Ouabain and Descending Vasa Recta Ca2+ Signaling

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The microcirculation of the renal medulla traps NaCI and urea deposited to the interstitium by the loops of Henle and collecting ducts and distributes blood flow to a hypoxic region of the kidney. Evidence links medullary perfusion to regulation of salt and water excretion, hypertension and genesis of acute renal failure. Descending vasa recta (DVR) are 15 mu m arteriolar microvessels through which blood flow reaches the renal medulla. DVR vasoactivity is controlled by contractile pericytes and adjacent endothelia. We have established methods to study Ca 2+ signaling and channel architecture in those cells. Past studies have shown that endothelial Ca 2+signaling is stimulated by vasodilators and inhibited by angiotensin II. Preliminary data verifies that maneuvers affecting cellular Na+ (Na+/K + ATPase inhibition and extracellular Na + reduction) strongly modulate DVR endothelial Ca 2+. We will test the hypothesis that high ouabain affinity Na + pump isoforms and Na+/Ca 2+ exchange (NCX) modulate DVR endothelial Ca 2+signaling, participate in Angll signaling and become deranged in the chronic ouabain hypertensive rat. In Aim 1, we will test whether Na+K+ATPase high affinity isoforms (alpha2/alpha3) and NCX are sequestered in subplasmalemmal microdomains that affect Ca 2+signaling. We will examine colocalization of signaling proteins with SR/ER using immunofluorescence. We will determine whether ouabain inhibition, alpha2Na+ pump deficiency, reduction of extracellular Na+ ([Na+]e) and NCX blockade modulate DVR endothelial [Ca 2+]CYT responses to vasodilators. In Aim 2, we will use low affinity fluorescent probes (furaFF, furaptra) to determine the effect of the maneuvers in Aim 1 on store Ca2+ sequestration. In Aim 3, we will investigate the mechanisms responsible for Angll inhibition of DVR endothelial [Ca2+]CYT responses to sarcoplasmic / endoplasmic release Ca 2+(SERCA) pump inhibition and vasodilators. We will characterize Ca 2+ entry pathways in DVR endothelia and test whether Angll inhibits them. We will test for roles of NCX and Ca 2+ store sequestration in the Angll induced reduction of endothelial [Ca2+]CYT. In Aim 4 we will follow up our observation that DVR endothelial dysfunction accompanies chronic ouabain hypertension (OH) in the rat. We will test whether endothelial Ca 2+ responses and NO release are altered in OH rats and whether Na+ pump isoforms are down-regulated. We will test whether DVR contractions to norepinephrine, Angll and KCI are increased, measure NO production and assess effects of OH on endothelial and pericyte Ca2+ signaling.