Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, Giessen, Germany.
Cardiovasc Res. 2010 Jul 15;87(2):375-84. doi: 10.1093/cvr/cvq065. Epub 2010 Mar 3.
Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied.
Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect.
cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.
cAMP 信号的激活可消除凝血酶诱导的通透性增加。这种保护作用的机制之一是使内皮收缩机制失活，内皮收缩机制是内皮屏障功能的主要决定因素之一，主要通过肌球蛋白轻链磷酸酶（MLCP）的激活来实现。迄今为止，cAMP 介导的 MLCP 激活机制仅部分被理解。本研究旨在研究两种 cAMP 效应器 PKA 和 Epac 在调节内皮收缩机制和屏障功能中的作用。
在培养的人脐静脉内皮细胞（HUVEC）中分析内皮收缩机制和屏障功能。使用 cAMP 类似物 8-CPT-cAMP 和 6-Bnz-cAMP 分别激活 Epac 和 PKA，并用 forskolin（FSK）激活腺苷酸环化酶。用凝血酶刺激细胞，通过 RhoA/Rock 途径抑制 MLCP。PKA 或 Epac 的激活部分阻断凝血酶诱导的通透性增加。PKA 和 Epac 的同时激活具有与 FSK 相当的相加作用。PKA 的激活而非 Epac 的激活抑制凝血酶诱导的 MLC 磷酸化和 MLCP 调节亚基 MYPT1 的磷酸化，部分通过抑制 RhoA/Rock 途径。FSK 通过去磷酸化和 PP1 抑制蛋白 CPI-17 的解离来激活 MLCP 催化亚基 PP1。FSK 减弱了凝血酶诱导的 CPI-17 磷酸化、CPI-17/PP1 复合物形成和 PP1 失活。CPI-17 的下调减弱了凝血酶诱导的通透性增加，并消除了 PKA 激活剂的拮抗作用，而 Epac 激活剂保留了其拮抗作用。
cAMP/PKA 通过抑制收缩机制来调节内皮屏障，主要通过抑制 CPI-17 和 RhoA/Rock 来激活 MLCP。cAMP/Epac 途径的降低通透性作用与 CPI-17 无关。
