α1-adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PKII (CaMKII) activation in rat ventricular myocytes

alpha(1)-Adrenoceptor stimulation (alpha(1)ARS) modulates cardiac muscle contraction under physiological conditions by means of changes in Ca2+ current through L-type channels (I-Ca,I-L) and Ca2+ sensitivity of the myofilaments. However, the cellular mechanisms of a1ARS are not fully clarified. In this study, we investigated the role of Ca2+/calmodulin-dependent PK II (CaMKII) in the regulation Of ICa,L during a1ARS in isolated adult rat ventricular myocytes by using the perforated patch-clamp technique. CaMKII inhibition with 0.5 mu M KN-93 abolished the potentiation in I-ca,I-L observed during a1ARS by 10 mu M phenylephrine. In the presence of PKC inhibitor (10 mu M chelerythrine), the potentiation of I-Ca,I-L by phenylephrine also disappeared. In Western immunoblotting analysis, phenylephrine (>= mu M) 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 alpha(1)ARS, active CaMKII was exclusively located just beneath the plasmalemma. However, after alpha(1)ARS, active CaMKII was localized close to transverse tubules, where most of L-type Ca2+ channels are located. From these results, we propose that CaMKII, which exists near transverse tubules, is activated and phosphorylated by alpha(1)ARS and that CaMKII activation directly potentiates I-Ca,I-L in rat ventricular myocytes.