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）在功能上相连，两种细胞之间的接触点，即肌内皮连接（MEJ），在血管功能的许多要素中起关键作用。然而，MEJ的位置和大小使得在体内研究非常困难。我们通过共培养VSMC和EC建立了MEJ模型，显示连接蛋白依赖性染料转移和Ca 2+偶联池。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被刺激，即细胞间第二信使信号传导被极化，这似乎模拟了体内观察到的信号传导模式。我们建议，以确定发生在MEJ，我们认为有重要的影响，在体内的MEJ操作的细胞间耦合的结构和分子基础。我们将确定哪些连接蛋白和第二信使参与细胞间信号传导，并将测试钙通讯的极化是否由MEJ处的差距连接的选择性渗透性或两种细胞类型中MEJ处第二信使受体的差异表达决定。我们提出了3个具体的实验目标：具体目标1 -使用基于光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.）兰尼碱受体，和c.）具体目标2 -测量差距连接蛋白组成的修饰对以下的影响：a.）Ca 2+和B.）最后，具体目标3 -评估a）兰尼碱受体和B）肌醇1，4，5三磷酸-受体的细胞特异性缺失对极化钙信号传导的影响。我们的实验应该提高对VSMC和EC协调的理解，并将为血管扩张控制和各种病理生理反应的基本问题提供见解。血管细胞的通讯方法是理解血管过程的关键，如高血压和血流控制。我们的模型和实验提出的第一个机会，调查的能力，肌内皮连接，并了解其在血管壁中的作用。我们建议研究血管细胞利用这种结构来维持血管功能的方式。

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.