NO/CGMP RELAXATION IN PULMONARY HYPERTENSION

NO/CGMP 缓解肺动脉高压

• 批准号: 6182649
• 负责人: BRIAN W FOUTY
• 金额: $ 11.9万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182649
• 关键词: CHO cells; cGMP dependent protein kinase; calcium flux; cyclic GMP; disease /disorder model; enzyme inhibitors; gene expression; laboratory rat; membrane channels; microcirculation; molecular pathology; nitric oxide; polymerase chain reaction; pulmonary hypertension; transfection; vascular resistance; vascular smooth muscle; vasodilatation; western blottings

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.

描述 （改编自申请人的摘要）肺动脉高压（PHT） 在美国代表了一个重要的临床问题。 增加 血管抗性通过增加音调的组合机制发生 和船只重塑。 一氧化氮（NO）是重要的内源性 血管扩张剂目前在ARDS中使用临床使用 新生儿的高血压和持续性肺动脉高压。 在血管内皮中合成，其血管肌效应是 被认为主要是通过CGMP依赖性减少介导的 血管平滑肌中的胞质[Ca ++]。 CGMP依赖性激酶 （PKG）被认为是CGMP诱导的减少的中心人物 [Ca ++] i。 支持此PKG依赖性途径的许多证据具有 在管道肺动脉或 但是，来自其他器官，而不是从肺循环中 NO/CGMP依赖性松弛的放松是独立于PKG发生的。 在 在PKG的激活中，CGMP也可以作用于膜结合离子 频道。 这些最近确定的离子通道是非选择性的 阳离子并直接结合CGMP，不需要PKG磷酸化。 申请人已经在大鼠肺微血管血管中鉴定出来 使用反向 转录酶/聚合酶链反应（RT/PCR）。 片段具有序列 与先前在肾脏中鉴定的CGMP抑制离子通道同源 上皮。 考虑到他们观察到CGMP依赖性的，但是 高血压肺循环中非pkg非依赖性的血管征 他们假设该CNG阳离子通道可能被上调，并且 在PHT中激活。 NO/CGMP依赖性血管舒张的重要组成部分 可能涉及关闭这些渠道。 这与 他们在高血压肺循环中的观察结果 依赖无依赖性的，但依赖pkg的血管征为依赖。 在这个 提案他们打算确定这一点的重要性和机制 肺循环中的PKG独立途径 强调CNG阳离子通道的潜在作用。 遗传和 将研究缺氧引起的PHT大鼠模型。 确定 NO/CGMP依赖放松的机制，他们将检查 选择性NO，CGMP和PKG抑制剂在肺血管耐药性上 孤立的灌注肺以及[Ca ++] I的变化 微血管平滑肌。 确定CNG的潜在作用 渠道他们将寻找基因和蛋白质表达增加的证据 在使用RT/PCR的高血压肺循环中，核糖核酸酶 保护测定法，蛋白质印迹和光性测定法。 确定 他们计划的通道的分子和生物物理特征 在中国仓鼠卵巢细胞中表达全长基因。