HYPOXIC CA+2 RELEASE IN PULMONARY ARTERY MYOCYTE

缺氧性肺血管收缩(HPV)作为一种重要的调节机制，在低氧时维持充足的动脉氧合，但也可导致肺动脉高压。虽然HPV的发病机制尚不完全清楚，但细胞内钙离子浓度([Ca~(2+)]_i)的升高在HPV的发生发展中起着关键作用。缺氧性[Ca~(2+)]i的升高是由于肌浆网(SR)的Ca~(2+)释放，以及通过电压依赖性的Ca~(2+)通道的Ca~(2+)内流。缺氧性钙内流可能是通过抑制钾电流和激活钙激活的氯离子电流而引起的。这两种缺氧效应可能都是钙离子释放的次要作用。然而，低氧与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证明，缺氧或代谢抑制后的钙释放可以通过耗尽肌质网钙离子来阻止。我们的初步数据表明，抑制NADPH氧化酶可以阻止缺氧性[Ca~(2+)]i的升高，而H_2O_2逆转了激动剂诱导的缺氧性增强的[Ca~(2+)]i升高和减缓钙衰变。我们还发现，还原剂模拟低氧的钙释放。因此，在这一应用中，我们将解决以下问题(具体目的)：(1)低氧钙释放是通过兰尼定受体还是通过三磷酸肌醇受体发生的？(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FK506结合蛋白是否介导低氧钙释放？(3)NADPH氧化酶是低氧钙释放的主要氧感受器吗？(4)H_2O_2、细胞内还原剂和蛋白激酶C在低氧钙释放中起信号转导作用吗？这些目标将通过同时测量单个电压钳制的肺阻力动脉肌细胞的膜电流和全细胞或局部[Ca~(2+)]i(Ca~(2+)火花)来实现。转基因小鼠，基因的过度表达和基因表达的抑制也将被用来确定缺氧与钙释放的偶联。这项拟议的研究结果将扩大我们对HPV发生的细胞和分子机制的理解，并可能导致找到一个潜在的治疗HPV的新靶点。

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.