Ca2+-independent, inhibitory effects of cyclic adenosine 5′-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase c2α, Rho, and myosin phosphatase in vascular smooth muscle

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 myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca2+-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 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 alpha-isoform of phosphoinositide 3-kinase ( PI3K-C2 alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2 alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) 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, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca2+-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2 alpha, Rho, and Rho kinase in a manner independent of Ca2+ and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.