Calcium Signaling in Vasa Recta Endothelium

描述（由申请人提供）：肾髓质的微循环通过亨利袢和集合管捕获沉积到间质的NaCl和尿素，并将血流分配到肾脏的缺氧区域。有证据表明髓质灌注与盐和水排泄的调节以及急性肾衰竭的发生有关。降直血管 (DVR) 是 15 μm 的小动脉微血管，血流通过它到达肾髓质。 DVR 血管活性由收缩周细胞和邻近内皮细胞控制。我们已经建立了研究这些细胞中 Ca 2 信号传导和通道结构的方法。过去的研究表明，内皮 Ca 2 信号传导受到血管扩张剂的刺激并受到血管紧张素 II 的抑制。初步数据证实，影响细胞 Na 的操作（Na /K ATP 酶抑制和细胞外 Na 减少）强烈调节 DVR 内皮 Ca 2 。我们将检验以下假设：高哇巴因亲和力 Na 泵亚型调节 DVR 内皮 Ca2 信号传导并参与 AngII 信号传导。在目标 1 中，我们将测试 Na K ATP 酶高亲和力异构体和 Na /Ca2 交换器是否被隔离在质膜下微域中并影响 Ca 2 信号传导。我们将使用免疫荧光检查信号蛋白与 SR/ER 的共定位。我们将使用整体细胞质和近膜 Ca2 探针来确定哇巴因抑制或细胞外 Na ([Na ]e) 的减少是否调节 DVR 内皮 [Ca2 ]CYT 对血管扩张剂的反应。在目标 2 中，我们将使用低亲和力荧光探针 furaFF 来确定类似操作对储存 Ca 2 封存的影响。在目标 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反应的机制。我们将描述 DVR 内皮细胞中 Ca2+ 进入途径的特征，并测试 Angll 是否抑制它们。我们将测试 Na /Ca 2 交换和 Ca 2 储存隔离以减少内皮 [Ca2 ]CYT 的作用。在目标 4 中，我们将跟踪我们的观察结果，即 DVR 内皮功能障碍伴随大鼠慢性哇巴因高血压 (OH)。我们将测试 OH 大鼠中内皮 Ca2+ 反应和 NO 释放是否改变以及 Na 泵亚型是否下调。我们将测试 DVR 对去甲肾上腺素、AngII 和 KCI 的收缩是否增加，测量 NO 的产生并评估 OH 对内皮和周细胞 Ca2 信号传导的影响。

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.