The long term goal of the proposed research is to provide a better understanding of the mechanism by which the endothelium modulates the reactivity of vascular smooth muscle. Although the role played by the vascular endothelium was not appreciated until recently, it is now readily apparent that this cell layer is an essential element in the control of vascular tone. It has recently been proposed that coupling of endothelium to smooth muscle cells may provide for direct electrical signalling between these two cell types. In addition, recent electrophysiologic data support the view that at least two types of ion channels in the endothelium act as transducers of hemodynamic forces to control the release of vasoactive substances. Thus, we propose to elucidate the electrophysiologic properties of endothelial cells to: characterize in greater detail the properties of the different outward and inward currents and the mechanism(s) of their regulation; quantitate the agonist mediated calcium influx through different pathways to establish whether these fluxes are adequate to account for the calcium transient observed; and identify whether differ- ences in properties exist in endothelial cells obtained from different vascular regions, so as to clarify the resulting functional implications. We therefore propose to address the following specific aims: (1) to characterize the different resting ionic currents in endothelial cells, (2) to relate the presence of a particular outward current to both the magnitude and time course of the evoked calcium transient and arachidonic acid released, (3) to characterize the electrophysiologic properties and basis of regulation of the different evoked inward and outward ionic currents in endothelial cells, (4) to ascertain the basis of the evoked calcium transient by measuring the magnitude and time course of the Ca2+ flux through the different pathways into the cytoplasm, (5) to determine if the electrophysiologic properties and response to different agonists are the same in endothelial cells obtained from the resistance vessels and microcirculation as those obtained from large arteries. While the immediate goals of this application are highly focused on examination of the electrophysiologic properties of endothelial cells and the biochemical coupling to vasoactive mediator release, we do not exclude the possibility that ion channels and ion transport systems play a larger role in the regulation of the biology of endothelial cells.
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