The studies in this amended proposal test the hypothesis that acute increases in coronary flow in vivo due to widened pulsatile perfusion are modulated by endothelial-dependent cellular signaling via nitric oxide, prostacyclin, pH, Ca2+, and inhibitory G-protein changes. Simultaneous activation of vasoconstriction responses due to endothelin and/or oxygen free radical generation will also be tested. Studies will further test whether subacute (6-48 hour) exposure to physiologic increases in pulsatile perfusion activate molecular signaling pathways linked to vascular smooth muscle proliferation, and whether repeat short-term exposure induces adaptations that limit these responses. In vivo studies utilize a custom designed servopump system to control mean and pulsatile perfusion pressure within an isolated vascular bed (coronary or carotid vessels). Selective pharmacologic manipulations will be used to test individual mechanisms. Companion in vitro studies will measure endothelial Ca2+ and pH responses to normal and abnormal pulsatile flow, relate these changes to altered NO release, test the role of G-linked signaling pathways. Molecular signaling in response to pulsatile perfusion will be studied both in vivo, in pig carotid arteries, and in vitro, using porcine and bovine smooth muscle cells and aortic endothelial cells cultured inside custom made clear distensible (Silastic) tubing. These tubes can be exposed to hours to several days of physiologic pulsatile perfusion, with independent control over the flow rate, mean perfusion pressure, and pulse pressure, using a modified version of the same servopump system. Cells are imaged through the tubing, for histologic and histochemical staining analysis.
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