alpha-adrenergic antagonists as possible calcium channel inhibitors.

Abstract

The effects of various organic Ca2+ channel inhibitors were investigated on the binding of the .alpha.1-antagonist 3H-labeled 2-[(2'',6''-dimethoxyphenoxyethyl)aminomethyl]-1,4-benzodioxane ([3H]WB-4101) to membranes from rat brain and neuroblastoma-glioma hybrid cells (NG108-15). As found by monitoring binding of [3H]WB-4101, the Ca2+ channel inhibitors methoxyverapamil (D600), verapamil and the nifedipine analog YC-93 [2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)]ethyl, 5-methyl ester.cntdot.HCl] bind to 2 different sites in rat brain: a high-affinity site (Kd = 2.9 nM and binding capacity B = 360 f[femto]mol/mg of protein) and a low-affinity site (Kd = 260 nM and B = 2700 fmol/mg of protein). In NG108-15 cells, where no .alpha.1 receptors were detected with [3H]WB-4101, the Ca2+ antagonists bind to nonadrenergic sites in the membrane with a capacity B = 976 fmol/mg of protein. The binding of Ca2+ antagonists to [3H]WB-4101 sites led to the investigation of WB-4101 as a Ca2+ inhibitor by electrophysiological techniques. WB-4101 depressed the amplitude and reduced the rate of rise of the Ca2+ spike with an affinity slightly greater than that observed for D600. The concentration for 50% inhibition of the Ca2+ spike amplitude was 48 .mu.M for WB-4101 and 80 .mu.M for D600. The WB-4101-induced blockade of the Ca2+ spike was antagonized by high Ca2+ concentrations, indicating a common site for Ca2+ and the .alpha.-antagonist. D600 and WB-4101 also inhibited voltage-dependent Na+ and K+ conductances. Ca2+ channels can account for a fraction of the sites labeled with [3H]WB-4101 in membrane preparations from brain and NG108-15 cells.