HYPOXIC CA+2 RELEASE IN PULMONARY ARTERY MYOCYTE

低氧性肺血管收缩（HPV）是一种重要的调节机制，在缺氧时维持足够的动脉氧合，但也可导致肺动脉高压。 虽然HPV的潜在机制尚未完全了解，但平滑肌细胞中细胞内钙浓度（[Ca 2 +]i）的增加在HPV的发展中起着关键作用。 缺氧[Ca 2 +]i增加是由于肌浆网（SR）释放Ca 2+以及通过电压依赖性Ca 2+通道的Ca 2+内流。 缺氧性Ca ~（2+）内流可能是由K ~+电流的抑制和Ca ~（2+）激活的Cl ~-电流的激活引起的。 这两种缺氧效应可能是继发于Ca 2+释放。 然而，耦合缺氧钙释放的分子过程仍然难以捉摸。 我们和其他研究人员已经表明，缺氧或代谢抑制后的Ca 2+释放被SR Ca 2+耗尽所阻断。 我们的初步数据表明，NADPH氧化酶的抑制块缺氧[Ca 2 +]i增加，和H2 O2逆转激动剂诱导的[Ca 2 +]i升高和钙衰减减慢缺氧增强。 我们还发现，还原剂模拟缺氧Ca 2+释放。 因此，在本申请中，我们将解决以下问题（特定目标）：（1）缺氧钙释放是否通过兰尼碱受体，三磷酸肌醇受体或两者发生？ (2)最近发现的内源性钙释放介质如环ADP-核糖、烟酸腺嘌呤二核苷酸磷酸和FK 506结合蛋白介导缺氧性钙释放吗？ (3)NADPH氧化酶是缺氧Ca ~（2+）释放的主要氧传感器吗？ (4)H_2O_2、细胞内还原剂和蛋白激酶C在缺氧Ca ~（2+）释放中是否起信号转导作用？ 这些目标将追求通过使用膜电流和全细胞或局部[Ca 2 +]i（Ca 2+火花）在单个电压钳位肺阻力动脉肌细胞的同时测量。 转基因小鼠、基因过表达和基因表达的抑制也将用于定义缺氧与Ca 2+释放的偶联。这项拟议研究的结果将扩展我们对HPV发展的细胞和分子机制的理解，并可能导致确定治疗HPV的潜在新靶点。

Hypoxic pulmonary vasoconstriction (HPV) serves as an important regulatory mechanism maintaining adequate arterial oxygenation in response to hypoxia, but can also result in pulmonary hypertension. While the mechanism underlying HPV is incompletely understood, an increase in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells plays a critical role in the development of HPV. The hypoxic [Ca2+]i increase is due to Ca2+ release from the sarcoplasmic reticulum (SR), as well as Ca2+ influx through voltage-dependent Ca2+ channels. The hypoxic Ca2+ influx is likely caused by inhibition of K+ currents and activation of Ca2+-activated Cl- currents. Both hypoxic effects may be secondary to Ca2+ release. However, the molecular processes coupling hypoxia to Ca2+ release remain elusive. We and other investigators have shown that Ca2+ release following hypoxia or metabolic inhibition is blocked by depletion of the SR Ca2+. Our preliminary data indicate that inhibition of the NADPH oxidase blocks hypoxic [Ca2+]i increase, and that H2O2 reverses hypoxic potentiation of agonist-induced [Ca2+]i rise and slowing of calcium decay. We have also found that reducing agents mimic the hypoxic Ca2+ release. Therefore, in this application we will address the following questions (specific aim): (1) does hypoxic Ca2+ release occur through ryanodine receptors, inositol triphosphate receptors or both? (2) do recently discovered endogenous Ca2+ releasing mediators such as cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate and FK506 binding protein mediate hypoxic Ca2+ release? (3) is the NADPH oxidase a primary oxygen sensor in hypoxic Ca2+ release? (4) do H2O2, intracellular reducing agents and protein kinase C serve as signal transducers in the hypoxic Ca2+ release? These aims will be pursued by using simultaneous measurements of membrane currents and whole-cell or local [Ca2+]i (Ca2+ sparks) in single voltage-clamped pulmonary resistance artery myocytes. Transgenic mice, gene overexpression and inhibition of gene expression will be also used to define the coupling of hypoxia to Ca2+ release. The findings of this proposed research will extend our understanding of cellular and molecular mechanisms responsible for the development of HPV, and may lead to identify a potential novel target to treat HPV.