enkephalin--leucine-2-alanine and fentanyl-isothiocyanate

The effects of fentanyl isothiocyanate (FIT) and pertussis toxin on the binding of [3H]D-Ala2, D-Leu5-enkephalin ([3H]DADLE) to rat brain membranes were compared. The site of action of pertussis toxin was confirmed by the labeling of a 41,000 dalton protein in the presence of [alpha-32P]NAD. Both reagents produced inhibition of [3H]DADLE binding when binding was assayed in 10 mM Tris-HCl buffer alone. FIT inhibited binding 91% whereas pertussis toxin treatment resulted in 27% inhibition. However, when binding was assayed in 10 mM Tris-HCl containing SMG (100 mM NaCl, 3 mM manganese acetate, and 2 microM guanosine triphosphate), inhibition due to both reagents was attenuated markedly: 66% for FIT and 5% for toxin. In addition, both reagents markedly potentiated enhancement of binding by SMG. Thus, the effects of FIT and pertussis toxin on [3H]DADLE binding were qualitatively similar. These results suggest that FIT and pertussis toxin affect binding of [3H]DADLE to the same population of delta receptors. This was further supported by the observation that treatment of membranes with FIT prior to pertussis toxin treatment blocked the effect of toxin on [3H]DADLE binding. FIT selectively eliminates the SMG-insensitive, mu-competitive [3H]DADLE binding site [Rothman et al., Neuropeptides 4, 201 (1984); Rothman et al., Molec. Pharmac. 27, 399 (1985)]. These results indicate that this site is coupled to G protein substrates for pertussis toxin and that it mediates the inhibitory effects of delta ligands on adenylate cyclase. The FIT-insensitive, SMG-sensitive mu-noncompetitive [3H]DADLE site appears not to be coupled to G protein substrates for pertussis toxin and may mediate some other biochemical effects of delta ligands.

Topics: Adenylate Cyclase Toxin; Alkylating Agents; Animals; Binding, Competitive; Brain; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Fentanyl; GTP-Binding Proteins; Intracellular Membranes; Isothiocyanates; Kinetics; Male; Pertussis Toxin; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Synaptosomes; Virulence Factors, Bordetella

Considerable evidence supports the notion that the prototypic delta agonist [3H]D-ala2-D-leu5-enkephalin labels two binding sites on brain membranes in vitro. Recent studies have demonstrated that treatment of brain membranes with the delta-selective, site-directed, alkylating agent, FIT (Rice et al., Science 220, 314-316, 1983) results in a membrane preparation devoid of detectable higher affinity [3H]D-ala2-D-leu5-enkephalin binding sites, but contain residual lower affinity binding sites at which mu-ligands are apparent noncompetitive inhibitors (Rothman et al., Neuropeptides 4:210-215, 1984). In this paper we extend these data by showing that although FIT eliminates the higher affinity binding site, it also alters the properties of the residual lower affinity binding sites.

Topics: Affinity Labels; Animals; Chemical Phenomena; Chemistry; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Fentanyl; In Vitro Techniques; Isothiocyanates; Oligopeptides; Rats; Receptors, Opioid; Receptors, Opioid, delta

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