ADRENAL-MEDULLARY OPIATE RECEPTORS - PHARMACOLOGICAL CHARACTERIZATION IN BOVINE ADRENAL-MEDULLA AND A HUMAN PHEOCHROMOCYTOMA

Abstract

The opiate binding sites on the membranes of bovine adrenal medulla and human pheochromocytoma were characterized using 3H-labeled D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC). Binding was stereoselective in both membrane preparations. Association and dissociation kinetics showed that steady state was achieved after 20-25 min of incubation at 37.degree.. Saturation experiments were performed in the absence or in the presence of morphiceptin (1 .mu.M), which masks the .mu. sites, D-Ser2-Leu-enkephalin-Thr6 (100 nM), which masks .delta. sites or DADLE (5 .mu.M), which masked the .delta., .mu. and benzomorphan receptor. Taking into consideration the affinities of the 3 radioligands used (DADLE identifying the .delta. and .mu. sites when used in the nmol range, etorphine identifying the .delta., .mu. and benzomorphan sites and EKC identifying the .delta., .mu., .kappa. and benzomorphan receptors), the opiate sites present on bovine and human membrane were pharmacologically characterized. Human pheochromocytoma membranes contained .mu. binding sites (15 fmol/mg of protein, Kd [3H]etorphine 1.0 nM, [3H]EKC 5.4 nM, [3H]DADLE 5.6 nM); .kappa. sites (41 fmol/mg of protein, Kd [3H]EKC 1.0 nM); and benzomorphan sites (115 fmol/mg of protein, Kd [3H]etorphine and [3H]EKC 1.0 nM). On bovine membranes the following were detected. .delta. binding sites (10 fmol/mg of protein, Kd [3H]DADLE 0.7 nM); .mu. sites (24 fmol/mg of protein, Kd [3H]DALDE 2.9 nM, [3H]etorphine 0.2 nM [3H.EPSILON.KC 3.4 nM); .kappa. sites (12 fmol/mg of protein, Kd[3H]EKC 0.4 nM); benzomorphian sites (80 fmol/mg of protein, Kd [3H]etorphine 0.2 nM, [3H]EKC 1.3 nM); and a residual high-affinity (20 fmol/mg of protein, Kd 0.2 nM) site identified by [3H]etorphine in the presence of 5 .mu.M DADLE. The relative proportions of benzomorpham sites were equal in both tissues (65% of the high-affinity sites) whereas .kappa.-receptors were more abundant on human membranes (25%) than on bovine membranes (9% of the high-affinity sites).