Effect of membrane depolarization on binding of [3H]nitrendipine to rat cardiac myocytes.

Binding of the dihydropyridine calcium antagonist [3H]nitrendipine was studied in cardiac myocytes incubated in normal and high potassium buffer so that we might examine the voltage dependence of dihydropyridine binding. Hearts were obtained from adult male Wistar rats, and isolated calcium tolerant myocytes were dissociated by enzymatic dispersion. Cells in 5.6 mM extracellular potassium showed specific binding of [3H]nitrendipine with KD 587 +/- 50 (mean +/- SE) pM and maximum receptor density, Bmax, 10.8 +/- 1.3 fmol/mg wet weight. Cells depolarized in 50 mM potassium showed no change in KD, 661 +/- 77 pM, but approximate doubling of Bmax 25.6 +/- 3.7 fmol/mg wet weight. Binding equilibrium was reached within 5 minutes at 37 degrees C, and the KD determined by kinetic analysis was in good agreement with KD determined by saturation experiments. Unlabeled nitrendipine and nifedipine completely inhibited [3H]nitrendipine binding with slope factors less than one, whereas verapamil and diltiazem only partially inhibited binding with slope factors substantially less than one. As a function of increasing extracellular potassium concentration from 2.4-54.1 mM, the number of nitrendipine-binding sites increased gradually 115%. Aconitine also produced a 58% increase in binding sites over a concentration range of 1-30 micrograms/ml. The potassium-induced changes in number of binding sites occurred rapidly and were rapidly reversible with changes in extracellular potassium concentration. There was no change in the number of [3H]nitrendipine-binding sites as a function of potassium or aconitine concentration in dead or digitonin-treated myocytes. We conclude that nitrendipine receptor density in viable myocytes is voltage dependent, but we detect no change in KD as a function of voltage.