morphine and naloxone as well as leu- and met-enkephalin and D-Ala 2 -substituted analogs at very high concentrations (10 −5 M) have no effect upon enzymic activity despite the fact that they totally block binding of the labeled enkephalin to receptors. The data strongly suggest that there is no correlation between receptor occupancy and rate of enkephalin degradation.

Biotinylated endothelin as a probe for the endothelin receptor

Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using 125I-labeled ET-1 revealed IC50 values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.