Abstract

In a modified Krebs buffer at 37 degrees C, the selective mu agonist [3H] D-Ala2,MePhe4,Gly-ol5]enkephalin [( 3H]DAMGO) and the nonselective mu/delta agonist human [125I]beta-endorphin [( 125I]beta-endH) bound to rat striatal membranes with a Kd of about 7 and 5 nM and a Bmax of about 95 and 260 fmol/mg of protein, respectively, consistent with labeling of mu receptors by the former ligand and labeling of both mu and delta receptors by the latter. The binding of 2 nM [125I]beta-endH was displaced by unlabeled DAMGO (IC50 30 nM), [D-Ala2-D-Leu5]enkephalin (IC50 60 nM) as well as by the selective delta agonists [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET, IC50 500 nM) and Tyr-Ala-Phe-Asp-Val-Val-Gly-NH2 (IC50 700 nM) in a monophasic manner within 2 to 3 log concentration units, suggesting an allosteric interaction between mu and delta sites labeled by [125I]beta-endH under these conditions. Accordingly, 500 nM DSTBULET caused almost 40% inhibition of the apparent Bmax without changing the apparent Kd of [3H] DAMGO. The kappa agonist U 50,488 was ineffective as competing ligand even at a concentration of 10 microM. Upon affinity cross-linking of [125I]beta-endH (2 nM) to rat striatal mu- and delta-opioid receptors, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized tissue under reducing conditions followed by autoradiography of the dried gels revealed a major broad band of covalently labeled protein with an apparent molecular weight of 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)