Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity

1 Tyrosine kinases have been proposed as regulators of voltage-operated cal cium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I-Ba) and pp60(c-src) kinase (c-src) activity in vitro. 2 I-Ba was measured in single myocytes isolated from rabbit ear artery by c onventional whole cell voltage-clamp techniques. The activity of purified h uman c-src was measured in vitro using a nonradioactive assay. 3 Bath application of tyrphostin-23 and genistein (non-selective PTK inhibi tors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src fa mily-selective inhibitor) inhibited IBa in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of pep tide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor pot ency series against I-Ba was PPI > genistein > tyrphostin 23 > bistyrphosti n. 4 Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state ina ctivation curves in a hyperpolarized direction without altering their slope . The inhibitors had no significant effects on IBa activation calculated fr om current-voltage relationships. 5 The agents inhibited c-sre activity in a concentration-dependent manner. The order of potency was PPI > genistein > peptide-A > tyrphostin-23 > bistyrphostin. The IC50 for inhibition of c-s rc activity was similar to the IC50 for inhibition of IBa in all cases. 6 Western blot analysis with a specific antibody to c-src showed the presen ce of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7 A range of structurally dissimilar inhibitors of PTKs inhibit I-Ba and c- src activity with similar potency. These data provide further evidence impl icating endogenous c-src in the modulation of L-type calcium channels in va scular smooth muscle cells.