Kwen-Jen Chang

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.

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.

An immunohistochemical and radioimmunological study of the distribution of [met5]- and [leu5]-enkephalin in the gastrointestinal tract.

Abstract The identification of opiate-like substances in extracts of the gastrointestinal tract and nervous system of vertebrates suggests that the known endogenous opiate-like peptides [Met 5 ]- and [Leu 5 ]-enkephalin might have a role in neurotransmission. In this study the gastrointestinal tract of guinea-pigs, rats and hamsters was examined by the immunoperoxidase-bridge method using specific antisera raised against [Met 5 ]- and [Leu 5 ]-enkephalin. Immunostained nerve fibers were most numerous in Meissners plexus of the duodenum and in the circular muscle layer of the stomach and rectum of the guinea-pig. Nerve fibers in the guinea-pig esophagus and cardia of the stomach stained with [Met 5 ]- but not with [Leu 5 ]-enkephalin antiserum. Staining was not observed in any epithelial cells. The regional distribution of these peptides was also examined by radioimmunoassay of extracts of the gut of guinea-pigs and rats. The highest concentrations of [Met 5 ]- and [Leu 5 ]-enkephalin were found in extracts of guinea-pig duodenum at a ratio of 11:1, respectively. These findings provide evidence for an enkephalinergic nervous system in the gastrointestinal tract.

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, beta-casomorphins, [D-Ala2, D-Leu5]enkephalin and Sandoz peptide, FK 33-824, were examined by intracerebroventricular administration in rats. Their relative potencies in vivo were compared with their receptors binding activities. The receptors binding affinities were determined from the competition curves against [3H]naloxone binding in the absence and presence of sodium ions for morphine (micro) receptors and against 125I-[D-A1A2, D-Leu]enkephalin binding for enkephalin (delta) receptors. A good correlation between analgesic activity and morphine (micro) receptor but not enkephalin (delta) receptor binding affinity was obtained. These data extend the hypothesis that morphine (micro) receptors mediate the major portion of the analgesic activity of opioids.

The metabolic stability of the enkephalins

Abstract [Met 5 ] and [Leu 5 ]-enkephalins inhibit 3 H-naloxone binding to brain opiate receptors much more effectively at 4°C than at 25°C. At 25°C several protease inhibitors potentiate the action of the enkephalins. Bacitracin is the most effective of these. In the presence of 100 μg/ml of bacitracin, the potencies of [Met 5 ]- and [Leu 5 ]-enkephalins are similar to those at 4°C. Some protease inhibitors, such as TLCK and TAME, are effective by themselves in inhibiting the binding of 3 H-naloxone. Enkephalins with D-amino acids in the 2-position are equally effective at 0°C and at 25°C and their action is not potentiated by bacitracin. In particular, [D-Ala 2 ]-enkephalins do not seem to be significantly degraded by the membrane enzymes which destroy the natural enkephalins.

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.

Down regulation of δ but not μ opioid receptors in the hippocampal slice associated with loss of physiological response

Abstract In rat hippocampal slices, opioids potentiate the synaptic activation of pyramidal neurons as revealed by the shift to the left in the input-output curve constructed by plotting the population spike as a function of the field EPSP. The peak effect was obtained within 12–25 min with D-Ala 2 , D-Leu 5 -enkephalin (DADLE), morphiceptin and morphine. However, the effect of both peptides declined during constant superfusion. About 60% peak effect was lost after 1 hr superfusion with morphiceptin or after 4 hr with DADLE. In contrast, the effect of morphine gradually increased over a 4 hr incubation. Following superfusion of the slices for 4 hr in DADLE or morphine, or 1.5 hr in morphiceptin, the membrane particulate fractions were prepared from the homogenate of slices. Opiate receptor binding activities were measured with 125 I-DADLE (δ-receptors) and 125 I-FK 33824 (μ-receptors). A significant reduction in δ- but not μ-receptor binding was detected in slices treated with DADLE. This seems to correlate to the development of desensitization to DADLE. Neither μ-receptor nor δ-receptor binding activity was altered by the superfusion of morphine or morphiceptin despite the development of desensitization to morphiceptin. These data suggest that there are differences in the regulation of μ- and δ-receptors in hippocampus. The down-regulation of δ-receptors may result in desensitization to δ-agonists and a different mechanism may be responsible for desensitization to μ-agonists.

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.

The antiproliferative effects of staurosporine are not exclusively mediated by inhibition of protein kinase C

The effects of the kinase inhibitor staurosporine on mitogenesis in NIH/3T3 fibroblasts were characterized. In the presence of serum, staurosporine caused dose- and time-dependent inhibitions of [3H]thymidine incorporation into DNA (IC50 approximately 0.1 nM after 24 hr). Depletion of protein kinase C (PKC) by treatment of the cells with phorbol myristate acetate (PMA) for 24 hr did not affect the rate of DNA synthesis either in the absence or presence of staurosporine. Down-regulation of PKC did not affect the basal rate of [3H]thymidine incorporation in serum-starved cells, or mitogenesis in response to serum or epidermal growth factor (EGF). Proliferation in response to PMA, platelet derived growth factor (PDGF), insulin and fibroblast growth factor (FGF) was inhibited by PKC-depletion. Dose response curves for staurosporine-mediated inhibition of DNA synthesis were essentially parallel for insulin, EGF, FGF, PDGF and PMA; however, mitogenesis in response to serum was more resistant to staurosporine. Therefore, staurosporine appears to be equally effective in inhibiting mitogenesis induced by activation of PKC and by activation of receptor tyrosine kinases.

Opioid peptides have actions on the immune system

Several recent reports indicate that the nervous system may have a role in the regulation of the immune system. Some neuropeptides, endogenous opioid peptides in particular, have been found to regulate the maturation or activation of natural killer (NK) cells 1 , the growth of tumors 2 and the proliferation of T-lymphocytes 3 . In addition, non-opiate, but β-endorphin-specific binding sites have also been described on lymphocytes 4 and the terminal complex of human complement 5,6 . β-Endorphin seems to share some actions and antigenetic cross-reactivity with human leukocyte interferon 7–9 .