Ouabain and Descending Vasa Recta Ca2+ Signaling

哇巴因和降支直肠 Ca2 信号传导

• 批准号: 6968176
• 负责人: THOMAS L PALLONE
• 金额: $ 37.85万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968176
• 关键词: angiotensin II; bradykinin; caffeine; calcium flux; clinical research; endoplasmic reticulum; essential hypertension; fluorescent dye /probe; genetically modified animals; immunofluorescence technique; laboratory mouse; laboratory rat; membrane transport proteins; microcirculation; muscle contraction; nitric oxide; ouabain; protein isoforms; protein localization; renal medulla; sarcoplasmic reticulum; sodium ion; sodium potassium exchanging ATPase; vascular endothelium; vasodilators; voltage /patch clamp

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.

肾髓质的微循环捕获通过Henle袢和集合管沉积到肾的NaCl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、高血压和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+。我们将验证在慢性哇巴因高血压大鼠中，高哇巴因亲和力的Na +泵亚型和Na+/Ca 2+交换（NCX）调节DVR内皮细胞Ca 2+信号转导，参与AngII信号转导，并使其发生紊乱的假说。在目的1中，我们将测试Na+K+ ATP酶高亲和力亚型（α 2/α 3）和NCX是否被隔离在影响Ca 2+信号传导的质膜下微区中。我们将使用免疫荧光检查信号蛋白与SR/ER的共定位。我们将确定哇巴因抑制、α 2 Na+泵缺乏、细胞外Na+（[Na+]e）减少和NCX阻断是否调节DVR内皮细胞的功能。 [Ca 2+]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，furaptra）来确定目标1中的操作对储存Ca 2+的影响。 隔离在目的3中，我们将研究负责AngII抑制DVR内皮[Ca 2 +]CYT对肌质/内质网释放Ca 2+（SERCA）泵抑制和血管舒张剂的反应的机制。我们将描述DVR内皮细胞中的Ca 2+进入途径并测试AngII是否抑制它们。我们将检测NCX和Ca 2+库隔离在AngII诱导的内皮细胞[Ca 2 +]CYT减少中的作用。在目标4中，我们将跟进我们的 观察到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。