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°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.