This project quantitates membrane excitation steps and Ca++ release which follows to trigger vascular muscle contraction. Intracellular Ca++ will be quantitated and localized by the birefringence signal intrinsic to the cells and by intracellular Ca++ indicators that will be introduced into the cells via liposomes or by tight seal pipettes. Extracellular Ca++ measurements will be made with optical indicators to indicate Ca++ influx to allow the fraction of Ca++ entering the cell to be separated from intracellular release in living cells. In addition to optical and electrical studies of activation, the role of Ca++ entry and voltage in activation of the contractile apparatus will be studied by manipulation of extracellular Ca++ and K+. Single ion channel recording by patch clamp techniques and whole single cell recording in voltage clamp will allow studies of Ca++ currents and K+ currents and provide information about the control of ion channels that determine membrane excitability. Those ion channels activated by intracellular Ca++ will be explored. The interaction between ouabain and other inhibitors of the Na+ pump with intracellular Ca++ will be studied. These measurements of ion currents will also allow the intracellular ion composition of vascular muscle cells to be controlled for the first time. Preparations include the neonatal rat azygous vein and adult rat cerebral and mesenteric arteries. In each case, studies of isolated single cells will be correlated with studies on pressurized perfused segments of the same blood vessels in vitro to allow cellular level mechanisms to be correlated with blood vessel excitation and contraction properties.
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