Cardiac Na⁺–Ca²⁺ exchanger current induced by tyrphostin tyrosine kinase inhibitors

Tyrosine kinase (TK) inhibitors genistein and tyrphostin A23 (A23) inhibited Ca(2+) currents in guinea-pig ventricular myocytes investigated under standard whole-cell conditions (K(+)-free Tyrode's superfusate; EGTA-buffered (pCa-10.5) Cs(+) dialysate). However, the inhibitors (100 microM) also induced membrane currents that reversed between -40 and 0 mV, and the objective of the present study was to characterize these currents. Genistein-induced current behaved like Cl(-) current, and was unaffected by either the addition of divalent cations (0.5 mM Cd(2+); 3 mM Ni(2+)) that block the Na(+)-Ca(2+) exchanger (NCX), or the removal of external Na(+) and Ca(2+). A23-induced current was independent of Cl(-) driving force, and strongly suppressed by addition of Cd(2+) and Ni(2+), and by removal of either external Na(+) or Ca(2+). These and other results suggested that A23 activated an NCX current driven by submembrane Na(+) and Ca(2+) concentrations higher than those in the bulk cytoplasm. Improved control of intracellular Na(+) and Ca(2+) concentrations was obtained by suppressing cation influx (10 microM verapamil) and raising dialysate Na(+) to 7 mM and dialysate pCa to 7. Under these conditions, stimulation by A23 was described by the Hill equation with EC(50) 68 +/- 4 microM and coefficient 1.1, tyrphostin A25 was as effective as A23, and TK-inactive tyrphostin A1 was ineffective. Phosphotyrosyl phosphatase inhibitor orthovanadate (1 mM) antagonized the action of 100 microM A23. The results suggest that activation of cardiac NCX by A23 is due to inhibition of genistein-insensitive TK.