title: Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes.
abstract: The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.
authors.affiliation: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio.
authors.forename: J F
authors.initials: JF
authors.lastname: French
authors.forename: M A
authors.initials: MA
authors.lastname: Matlib
mesh_terms: D000818:Animals; D001011:Aorta / Q000032:analysis; D001570:Benzodiazepinones / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000032:analysis*; D009568:Nitrendipine / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000032:analysis* / Q000502:physiology
chemical_list: D001570:Benzodiazepinones; D011963:Receptors, GABA-A; C028513:4'-chlorodiazepam; D009568:Nitrendipine