enkephalin--leucine-2-alanine and 2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole

This study examined the effect of beta-funaltrexamine (beta-FNA), an irreversible mu-receptor antagonist, on naltrexone-induced upregulation of mu-(mu cx + mu nex) and delta nex-opioid receptors. [The subscripts 'cx' and 'nex' denote binding sites 'in' (cx) and 'not in' (nex) the opioid receptor complex.] Rats were treated according to the following protocol. Two naltrexone or two placebo pellets were implanted subcutaneously in a nylon mesh on day 1. and were removed intact on day 8. Rats were given either saline or 20 nmol of beta-FNA in 10 microliters of saline (i.c.v.) on days 1, 3, 5 and 6, 60 min prior to implantation of the pellet. On day 9 frozen lysed-P2 membranes were prepared for assay of mu binding sites. In other experiments, membranes were depleted of mu-receptors by pretreatment with the site-directed acylating agent 2-(4-ethoxybenzyl)-l-diethylaminoethyl-5-isothiocyanatobenzimid azole.HCl (BIT) for assay of delta nex binding sites, using [3H] [D-ala2, D-leu5]enkephalin. The results demonstrated that beta-FNA did not upregulate the mu binding sites and also did not prevent naltrexone-induced upregulation of mu binding sites. Both beta-FNA and naltrexone increased the Bmax of delta nex binding sites and their effects were additive. These data suggest that the mechanism(s) responsible for antagonist-induced upregulation of opioid receptors are more complex than previously appreciated.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Isothiocyanates; Kinetics; Naltrexone; Narcotic Antagonists; Oxymorphone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Thiocyanates; Up-Regulation

Although it is widely accepted that radiolabeled prototypic delta receptor agonists label two binding sites in vitro, the mechanism by which mu ligands inhibit peptide binding as well as the identity of the binding sites remains unsettled (Rothman and Westfall, Mol. Pharmacol. 21:538-547, 1982 ; Bowen et al., Proc. Natl. Acad. Sci. U.S.A. 78:4818-4822, 1981). Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al., Science 220:314-316, 1983), we describe the preparation of membranes devoid of high affinity binding sites and demonstrate that the mu agonist oxymorphone noncompetitively inhibits the binding of [3H]DADL to the residual lower affinity binding sites.

Topics: Alkylating Agents; Allosteric Regulation; Animals; Brain; Cell Membrane; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Fentanyl; Isothiocyanates; Kinetics; Male; Oxymorphone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Thiocyanates

It is widely accepted that the prototypic delta agonist DADL (D-ala2-D-leu5-enkephalin) labels two binding sites in vitro. Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al.. Science 220:314-316, 1983), we recently described (Rothman et al, Neuropeptides, in press) the preparation of membranes possessing only lower affinity 3H-DADL binding sites (FIT-treated membranes, type-I delta sites) as well as membranes greatly enriched with higher affinity binding sites (BIT-treated membranes, type-II delta sites). In this paper we report that ionic conditions differentially affect the binding of 3H-DADL to FIT- and BIT-treated membranes, supporting the notion that 3-H-DADL labels two distinct delta binding sites.

Topics: Animals; Brain; Calcium; Cell Membrane; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Fentanyl; Isothiocyanates; Kinetics; Manganese; Osmolar Concentration; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Sodium; Thiocyanates

In this paper we examine the binding of [3H]FOXY (tritiated-6-beta-fluoro-6-desoxy-oxymorphone) to membranes of rat brain. Using the site-directed alkylating agents BIT and FIT, evidence is presented that [3H]FOXY selectively labels mu opiate binding sites in vitro. Further, BIT and FIT did not significantly affect [3H]bremazocine binding to kappa receptors. Scatchard plots of [3H]FOXY binding were somewhat curvilinear, suggesting the presence of two classes of mu binding sites. At concentrations up to 19 nM, 90 percent of the total binding was specific. The combination of high mu-selectivity and low nonspecific binding suggests the [3H]FOXY may prove to be a powerful tool for studying the opiate mu receptor.

Topics: Animals; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Fentanyl; Hydromorphone; Isothiocyanates; Kinetics; Oxymorphone; Rats; Receptors, Opioid; Receptors, Opioid, mu; Thiocyanates; Tomography, Emission-Computed