84 日本生理学会 2007年3月20日〜22日(大阪

Ca
2+-independent, inhibitory effects of cyclic AMP on Ca2+ regulation of phosphoinositide 3-kinase C2, Rho and myosin phosphatase in vascular smooth muscle

Mohammed Ali Azam, Kazuaki Yoshioka, Shinsuke Ohkura, Noriko Takuwa, Naotoshi Sugimoto, Koichi Sato, and Yoh Takuwa

Dept. Physiol., Grad. Med., Kanazawa Univ.
ABSTRACT
We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca2+-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca2+-induced Ca2+-sensitization) through the mechanisms involving phosphorylation of MYPT1 and CPI-17. In the present study, we investigated whether and how cyclic AMP affected Ca2+-dependent MLCP inhibiton by examining the effects of forskolin, cell permeable dibutyryl cyclic AMP (dbcAMP) and isoproterenol. Forskolin, but not its inactive analogue 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20 kDa myosin light chain (MLC) phosphorylation without inhibiting Ca2+ mobilization in rabbit aortic VSM. DbcAMP mimicked these forskolin effects. We recently suggested that Ca2+-mediated Rho activation is dependent on class II -isoform of phosphoinositide 3-kinase (PI3K-C2). Forskolin inhibited KCl-induced stimulation of PI3K-C2 activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC (protein kinase C), and stimulated phosphorylation of MYPT1 at Thr850 and CPI-17 at Thr38 in manners dependent on both PI3K and Rho-kinase, but not PKC. Forskolin, dbcAMP and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, either forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced di-phosphorylation of MLC. These observations indicate that cyclic AMP inhibits Ca2+-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2, Rho, and Rho kinase in a manner independent of Ca2+, and point to the novel mechanism of the cyclic AMP actions in the regulation of vascular smooth muscle contraction.