The central hypothesis of this proposal is that in vascular smooth muscle cells (VSMCs) of the spontaneously hypertensive rat (SHR), elevated intracellular Ca2+ (Cai2+) or increased Ca2+ turnover rate lead to increased Na+/H+ antiport activity. These perturbations in Ca2+ result in: a) increased metabolic demands leading to accelerated H+ production, and b) increased Ca2+-ATPase activity (Ca2+-H+ exchange) and the consequent enhanced H+ entry. Thus, increased Na+/H+ exchange in VSMCs of the SHR represents a regulatory mechanism that attenuates intracellular pH (pHi) acidification. Increased Na+/H+ antiport activity can be accomplished without cytosolic acidifications by an alkaline shift in the pH set point for activation of the exchanger. This shift in the pH set point and augmented transport by the exchanger may relate to elevated Cai2+, altered protein kinase C (PKC) activity, and/or effects of second messengers such as cyclic nucleotides and diacylglycerol (DAG). To investigate the role of these variables in the increased activity of the Na+/H+ antiport, the relation between intracellular signals and the exchanger in primary cultures of VSMCs will be examined in the adult SHR and two normotensive strains - Wistar Kyoto (WKY) and Wistar (WIS) rats. In some studies, Na+/H+ exchange in VSMCs from prehypertensive rats will be assessed to determine genetic and age related effects on antiport activity. Na+/H+ antiport activity in monolayers of VSMCs will be measured with the pH-sensitive fluorescent probe 2',7'-bis (carboxyethyl)-5,6- carboxyfluorescein (BCECF). Kinetic parameters of this transport system will be estimated from experiments that monitor the initial pHi recovery of Na+/H+ exchange. The effects of second messengers will be assessed during activation of the Na+/H+ antiport by cellular acidification, osmotic shrinkage, and alkalinization resulting from stimulation of PKC-dependent and -independent mechanisms. The profiles of Cai2+ and pHi will be compared in VSMCs from the three strains following treatment with agents that alter cellular cAMP, cGMP and DAG concentrations. These second messengers have been selected because of their ability to modulate Na+/H+ antiport activity. Cai2+ will be monitored with the fluorescent probe fura-2, under experimental conditions designed to manipulate Cai2+ levels. PKC activity will be correlated with Cai2+ and Na+/H+ antiport activity under the aforementioned conditions and experimental maneuvers. The main objective is to identify the specific factors that cause increased Na+/H+ antiport activity in primary hypertension. This information is required for initiating strategies for the prevention and/or management of essential hypertension.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Experimental Cardiovascular Sciences Study Section (ECS)
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University of Medicine & Dentistry of NJ
Schools of Medicine
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