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

Abstract

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.

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

“…1A. In contrast to ␣ 1 -adrenoceptor stimulation (22), negative effect (a decrease in the I Ca ) was not apparent. After 15 min, ET-1 significantly increased the I Ca from 1.21 Ϯ 0.17 to 1.53 Ϯ 0.19 nA (28.8 Ϯ 6.8% increase compared with the current before stimulation, P ϭ 0.002, N ϭ 6), without changing the shape of currentvoltage relationship.…”

“…For measurement of the I Ca, the holding potential was set Ϫ40 mV to inactivate Na ϩ current, and 200-ms depolarization pulse to 0 mV was applied every 10 s. The amplitude of the current was defined as the difference between peak inward current and the current remaining at the end of pulses. The amplitude of ICa recorded with the perforated patch is stable (no significant run down and/or up) for up to 15 min (22,23). The holding current and remaining current were not altered throughout the experiments.…”

“…Protein concentration was determined by bicinchoninic acid methods. Each protein was separated by SDS-PAGE and analyzed by Western immunoblotting (22,23). Immunoreactive bands were visualized by the ECL-plus detection kit (GE Healthcare UK, Amersham, UK).…”

“…The patch-clamp amplifier EPC-7Plus (HEKA Electronik, Lambrecht/Pfalz, Germany) was controlled by a personal computer through an analog-to-digital interface (ITC-16, Instrutech, Long Island, NY). The data acquisition was performed using the Pulse Pulsefit software program (HEKA Electronik) (22,23). Electrodes were made from glass capillaries (Harvard Apparatus, Kent, UK) using an electric puller Model P-97 (Sutter Instrument, Novato, CA).…”

“…The ET A receptor is one of the G proteincoupled receptors, and it couples with G q , which is the same as AT 1 receptor and ␣ 1A -adrenoceptor. ␣ 1A -Adrenoceptor stimulation increases the I Ca via the activation of PKC and Ca 2ϩ / calmodulin-dependent protein kinase II (CaMKII) in rat ventricular myocytes (22,23). Therefore, we hypothesized that ET-1 increases I Ca via the same mechanism as ␣ 1A -adrenoceptor stimulation.…”