Intercellular Communication in Microvessels

在这次更新中，我们建议为连接蛋白在微血管中细胞间通讯中的作用建立一个更机械的基础。我们将研究小动脉的径向和纵向细胞-细胞通讯，强调平滑肌和内皮细胞通过肌内皮细胞连接而形成一个功能单位的想法。这一应用充分利用了我们在体外和体内系统中使用钙和电压成像方面的广泛理论和技术发展。我们将系统地测试这一想法，即平滑肌和内皮的钙池的联系对小动脉的行为赋予了独特的反应模式。这一建议的一个独特方面是对三个已知具有完全不同微动脉反应的物种的微动脉进行比较研究。利用分子遗传学的工具，我们还开始了识别缝隙连接通讯的特定分子贡献者的过程。我们将包括连接蛋白37和40基因敲除的实验，以及新开发的连接蛋白43的平滑肌和内皮细胞特异性基因敲除的实验。我们试图回答三个广泛的问题。首先，平滑肌和内皮细胞之间的细胞-细胞偶联如何调节小动脉的反应性？第二，参与传导的血管舒缩反应的细胞间通路是什么？第三，缝隙连接交流在生理上重要吗？几个相互关联的假设将得到检验。它们是：假设1-膜电位和钙池的肌内皮细胞偶联在决定局部对刺激的反应中起关键作用假设2-膜电位的变化而不是内皮细胞钙的变化介导传导的血管运动反应的肌内皮偶联假说3-体外和体内存在相似的细胞-细胞偶联途径假说4-内皮细胞作为染料和电偶联的优先途径发挥作用。假说5-细胞间通讯由多个连接蛋白介导假说6-缝隙连接通讯在建立正常的血管舒缩张力中起作用

In this renewal we propose to establish a more mechanistic basis for the roles of the connexins in cell-cell communication in the microvasculature. We will study radial and longitudinal cell-cell communication in the arterioles, emphasizing the idea that the smooth muscle and the endothelial cell are united to form a functional unit via the myoendothelial cell junctions. The application takes advantage of our extensive theoretical and technical developments in the use of calcium and voltage imaging on in vitro and in vivo systems. We will systematically test the idea the linkage of the calcium pools of the smooth muscle and the endothelium confers unique response patterns on the behavior of arterioles. A unique aspect of this proposal is a comparative study of the arterioles from three species known to have quite different arteriolar responses. Using the tools of molecular genetics we have also begun the process of the process of identification of specific molecular contributors to gap junctional communication. We will include experiments on connexin 37 and 40 knockouts and newly developed smooth muscle and endothelial-cell specific knockouts of connexin 43. We seek to answer three broad questions. First, How does cell-cell coupling between smooth muscle and endothelial cell modulate arteriolar reactivity? Second, what are the intercellular pathways that are involved in the conducted vasomotor response? Third, is gap junctional communication physiologically important? Several interrelated hypotheses will be tested. These are: Hypothesis 1 - Myoendothelial coupling of both membrane potential and calcium pools play a critical role in determining local responses to stimulation Hypothesis 2 - Myoendothelial coupling of changes in membrane potential and not changes in the endothelial cell calcium mediate the conducted vasomotor responses Hypothesis 3 - Similar pathways for cell-cell coupling exist in vitro and in vivo Hypothesis 4 - The endothelium functions as a preferential pathway for dye and electrical coupling. Hypothesis 5 - Intercellular communication is mediated by multiple connexins Hypothesis 6- Gap junctional communication plays a role in the establishment of normal vasomotor tone