Ouabain and Descending Vasa Recta Ca2+ Signaling

哇巴因和降支直肠 Ca2 信号传导

• 批准号: 7644867
• 负责人: THOMAS L PALLONE
• 金额: $ 41.02万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7644867
• 关键词: ATP phosphohydrolase; ATP2A2; Abbreviations; Acetylcholine; Acute; Acute Kidney Failure; Adrenal Glands; Affect; Affinity; Angiotensin II; Animal Model; Animals; Architecture; Blood Pressure; Blood flow; Bradykinin; Ca(2+)-Transporting ATPase; Caffeine; Calcium Channel; Calmodulin; Cations; Cell membrane; Cells; Chronic; Computing Methodologies; Data; Databases; Deposition; Dose; Duct (organ) structure; Electrophysiology (science); Endoplasmic Reticulum; Endothelium; Endothelium-Dependent Relaxing Factors; Essential Hypertension; Event; Excretory function; Exposure to; Fluorescence; Fluorescent Probes; Functional disorder; Generations; Henle' s loop; Hypertension; Hypoxia; Image; Immunofluorescence Immunologic; Individual; Kidney; Link; Literature; Localized; Measures; Mediating; Mesenteric Arteries; Methods; Microcirculation; Mus; Muscle Cells; Na(+)-K(+)-Exchanging ATPase; Nitric Oxide; Norepinephrine; Ouabain; Pathway interactions; Perfusion; Pericytes; Physiological; Physiology; Process; Production; Prostaglandins; Protein Isoforms; Protein Overexpression; Pump; Rattus; Rectum; Regulation; Research Personnel; Role; Running; Ryanodine; SEA 0400; Signal Transduction; Signaling Protein; Smooth Muscle; Sodium Chloride; Sodium-Calcium Exchanger; Testing; Tissues; Transgenic Model; Urea; Vasodilator Agents; Water; cyclopiazonic acid; extracellular; follow-up; furaptra; inhibitor/antagonist; kidney medulla; large-conductance calcium-activated potassium channels; neuromuscular transmission; prevent; programs; relating to nervous system; research study; response; sensor; sodium calcium ATPase; vasoconstriction

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.

肾脏髓质的微循环捕获Naci和尿素，并通过Henle的环沉积在间隙中，收集管道并将血液流动到肾脏的低氧区域。证据将髓样灌注与盐和水排泄的调节，急性肾衰竭的高血压和起源有关。下降的Vasa Recta（DVR）是15 mu M动脉微血管，血流到达肾脏髓质。 DVR血管活性由收缩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Ca 2+信号传导和通道结构的方法。过去的研究表明，血管扩张剂刺激内皮Ca 2+信号传导并被血管紧张素II抑制。 初步数据验证了影响细胞Na+（Na+/K+ ATPase抑制和细胞外Na+还原）的操纵强烈调节DVR内皮Ca 2+。我们将检验以下假设：高ouabain亲和力Na+泵同工型和Na+/Ca 2+交换（NCX）调节DVR内皮Ca 2+信号传导，参与Angll信号传导并在慢性Ouabain Hypertensive Hypertensive大鼠中被危险。在AIM 1中，我们将测试Na+K+ATPase高亲和力同工型（alpha2/alpha3）和NCX是否在影响Ca 2+信号传导的亚质量微区中隔离。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ouabain抑制作用，α2NA+泵缺乏症，细胞外Na+（[Na+] e）的减少和NCX阻滞调节DVR内皮 [Ca 2+]对血管扩张剂的响应。在AIM 2中，我们将使用低亲和力荧光探针（Furaff，furaptra）来确定AIM 1对AIM 1对存储CA2+固存的影响。在AIM 3中，我们将研究负责DVR内皮抑制[Ca2+] Cyt对肌胞浆 /内质释放Ca 2+（SERCA）泵抑制和血管扩张剂的机制。我们将表征DVR内皮中的Ca 2+进入途径，并测试Angll是否抑制它们。我们将测试NCX和Ca 2+储存在Angll诱导的内皮[Ca2+] Cyt的降低中的作用。在AIM 4中，我们将跟进我们的 观察到DVR内皮功能障碍伴随着大鼠的慢性Ouabain高血压（OH）。我们将测试OH大鼠的内皮Ca 2+反应和没有释放的反应以及Na+泵的同工型是否下调。我们将测试是否增加了与去甲肾上腺素，Angll和KCI的DVR收缩，测量OH对OH对内皮和周细胞CA2+信号传导的影响。