Hemodynamic shear stress acts through the endothelium to modulate basal vascular tone by endothelial production of vasoactive factors, including nitric oxide (NO). However, our understanding of how the endothelium transduces shear stress into NO production is quite limited. The Central Hypotheses of the proposal is that increases in shear stress lead to both Ca2+-dependent and - independent regulation of NO production; the former are mediated through ion channels, the latter are mediated primarily by regulation of intracellular kinase systems. To test this hypothesis, two preparations will be used to study the endothelium of porcine coronary arterioles (100-200 mum, ID): 1) cultured endothelial cells will be superfused in a novel chamber that allows shear-stress to be controlled while single cells are patch clamped and NO is continuously measured with a polarigraphic microelectrode; 2) isolated, cannulated arteriolar segments will be perfused to control shear stress while the biological response to NO (vasodilation) is measured. A variety of techniques will be employed in these studies: endothelial cell [Ca2+]i and pHi changes will be measured using fluorescent dyes; membrane potential and ionic currents will be measured using patch clamp techniques.
Three specific aims are proposed: A. To determine the role of K+ and Ca2+ channels in the initial, Ca2+-dependent phase of shear stress-induced NO production. B. To determine how changes in PKC and pHi regulate the sustained, Ca2+- independent phase of NO production in response to shear stress. C. To determine how endothelial cell interactions with the extracellular matrix regulate sustained NO production in response to shear stress. Data from these studies will provide essential information regarding the mechanisms of shear-stress induced NO release from endothelium, and the results will be particularly relevant to the coronary microcirculation. Endothelium-derived NO production is impaired in a number of pathological states, particularly in the heart, and this study will be an important step in the future development of therapeutic agents targeted specifically at the endothelium.
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