Mechanisms of Heterocellular Signaling at the Myoendothelial Junction

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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.