Characterization of binding of the Ca++ channel antagonist, [3H]nitrendipine, to guinea-pig ileal smooth muscle.

A chemically heterogeneous group of compounds, the Ca++ channel antagonists, which includes verapamil, diltiazem and nifedipine inhibits excitation-contraction coupling in smooth and cardiac muscle by blocking Ca+ entry at a specific class of Ca++ channels. The binding of the nifedipine analog, [3H]nitrendipine, to a microsomal fraction from guinea-pig longitudinal smooth muscle has been characterized. Specific binding was saturable, linear with protein concentration and reversible. The apparent equilibrium dissociation constant was 1.63 +/- 0.06 X 10(-10)M and the maximum site density was 1.13 +/- 0.03 pmol/mg of protein determined from Scatchard analysis of equilibrium binding at 25 degrees C. Inhibition of binding was specific and stereoselective for Ca++ channel antagonist drugs and was unaffected by a variety of receptor active ligands. Correlations between binding and inhibition of mechanical response to methylfurmethide- and K+-stimulation in a series of nifedipine analogs were determined. A 1:1 correlation was found for the K+ tonic response, but for the phasic component of the K+ response and for both components of the methylfurmethide response the antagonists were more active as inhibitors of [3H]nitrendipine binding than as inhibitors of mechanical response. [3H]Nitrendipine binding was sensitive to other Ca++ channel antagonists including verapamil, D-600, diltiazem, flunarizine, lidoflazine and bepridil. Interaction with these agents suggests, consistent with previous reports, that more than one binding site for Ca++ antagonists exists. A variety of inorganic divalent and trivalent cations (Mn++, Co++, Ni++, Pb++, UO2++, Zn++, Cd++, Cu++, Tm+++ and La+++) inhibit specific [3H]nitrendipine binding. The data suggest that [3H]nitrendipine binding in smooth muscle is to a site which mediates the pharmacologic response.