Ouabain and Descending Vasa Recta Ca2+ Signaling

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，骨髓灌注与调节盐和水的排泄、高血压和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的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抑制血管内皮细胞钙信号。 初步数据证实，影响细胞Na+(Na+/K+ATPase抑制和细胞外Na+还原)的动作对DVR内皮细胞钙离子有强烈的调节作用。我们将在慢性哇巴因高血压大鼠中验证高亲和力Na+泵异构体和Na+/Ca2+交换(NCX)调节DVR内皮细胞钙信号，参与Angll信号转导并变得疯狂的假说。在目标1中，我们将测试Na+K+ATPase高亲和力亚型(α2/α3)和NCX是否隔离在影响钙信号转导的亚质膜微域中。我们将使用免疫荧光来检测信号蛋白与SR/ER的共存。我们将确定哇巴因抑制、α2Na+泵缺乏、细胞外Na+([Na+]e)减少和NCX阻断是否调节DVR内皮 [Ca2+]细胞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，Furaptra)来确定目标1中的动作对钙储存的影响。在目标3中，我们将探讨肌浆/内质释放钙泵(SERCA)抑制和血管扩张剂抑制DVR内皮细胞[Ca~(2+)]CyT反应的机制。我们将研究DVR血管内皮细胞的钙内流途径，并检测Ang11是否对其有抑制作用。我们将测试NCX和钙库封存在Ang11诱导的内皮细胞[Ca~(2+)]CyT降低中的作用。在目标4中，我们将跟进我们的 慢性哇巴因高血压大鼠DVR内皮功能障碍的观察我们将测试OH大鼠血管内皮细胞的钙反应和NO释放是否改变，以及Na+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。

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, hypertension 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 and Na+/Ca 2+ exchange (NCX) modulate DVR endothelial Ca 2+signaling, participate in Angll signaling and become deranged in the chronic ouabain hypertensive rat. In Aim 1, we will test whether Na+K+ATPase high affinity isoforms (alpha2/alpha3) and NCX are sequestered in subplasmalemmal microdomains that affect Ca 2+signaling. We will examine colocalization of signaling proteins with SR/ER using immunofluorescence. We will determine whether ouabain inhibition, alpha2Na+ pump deficiency, reduction of extracellular Na+ ([Na+]e) and NCX blockade modulate DVR endothelial [Ca 2+]CYT responses to vasodilators. In Aim 2, we will use low affinity fluorescent probes (furaFF, furaptra) to determine the effect of the maneuvers in Aim 1 on store Ca2+ sequestration. In Aim 3, we will investigate the mechanisms responsible for Angll inhibition of DVR endothelial [Ca2+]CYT responses to sarcoplasmic / endoplasmic release Ca 2+(SERCA) pump inhibition and vasodilators. We will characterize Ca 2+ entry pathways in DVR endothelia and test whether Angll inhibits them. We will test for roles of NCX and Ca 2+ store sequestration in the Angll induced reduction of 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 Ca 2+ 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.