Phosphorylation of ventricular sarcolemmal membranes does not alter binding properties of nitrendipine

Isoproterenol increased contractility in isolated cat papillary muscles 2-fold with an EC50 of 6.3 X 10(-8) M. Nifedipine (3 X 10(-7) M) reduced contractility in control muscles by 43%; however, inotropic state was restored by isoproterenol with a comparable EC50 of 5 X 10(-8) M. To test the hypothesis that this effect might result from cAMP-dependent phosphorylation of a Ca2+ channel-associated protein, [3H]nitrendipine binding was used to probe the high-affinity 1,4-dihydropyridine site in either phosphorylated or dephosphorylated sarcolemmal vesicles. Kd and Bmax values for binding to phosphorylated sarcolemmal vesicles (0.14 +/- 0.027 nM and 479 +/- 62 fmol/mg protein, respectively) were not significantly different from control values P greater than 0.4). Similarly, dephosphorylation of sarcolemmal vesicles did not alter binding parameters. These data demonstrate that phosphorylation of sarcolemmal vesicles neither alters the binding affinity for [3H]nitrendipine nor promotes an interconversion of dihydropyridine-binding sites from high to low affinity or vice versa. While phosphorylation may regulate the slow Ca2+ channel, this is not reflected as changes in [3H]nitrendipine-binding parameters determined in vitro. Furthermore, the cyclic AMP-dependent phosphorylation state of sarcolemmal proteins does not appear to account for wide variations (more than 100-fold) between Kd values from binding studies and IC50 values determined in pharmacological investigations.