Calcium Signaling in Vasa Recta Endothelium

University Of Maryland Baltimore, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): 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 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 modulate DVR endothelial Ca2+ signaling and participate in Angll signaling. In Aim 1, we will test whether Na+K+ATPase high affinity isoforms and Na+/Ca2+ exchanger are sequestered in subplasmalemmal microdomains and affect Ca 2+ signaling. We will examine colocalization of signaling proteins with SR/ER using immunofluorescence. We will use global cytoplasmic and near membrane Ca2+ probes to determine whether ouabain inhibition or reduction of extracellular Na+ ([Na+]e) modulates DVR endothelial [Ca2+]CYT responses to vasodilators. In Aim 2, we will use low affinity fluorescent probe, furaFF to determine the effect of similar maneuvers on store Ca 2+ sequestration. In Aim 3, we will investigate the mechanisms responsible for Angll inhibition of DVR endothelial [Ca2+]CYT responses to sarcoplasmic/endoplasmic release Ca2+ (SERCA) pump inhibition and vasodilators. We will characterize Ca2+ entry pathways in DVR endothelia and test whether Angll inhibits them. We will test for roles of Na+/Ca 2+exchange and Ca 2+ store sequestration to reduce 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 Ca2+ 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.