Parenchymal arterioles (PAs) play a critical role in assuring that appropriate local cerebral blood flow and perfusion pressure are maintained under a variety of conditions. This essential physiological function is regulated by tight communication among the various cells that form the cerebrovascular unit (endothelium, smooth muscle, and astrocytes) and clearly involves the dynamic regulation of intracellular [Ca^""""""""*] in each cell type. The focus of this project is on parenchymal arteriolar smooth muscle cell calcium (Ca^*) signaling, which is the ultimate determinant of myocyte excitability, vasomotor tone, and blood flow in the microcirculation of the brain. Voltage-dependent Ca^* (Cav) channels are central integrators of both vasodilator and vasoconstrictor stimuli in the cerebral circulation. In addition, Transient Receptor Potential (TRP) channels transduce vasoactive signals, including intravascular pressure and receptor activation, to directly and indirectly modulate intracellular Ca^* in the vasculature. However, virtually nothing is known about the functional contributions of these channels in PAs. Thus the overarching goal of this project is to reveal the molecular mechanisms of vascular control of PAs involving Cav and TRP channels. Based on our preliminary data we have formulated a model of excitation-contraction coupling in arteriolar smooth muscle in which E-C coupling is facilitated in two ways: 1) High activity of Ca^* entry pathways mediated or modulated by Cav, TRPC6, TRPM4, and TRPV4 channels, and by PCK, and 2) suppressed negative feedback input normally provided by Ca^* spark and BK channel activity. To assess the specific roles of Cav and TRP channels in generating Ca^* signals, Ca^* will be measured with fluorescent dyes using confocal and TIRF microscopy, approaches developed or implemented by our team. Vasomotor function will be measured in isolated, pressurized parenchymal arteriole segments, and ion channel function will be studied using patch clamp approaches. Information and insights gained will be coordinated with studies focused on normal endothelial cell function in PAs (M. Nelson, Project 1) and used to understand how arteriolar smooth muscle function, in general, and the expression and activity of various vasoconstrictor mechanisms, in particular, may be altered following ischemia and reperfusion (M. Cipolla, Project 3) and subarachnoid hemorrhage (G. Wellman, Project 4). Elucidation of the roles of TRP channels and their interaction with Cav channels in smooth muscle function and dysfunction as detailed in this project represents a unique opportunity to define a new set of pharmacologically relevant drug targets aimed at treating and preventing cerebrovascular disorders.
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