application) The long-term goal of this proposal is to elucidate mechanisms that underlie altered membrane function associated with the pathogenesis of hypertension. It will focus on altered Ca-regulation in vascular smooth muscle (VSM) from aldosterone-salt hypertensive rats (AHR), and the role played by the superficial buffer barrier (SBB) and high conductance Ca- activated channels (BKCa) as part of a negative feedback mechanism to control membrane excitability. This proposal will test the hypothesis that aldosterone-salt treatment modulates the expression of BKCa leading to increased K currents (IK) under basal and depolarizing conditions. Single cells from aorta and femoral arteries will be used for function measures of: BKCa activity by isolated and cell attached patch-clamp techniques, BKca number and distribution by digital imaging fluorescent microscopy of a charybdotoxin - 19 cysteine mutant (CTX-R19C) fluorophore conjugate, and cellular Ca by means of fluorescent probes selective for cytosol (Fura-2 and Fura-red) and sarcoplasmic reticulum (DiOC6). Intact aorta and femoral rings will be used to measure integrated contractile and Ca responses.
The Specific Aims will: 1) determine parameters that govern the probability of BKCa openings; 2) determine the number of BKCa per cell and their distribution relative to sarcoplasmic reticulum (SR); 3) determine the frequency, time course and magnitude of spontaneous increases in Ca (Ca sparks) and to relate these to spontaneous transient outward currents (STOCs); 4) determine the rate of SR Ca unloading from cells after exposure to conditions (e.g. depolarization) that significantly increase SR Ca stores; and, 5) determine the effects of protein kinase and phosphatase inhibitors on BKCa parameters in the absence of agonist stimulation. Achievement of these aims will provide definitive information concerning mechanisms that underlie increased BKCa activity in VSM from AHR, and the modulatory role it has with SBB in membrane regulation during hypertension. Important information will also be forthcoming concerning altered Ca metabolism at the membrane and subcellular level which will allow us to dissect cause and effect processes in the pathogenesis of hypertension. Moreover, this model which appears to be critically dependent on SBB will allow us to test concepts that have wide applicability to other cell systems which have significant interactions between plasma and SR membranes.
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