NO/CGMP RELAXATION IN PULMONARY HYPERTENSION

描述 （改编自申请人摘要）肺动脉高压（PHT） 在美国是一个重要的临床问题。 增加 血管阻力是通过张力增加和张力降低的联合机制发生的 和血管重塑 一氧化氮（NO）是一种重要的内源性 目前临床上用于ARDS的血管扩张剂，原发性肺动脉高压 高血压和新生儿持续性肺动脉高压。 在血管内皮中合成，其血管舒张作用是 被认为主要通过cGMP依赖性降低介导， 血管平滑肌细胞内[Ca++]。 cGMP依赖性激酶 (PKG)被认为是cGMP诱导的细胞凋亡减少的中心人物。 [Ca++]I. 支持这种PKG依赖性途径的许多证据 在来自导管肺动脉的平滑肌细胞中测定，或 然而，从其他器官，而不是从肺循环， NO/cGMP依赖性舒张的发生独立于PKG。 在 cGMP除了激活PKG外，还可作用于膜结合离子 渠道 这些最近鉴定的离子通道对 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中鉴定了一种 该环核苷酸门控（CNG）离子通道的片段，使用反向 逆转录酶/聚合酶链反应（RT/PCR）。 片段有序列 与先前在肾脏中鉴定的cGMP抑制离子通道同源 上皮 鉴于他们观察到cGMP依赖性， 高血压肺循环中PKG非依赖性血管舒张 他们假设这种CNG阳离子通道可能被上调， 在PHT中激活。 NO/cGMP依赖性血管舒张的重要组成部分 可能会导致这些渠道的关闭。 这符合 他们在高血压肺循环中的观察结果表明， 依赖于NO依赖性，但PKG非依赖性血管舒张。 在这 他们打算确定这一建议的重要性和机制， 肺循环中的PKG非依赖性途径， 强调CNG阳离子通道的潜在作用。 遗传和 将研究缺氧诱导的PHT大鼠模型。 确定 NO/cGMP依赖性舒张的机制，他们将检查 选择性NO、cGMP和PKG抑制剂对肺血管阻力的影响 离体灌流肺，也对[Ca++]I的变化，在水平的 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， 在高血压肺循环中使用RT/PCR，核糖核酸酶 保护测定、蛋白质印迹和光亲和测定。 确定 分子和生物物理特性的通道，他们计划， 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.