Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2011 May 13;286(19):16681-92. doi: 10.1074/jbc.M110.205062. Epub 2011 Mar 25.
Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.
激动剂激活小分子 GTP 酶 RhoA 及其效应物 Rho 激酶导致平滑肌 (SM) 肌球蛋白轻链磷酸酶活性下调，肌球蛋白轻链 (RLC(20)) 磷酸化和力增加。环核苷酸可以逆转这个过程。我们报告了一种新的 cAMP 介导的松弛机制，通过 Epac，一种小分子 GTP 酶 Rap1 的 GTP 交换因子，导致 Rap1 活性增加和 RhoA 活性抑制。一种 Epac 选择性 cAMP 类似物，8-pCPT-2'-O-Me-cAMP("007")，显著降低了激动剂诱导的收缩力、RLC(20)和肌球蛋白轻链磷酸酶磷酸化，无论是在完整的还是通透的血管、肠道和气道 SM 中，都独立于 PKA 和 PKG。血管扩张剂 PGI(2)类似物西卡前列素在完整的 SM 中增加了 Rap1 活性并降低了 RhoA 活性。forskolin、磷酸二酯酶抑制剂异丁基甲基黄嘌呤和异丙肾上腺素也显著增加了大鼠主动脉 SM 细胞中的 Rap1-GTP。PKA 抑制剂 H89 对 007 诱导的 Rap1-GTP 增加没有影响。在 WT 但不是 Rap1B 缺失成纤维细胞中，用 007 处理可降低溶血磷脂酸诱导的 RhoA 活性，这与 Epac 通过 Rap1B 信号转导来下调 RhoA 活性一致。在大鼠主动脉 SM 细胞中沉默 Epac1 可抑制异丙肾上腺素诱导的 Rap1 活性增加。有证据表明，cAMP 激活 PKA 和 Epac 的协同作用有助于 SM 的松弛。我们的研究结果表明，一种 cAMP 介导的信号机制，即 Epac 的激活导致 PKA 独立、Rap1 依赖性的 SM 力的 Ca(2+)脱敏，通过下调 RhoA 活性。环 AMP 对 RhoA 的抑制是通过激活 Epac 和 PKA 介导的。
