Mechanisms of Heterocellular Signaling at the Myoendothelial Junction

描述（由申请人提供）：阻力血管中的血管平滑肌细胞（VSMC）和内皮细胞（EC）在功能上是相连的，两个细胞之间的接触点，肌内皮连接（MEJ），在血管功能的许多要素中起着关键作用。然而，MEJ的位置和大小使得在体内研究极其困难。我们通过共培养VSMC和EC建立了MEJ模型，该模型显示了连接蛋白依赖的染料转移和Ca2+的耦合池。此外，细胞间钙信号传导的模式取决于两种细胞类型中的哪一种受到刺激，即细胞间第二信使信号是极化的，这似乎模拟了在体内看到的信号传导模式。我们建议确定发生在MEJ的细胞间偶联的结构和分子基础，我们认为这对MEJ在体内的运作具有重要意义。我们将确定哪些连接蛋白和第二信使参与细胞间信号传递，并将测试钙通讯的极化是否由MEJ间隙连接的选择性通透性或MEJ第二信使受体在两种细胞类型中的差异表达决定。我们提出了3个特定的实验目的：1 -使用基于光学和电子显微镜的免疫细胞化学来评估和比较体内和体外放置：a.)连接蛋白，b.) ryanodine受体，c.)肌醇1,4,5三磷酸受体在MEJ处；具体目标2 -测量间隙连接素组成的改变对以下方面的影响：Ca2+和b.)肌醇1,4,5三磷酸介导的细胞间信号传导；最后，特异性目标3 -评估细胞特异性缺失a) ryanodine受体和b)肌醇1,4,5三磷酸受体对极化钙信号传导的影响。我们的实验将加深对VSMC和EC协调的理解，并将为血管舒缩控制和各种病理生理反应的基本问题提供见解。血管细胞沟通的方法是理解血管过程的关键，如高血压和血流控制。我们的模型和提出的实验为研究肌内皮连接的能力和了解其在血管壁中的作用提供了第一次机会。我们建议研究血管细胞如何利用这种结构来维持血管功能。

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (VSMC) and endothelial cells (EC) in the resistance vessels are functionally linked, and the point of contact between the two cells, the myoendothelial junction (MEJ), plays a key role in many elements of vascular function. However, the location and size of the MEJ have made it extremely difficult to study in vivo. We have developed a model of the MEJ by co-culturing VSMC and EC that show connexin- dependent dye transfer and a coupled pool of Ca2+. Moreover, the mode of intercellular calcium signaling depends on which of the two cell types is stimulated, i.e. intercellular second messenger signaling is polarized, and this appears to mimic signaling patterns seen in vivo. We propose to determine the structural and molecular basis for the intercellular coupling that occurs at the MEJ, which we believe has important implications for the operation of the MEJ in vivo. We will determine which connexins and second messengers are involved in intercellular signaling, and will test whether polarization of calcium communication is determined by selective permeability of the gap junctions at the MEJ or differential expression of second messenger receptors at the MEJ in the two cell types. We propose 3 specific experimental aims: Specific Aim 1 - Use light and electron microscopy-based immunocytochemistry to assess and compare the in vivo and in vitro placement of: a.) connexins, b.) ryanodine receptors, and c.) inositol 1,4,5 triphosphate-receptors at the MEJ; Specific Aim 2 - Measure the effect of modification of the gap junctional connexin composition on: a.) Ca2+ and b.) inositol 1,4,5 triphosphate -mediated intercellular signaling; and lastly, Specific Aim 3 - Assess the effects of cell specific deletion of a) ryanodine receptors and b) inositol 1,4,5 triphosphate-receptors on polarized calcium signaling. Our experiments should enhance understanding of the coordination of VSMC and EC and will provide insights into basic questions of vasomotor control and a variety of pathophysiological responses. The methods by which vascular cells communicate are key for understanding vascular processes such as hypertension and control of blood flow. Our model and the experiments proposed offer the first opportunity to investigate the capabilities of the myoendothelial junction and to understand its role in the vessel wall. We propose to study the ways in which vascular cells utilize this structure to maintain vascular function.