title: (+)-PN200-110 and ouabain binding sites in purified bovine adrenomedullary plasma membranes and chromaffin cells.
abstract: Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Departamento de Farmacologia, Universidad Autonoma de Madrid, Facultad de Medicina, Madrid, Spain.
authors.forename: C J
authors.initials: CJ
authors.lastname: Castillo
authors.forename: R I
authors.initials: RI
authors.lastname: Fonteríz
authors.forename: M G
authors.initials: MG
authors.lastname: López
authors.forename: K
authors.initials: K
authors.lastname: Rosenheck
authors.forename: A G
authors.initials: AG
authors.lastname: García
mesh_terms: D000313:Adrenal Medulla / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism; D017275:Isradipine; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D010069:Oxadiazoles; D010042:Ouabain; C038806:1,4-dihydropyridine; D017275:Isradipine