NO/CGMP RELAXATION IN PULMONARY HYPERTENSION

描述 (改编自申请人的摘要)肺动脉高压(PHT) 在美国代表着一个重要的临床问题。增加了 血管阻力是通过张力增加的综合机制发生的 和血管重塑。一氧化氮(NO)是一种重要的内源性 血管扩张剂目前临床用于ARDS，原发性肺 新生儿高血压和持续性肺动脉高压。 在血管内皮细胞中合成，其血管松弛作用是 被认为主要是通过cGMP依赖的减少在 血管平滑肌胞浆[Ca~(++)]。一种依赖cGMP的激酶 (PKG)被认为是cGMP诱导的 [Ca++]I.支持这种PKG依赖途径的许多证据 在导管肺动脉的平滑肌细胞中被检测到 然而，从其他器官，而不是从肺循环，大多数 依赖NO/cGMP的松弛作用不依赖于PKG。在……里面 CGMP除了能激活PKG外，还能作用于膜结合离子 频道。这些新近发现的离子通道对 阳离子并直接与cGMP结合，不需要PKG的磷酸化。 申请人已经在大鼠肺微血管中发现了一种 该环核苷酸门控(CNG)离子通道的片段 转录酶/聚合酶链式反应(RT/PCR)。该片段具有序列 与先前在肾脏中发现的cGMP抑制的离子通道同源 上皮组织。鉴于他们观察到cGMP依赖，但 高血压性肺循环中的PKG非依赖性血管松弛 他们推测，这种CNG阳离子通道可能上调并 在PHT中激活。NO/cGMP依赖的血管扩张的重要成分 可能涉及关闭这些渠道。这将与 他们观察到高血压病患者肺循环增加。 依赖于NO依赖，但不依赖PKG的血管松弛。在这 他们打算确定这一点的重要性和机制 特发性肺循环中的PKG非依赖性通路 强调CNG阳离子通道的潜在作用。遗传和 将对低氧诱导的大鼠PHT模型进行研究。要确定 NO/cGMP依赖的松弛机制他们将研究 选择性NO、cGMP和PKG抑制剂对大鼠肺血管阻力的影响 离体肺灌流后[Ca~(++)]i的变化 微血管平滑肌。确定CNG的潜在作用 他们将寻找基因和蛋白质表达增加的证据 高血压性肺循环中核糖核酸酶的RT/PCR检测 保护试验、免疫印迹和光亲和试验。要确定 他们计划建立的通道的分子和生物物理特性 在中国仓鼠卵巢细胞中表达全长基因。

DESCRIPTION (Adapted from the applicant's abstract) Pulmonary hypertension (PHT) represents an important clinical problem in the United States. Increased vascular resistance occurs through the combined mechanisms of increased tone and vessel remodeling. Nitric oxide (NO) is an important endogenous vasodilator currently used clinically in ARDS, primary pulmonary hypertension, and persistent pulmonary hypertension of the newborn. Synthesized in the vascular endothelium, its vasorelaxant effects are thought to be primarily mediated through cGMP-dependent reductions in cytosolic [Ca++] in the vascular smooth muscle. A cGMP-dependent kinase (PKG) is thought to be the central figure in cGMP-induced reductions in [Ca++]I. Much of the evidence to support this PKG-dependent pathway has been determined in smooth muscle cells from conduit pulmonary arteries or from other organs, however, and not from the pulmonary circulation that most of the NO/cGMP-dependent relaxation occurs independently of PKG. In addition to activation of PKG, cGMP can also act on membrane bound ion channels. These recently identified ion channels are nonselective to cations and directly bind cGMP and do not require phosphorylation by PKG. The applicants have identified in rat pulmonary microvascular vessels a fragment of this cyclic nucleotide gated (CNG) ion channel using reverse transcriptase/polymerase chain reaction (RT/PCR). The fragment has sequence homology to a cGMP-inhibited ion channel previously identified in the renal epithelium. Given their observation of a cGMP-dependent, but PKG-independent vasorelaxation in the hypertensive pulmonary circulation they hypothesize that this CNG cation channel may be upregulated and activated in PHT. An important component of NO/cGMP dependent vasodilation could involve closure of these channels. This would be consistent with their observations in the hypertensive pulmonary circulation of an increased reliance on NO-dependent, but PKG-independent vasorelaxation. In this proposal they intend to determine the importance and mechanism of this PKG-independent pathway in the pulmonary circulation with particular emphasis on the potential role of the CNG cation channel. Genetic and hypoxia-induced rat models of PHT will be studied. To determine the mechanism of NO/cGMP dependent relaxation they will examine the effect of selective NO, cGMP, and PKG inhibitors on pulmonary vascular resistance in isolated perfused lungs and also on changes in [Ca++]I at the level of the microvascular smooth muscle. To determine the potential role of the CNG channel they will look for evidence of increased gene and protein expression in the hypertensive pulmonary circulation using RT/PCR, ribonuclease protection assay, western blots, and photoaffinity assays. To determine the molecular and biophysical characteristics of the channel they plan to express the full length gene in Chinese hamster ovary cells.