Calcium Signaling in Vasa Recta Endothelium

描述(申请人提供)：肾髓质的微循环通过Henle环和集合管将NaCI和尿素沉积到间质中，并将血流分配到肾脏的缺氧区。有证据表明，骨髓灌流与盐和水排泄的调节以及急性肾功能衰竭的发生有关。直肠降血管(DVR)是一种15微米长的微血管，血流通过这些微血管到达肾髓质。DVR的血管活动由收缩的周细胞和邻近的内皮控制。我们已经建立了研究这些细胞中钙信号和通道结构的方法。过去的研究表明，血管扩张剂刺激内皮细胞钙信号，而血管紧张素II则抑制内皮细胞钙信号。初步数据证实，影响细胞Na(Na/K-ATPase抑制和细胞外Na还原)的动作强烈地调节DVR内皮细胞的钙。我们将验证高哇巴因亲和力钠泵异构体调节DVR内皮细胞钙信号并参与Ang11信号转导的假设。在目标1中，我们将测试Na-K-ATPase高亲和力异构体和Na/Ca~(2+)交换器是否隔离在亚质膜微区并影响钙信号转导。我们将使用免疫荧光来检测信号蛋白与SR/ER的共存。我们将使用整体细胞质和膜附近的钙探针来确定哇巴因抑制或减少细胞外Na([Na]e)是否调节DVR内皮[钙]细胞对血管扩张剂的细胞毒性反应。在目标2中，我们将使用低亲和力荧光探针FuraFf来确定类似动作对STORE钙固定的影响。在目标3中，我们将探讨肌浆/内质释放钙(SERCA)泵抑制和血管扩张剂对DVR内皮细胞[Ca~(2+)]CyT反应的Angll抑制机制。我们将研究DVR血管内皮细胞中钙离子的进入途径，并检测Ang11是否抑制这些途径。我们将测试钠/钙交换和钙储存隔离在降低内皮细胞[Ca~(2+)]CyT中的作用。在目标4中，我们将跟踪观察到DVR内皮功能障碍伴随着慢性哇巴因高血压(OH)。我们将测试OH大鼠血管内皮细胞的钙反应和NO释放是否改变，以及钠泵异构体是否下调。我们将测试DVR对去甲肾上腺素、血管紧张素转换酶和KCI的收缩是否增加，测量NO的产生，并评估OH对内皮和周细胞钙信号的影响。

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.