Ca²⁺-independent, inhibitory effects of cyclic AMP on Ca²⁺ regulation of phosphoinositide 3-kinase C2α, Rho and myosin phosphatase in vascular smooth muscle

Abstract

We have recently demonstrated in vascular smooth muscle (VSM) that high KCl induces Ca²⁺-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca²⁺-induced Ca²⁺-sensitization) through phosphorylating MYPT1 and CPI-17. 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 inhibited KCl-induced contraction and the 20 kDa myosin light chain (MLC) phosphorylation without inhibiting Ca²⁺ mobilization in VSM. DbcAMP mimicked these forskolin effects. Ca²⁺-mediated Rho activation is dependent on phosphoinositide 3-kinase C2α (PI3K-C2α). Forskolin inhibited KCl-induced stimulation of PI3K-C2α activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC, 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. Thus, cyclic AMP prevents Ca²⁺-mediated MLCP inhibition through mechanisms involving Ca²⁺-independent PI3K-C2α suppression. [J Physiol Sci. 2007;57 Suppl:S66]