The objective of this project is to test the hypothesis that platelets of patients with essential hypertension exhibit increased sensitivity to agonists that exert their effect through the Ca messenger system. This sensitivity would be expressed by a higher cytosolic free Ca (Cai) response. It may reflect a higher agonist mediated Ca entry or mobilization, inappropriate response of the Ca-ATPase and the Na-H antiport feedback system or both. To explore these alternatives, platelets and their plasma membrane fractions (i.e., the external plasma and dense tabular system membranes) from hypertensives and nonmotensives will be examined under basal conditions and after treatments by agonists and experimental perturbations that are known to: 1) raise Cai, 2) stimulate phospholipase C (PLC), and 3) activate the Na-H antiport via protein kinase C dependent and independent mechanisms. The role of extracellular and intracellular Ca in possible differences of Cai homeostasis between platelets from hypertensives and normotensives will be evaluated by performing experiments in the presence and absence of extracellular Ca, in the presence of Ca channel blockers and Ca ionophores, and after the quenching of cellular Ca. Kinetics of activation of Ca-ATPase in the external plasma membrane, activation of the Na-H antiport and measurement of Ca in intact platelets, will be performed using fluorescent methods. The fluorescent probes 2', 7'-bis (carboxyethyl)-5, 6'carboxyfluorescein (BCECF) and fura-2 will be respectively used for the measurements of cytosolic pH and Cai. Results of these studies will be instrumental in gaining a better insight, at the cellular level, into the pathophysiology of essential hypertension. Understanding the links between abnormalities in the cellular regulation of Cai and elevated blood pressure will aid in formulating the appropriate approaches to prevent and treat essential hypertension.
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