HYPOXIC CA+2 RELEASE IN PULMONARY ARTERY MYOCYTE

低氧性肺血管收缩（HPV）是维持缺氧时动脉氧合充足的重要调节机制，但也可导致肺动脉高压。虽然HPV的机制尚不完全清楚，平滑肌细胞内钙浓度（[Ca2+]i）的增加在HPV的发展中起着关键作用。低氧[Ca2+]i增加是由于Ca2+从肌浆网（SR）释放，以及Ca2+通过电压依赖性Ca2+通道内流。低氧Ca2+内流可能是由抑制K+电流和激活Ca2+激活的Cl-电流引起的。这两种缺氧作用都可能继发于Ca2+释放。然而，分子过程耦合缺氧Ca2+释放仍然难以捉摸。我们和其他研究人员已经表明，缺氧或代谢抑制后的Ca2+释放被SR Ca2+的耗尽所阻断。我们的初步数据表明，抑制NADPH氧化酶可以阻断缺氧[Ca2+]i的增加，H2O2可以逆转激动剂诱导的[Ca2+]i升高的缺氧增强和钙衰变的减缓。我们还发现还原剂模拟缺氧Ca2+释放。因此，在本应用中，我们将解决以下问题（具体目标）：(1)缺氧Ca2+释放是否通过ryanodine受体，肌醇三磷酸受体或两者发生？(2)最近发现的内源性Ca2+释放介质如环adp核糖、烟酸腺嘌呤二核苷酸磷酸和FK506结合蛋白是否介导缺氧Ca2+释放？(3) NADPH氧化酶是缺氧Ca2+释放的主要氧传感器吗？(4) H2O2、细胞内还原剂和蛋白激酶C是否在缺氧Ca2+释放中起信号转导作用？这些目标将通过同时测量膜电流和全细胞或局部[Ca2+]i （Ca2+火花）在单个电压夹住的肺阻力动脉肌细胞来实现。转基因小鼠，基因过表达和基因表达抑制也将被用来定义缺氧与Ca2+释放的耦合。这项研究的发现将扩展我们对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.