Calcium Signaling in Vasa Recta Endothelium

描述（由申请人提供）：肾髓质的微循环捕获通过Henle袢和集管沉积到间质中的NaCI和尿素，并将血流分配到肾的缺氧区域。有证据表明髓质灌注与盐和水排泄的调节和急性肾衰竭的发生有关。直降血管（DVR）是15 μ m的小动脉微血管，血流通过它到达肾髓质。DVR血管活性受收缩的周细胞和邻近的内皮细胞控制。我们已经建立了研究这些细胞中的ca2 +信号和通道结构的方法。过去的研究表明，血管扩张剂可以刺激内皮细胞ca2 +信号，而血管紧张素II可以抑制内皮细胞ca2 +信号。初步数据证实，影响细胞Na+的运动（Na+/K+ atp酶抑制和细胞外Na+还原）强烈调节DVR内皮ca2 +。我们将验证高瓦巴因亲和Na+泵异构体调节DVR内皮Ca2+信号并参与Angll信号传导的假设。在Aim 1中，我们将测试Na+K+ atp酶高亲和异构体和Na+/Ca2+交换器是否被隔离在质下微域并影响Ca2+信号传导。我们将使用免疫荧光检测信号蛋白与SR/ER的共定位。我们将使用全局细胞质和近膜Ca2+探针来确定瓦巴因抑制或减少细胞外Na+ ([Na+]e)是否会调节DVR内皮细胞对血管扩张剂的反应。在Aim 2中，我们将使用低亲和力荧光探针furaFF来确定类似操作对存储ca2 +封存的影响。在Aim 3中，我们将研究Angll抑制DVR内皮[Ca2+]CYT对肌浆/内质释放Ca2+ (SERCA)泵抑制和血管扩张剂的反应的机制。我们将表征Ca2+进入DVR内皮的途径，并测试Angll是否抑制它们。我们将测试Na+/ Ca2+交换和Ca2+储存封存在减少内皮细胞[Ca2+]CYT中的作用。在Aim 4中，我们将继续观察大鼠慢性瓦巴因高血压（OH）并发DVR内皮功能障碍的情况。我们将测试内皮Ca2+反应和NO释放是否在OH大鼠中改变，以及Na+泵异构体是否下调。我们将测试DVR对去甲肾上腺素、Angll和KCI的收缩是否增加，测量NO的产生，并评估OH对内皮细胞和周细胞Ca2+信号的影响。

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.