(+)‐PN200‐l 10 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) was 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 .times. 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 45 M, 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 .mu.M, and diltiazem increased it by 20% at 10 .mu.M. [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/106 cells. This figure, together with the plasma membrane ouabain/(+)-PN200-110 ratio of 80, allows the calculation of 1,884 (+)-PN200-110 binding sites/cell. Assuming a 1:1 stoichiometry, this means that intact chromaffin cells contain .apprx. 1.5 L-type, dihydropyridine-associated, voltage-dependent Ca2+ channels/.mu.m2 of surface area.