Role of Ca(2+)/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca(2+) current during endothelin-1 stimulation.

Endothelin-1 (ET-1) shows a positive inotropic effect on cardiac muscle. Although the L-type Ca(2+) 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 ET(A)-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 Ca(2+)/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.