Calcium Signaling in Vasa Recta Endothelium

描述（由申请方提供）：肾髓质的微循环通过Henle袢和集合管捕获沉积到肾小管的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。直降血管（DVR）是直径为15 μ m的微动脉血管，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内皮细胞控制。我们已经建立了研究这些细胞中Ca 2+信号和通道结构的方法。过去的研究表明，内皮细胞Ca 2+信号被血管扩张剂刺激，被血管紧张素II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+还原）强烈调节DVR内皮Ca 2+。我们将检验高哇巴因亲和力Na+泵同种型调节DVR内皮Ca 2+信号传导并参与AngII信号传导的假设。目的1：探讨Na+K+ ATP酶高亲和力亚型和Na+/Ca 2+交换体是否存在于质膜下微区，并影响Ca 2+信号转导。我们将使用免疫荧光检查信号蛋白与SR/ER的共定位。我们将使用全球细胞质和近膜Ca 2+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内皮细胞[Ca 2 +]CYT对血管扩张剂的反应。目的二是利用低亲和力荧光探针furaFF来检测类似的操作对钙库固定的影响。在目的3中，我们将研究负责AngII抑制DVR内皮[Ca 2 +]CYT对肌质/内质释放Ca 2+（SERCA）泵抑制和血管舒张剂的响应的机制。我们将表征DVR内皮中的Ca 2+进入途径并测试AngII是否抑制它们。我们将测试Na+/Ca 2+交换和Ca 2+储存螯合在降低内皮细胞[Ca 2 +]CYT中的作用。在目的4中，我们将跟踪我们的观察，DVR内皮功能障碍伴随慢性哇巴因高血压（OH）大鼠。我们将测试是否内皮细胞的Ca 2+反应和NO的释放在OH大鼠的改变，是否Na+泵亚型下调。我们将测试DVR对去甲肾上腺素、AngII和KCl的收缩是否增加，测量NO产生并评估OH对内皮和周细胞Ca 2+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.