α(1)-Adrenoceptor stimulation potentiates L-type Ca(2+) current through Ca(2+)/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes

α(1)-Adrenoceptor stimulation (α(1)ARS) modulates cardiac muscle contraction under physiological conditions by means of changes in Ca(2+) current through L-type channels (I(Ca,L)) and Ca(2+) ensitivity of the myofilaments. However, the cellular mechanisms of α(1)ARS are not fully clarified. In this study, we investigated the role of Ca(2+)/calmodulin-dependent PK II (CaMKII) in the regulation of I(Ca,L) during α(1)ARS in isolated adult rat ventricular myocytes by using the perforated patch–clamp technique. CaMKII inhibition with 0.5 μM KN-93 abolished the potentiation in I(Ca,L) observed during α(1)ARS by 10 μM phenylephrine. In the presence of PKC inhibitor (10 μM chelerythrine), the potentiation of I(Ca,L) by phenylephrine also disappeared. In Western immunoblotting analysis, phenylephrine (≥1 μM) increased the amount of autophosphorylated CaMKII (active CaMKII) significantly, and this increase was abolished by CaMKII inhibition or PKC inhibition. Also, we investigated changes in the subcellular localization of active CaMKII by using immunofluorescence microscopy and immunoelectron microscopy. Before α(1)ARS, active CaMKII was exclusively located just beneath the plasmalemma. However, after α(1)ARS, active CaMKII was localized close to transverse tubules, where most of L-type Ca(2+) channels are located. From these results, we propose that CaMKII, which exists near transverse tubules, is activated and phosphorylated by α(1)ARS and that CaMKII activation directly potentiates I(Ca,L) in rat ventricular myocytes