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

Abstract

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca 2ϩ -dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca 2ϩ -induced Ca 2ϩ -sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 . In the present study, we investigated whether and how cAMP affected Ca 2ϩ -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 Ca 2ϩ mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca 2ϩ -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 and stimulated phosphorylation of MYPT1 at Thr 850 and CPI-17 at Thr 38 in manners dependent on both PI3K and Rhokinase, 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 Ca 2ϩ -mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2␣, Rho, and Rho kinase in a manner independent of Ca 2ϩ and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction. Ca2ϩ plays a primary role in the regulation of smooth muscle contraction (Somlyo and Somlyo, 1994). Excitatory receptor agonist stimulation and membrane depolarization induce an increase in the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) through phospholipase C-catalyzed generation of inositol-1,4,5-trisphosphate and/or gating of plasma membrane Ca 2ϩ channels, resulting in the activation of the Ca 2ϩ -and calmodulin-dependent enzyme myosin light-chain kinase (MLCK). Receptor agonists also activate Rho and Rho kinase, which leads to inhibition of myosin phosphatase (MLCP) through mechanisms involving Rho kinase-dependent phosphorylation of the MLCP-regulatory proteins MYPT1 and CPI-17 (Noda et al., 1995;Pfitzer, 2001;Somlyo and Somlyo, 2003;Hartshorne et al., 2004). CPI-17 is phosphorylated by the protein kinase C-dependent mechanism as well (Eto et al., 1995). We and others previously dem-

Enhanced Ca 2+ -Dependent Activation of Phosphoinositide 3-Kinase Class IIα Isoform-Rho Axis in Blood Vessels of Spontaneously Hypertensive Rats

“…11,22 We tested the effects of WMN infusion on blood pressure and vascular activities of PI3K-C2␣, Rho, and MLCP in SHRs. WMN substantially lowered systolic blood pressure with the maximal decline of Ϸ70 mm Hg at 10 minutes after infusion in 12-week-old SHRs ( Figure 5A).…”

Current Topics in the Regulatory Mechanism Underlying the Ca2+ Sensitization of the Contractile Apparatus in Vascular Smooth Muscle

“…2). The agonist-induced phosphorylation of CPI-17 has been shown to be inhibited by either RhoK inhibitors or PKC inhibitors, depending on the type of agonist and the vascular tissues (22,29,30). Furthermore, the contribution of RhoK and PKC to the CPI-17 phosphorylation differed with the phase of contraction, i.e., early vs late phase (9).…”

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

“…The key component of generating and sustaining vascular tone is the phosphorylation state of the smooth muscle contractile apparatus myosin light chain (MLC) (34). It has been reported that cAMP decreases MLC phosphorylation by activating the MLC phosphatase (MLCP) (3,4); however, to the best of our knowledge, a potential link between estrogen, cAMP, and MLC has not been investigated. In this study described here, we tested the hypothesis that GPER activation induces coronary relaxation by activating MLCP via cAMP/PKA signaling.…”