Rac1 Stimulation in Adenosine-Induced Barrier Protection

项目总结（见说明）： 内皮细胞（EC）在血管内部空间和下面的组织之间形成半渗透屏障。在急性肺损伤中，EC屏障被削弱，导致渗透性增加。在调节EC屏障功能的机制中，Rho/VRad平衡似乎是非常重要的，RhoA激活是屏障破坏性的，而Rad激活是屏障保护性的。本项目将阐明Rad屏障保护作用的机制。我们发现，腺苷诱导快速增加的拉德激活，这与一个显着的衰减LPS诱导的EC渗透性在体外和体内。腺苷增强屏障功能的能力似乎依赖于MLCP，并与cAMP水平升高相关。然而，cAMP增加与Rad激活、MLCP刺激和屏障保护的机制联系尚不清楚。 因此，在这个项目中，我们建议研究一种新的机制，腺苷诱导的EC屏障增强/保护通过协调激活，和相互作用，Rad和MLCP介导的Epac 1-Rap 1-和蛋白激酶A（PKA）-信号。我们还将确定Rad和MYPT 1之间的相互作用是否参与调节MLCP激活，以及Epac 1-Rap 1和PKA在此过程中的作用。最后，我们将测试的假设，MLCP激活导致EC屏障增强/保护通过去磷酸化的新的细胞骨架蛋白：ERM蛋白（埃兹蛋白，根蛋白，膜突蛋白），和钙调蛋白。因此，本项目的总体目标是阐明 Rad通过其参与响应腺苷的EC屏障增强/保护的分子机制。具体目标如下： SA #1。确定腺苷诱导的Rad和MLCP激活之间的联系，并确定它们在体外和体内EC屏障保护中的作用SA #2。确定MLCP细胞骨架靶点在体外和体内参与放射性诱导的EC屏障增强/保护。

PROJECT SUMMARY (See instructions): Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury the EC barrier is weakened leading to increased permeability. Among the mechanisms regulating EC barrier function, Rho/VRad balance appears to be of major importance with RhoA activation being barrier-destructive and Rad activation is barrier-protective. This Project will elucidate the mechanisms underlying the barrier protective effects of Rad. We show that adenosine induces rapid increases in Rad activation and this correlates with a significant attenuation of LPS-induced EC permeability in vitro and in vivo. The ability of adenosine to enhance barrier function appears to be dependent on MLCP and correlates with increased levels of cAMP. However, the mechanistic links coupling increases in cAMP with the activation of Rad, MLCP stimulation and barrier protection are unknown. Thus, in this project we propose to investigate a novel mechanism of adenosine-induced EC barrier enhancement/protection via the coordinated activation, and interaction, of Rad and MLCP that is mediated by Epac1-Rap1- and protein kinase A (PKA)-signaling. We will also determine if the interactions between Rad and MYPT1 are involved in regulating MLCP activation and if the role of Epac1-Rap1 and PKA in this process. Finally, we will test the hypothesis that MLCP activation leads to EC barrier enhancement/protection via the dephosphorylation of the novel cytoskeletal proteins: the ERM proteins (ezrin, radixin, and moesin), and caldesmon. Thus, the overall objective of this project is to elucidate the molecular mechanisms through which Rad is involved in EC barrier enhancement/protection in response to adenosine. The detailed specific aims are: SA #1. To define the links between adenosine-induced Rad and MLCP activation and determine their role in EC barrier protection in vitro and in vivo SA #2. To define the involvement of MLCP cytoskeletal targets in Rad-induced EC barrier enhancement/protection in vitro and in vivo.