Role of Ca²⁺/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca²⁺ current during endothelin-1 stimulation

Endothelin-1 (ET-1) shows a positive inotropic effect on cardiac muscle. Although the L-type Ca²⁺ current (I_Ca) is one of the important determinants of cardiac excitation-contraction coupling, the effect of ET-1 on the I_Ca is not always clear. The controversial results appear to be due to different patch-clamp methods. The present study measured the effect of ET-1 on the I_Ca of rat ventricular myocytes using the perforated patch-clamp technique. The holding potential was set to -40 mV, and depolarization was applied every 10 s. ET-1 (10 nM) increased the I_Ca in a monophasic manner. The current reached a steady state 15 min after the application of ET-1, when the measurement was done. Endothelin receptor subtype expression was also investigated using Western immunoblotting. ET _A-receptor protein was expressed, but ET_B-receptor protein was not expressed, in the cell membranes of rat ventricular myocytes. The effect of ET-1 on the I_Ca was inhibited by a selective ETA-receptor antagonist, BQ-123, but not by a selective ET_B-receptor antagonist, BQ-788. The effect was inhibited by protein kinase C (PKC) inhibitor chelerythrine and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93, but not by its inactive analog KN-92. The effect of ET-1 was also blocked by another CaMKII inhibitor, autocamtide-2-related inhibitory peptide. These results suggest that ET-1 increases the I_Ca via the ET_A-receptor-PKC-CaMKII pathway.