Ca regulation in chronic hypoxic pulmonary hypertension

慢性缺氧性肺动脉高压的钙离子调节

• 批准号: 7019165
• 负责人: JAMES SK SHAM
• 金额: $ 36.81万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-08 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7019165
• 关键词: biological signal transduction; calcium channel; calcium ion; complementary DNA; confocal scanning microscopy; electrophysiology; flash photolysis; fluorescence microscopy; gene expression; hypoxia; laboratory rat; microarray technology; muscle cells; polymerase chain reaction; protein protein interaction; pulmonary artery; pulmonary hypertension; ryanodine; sarcoplasmic reticulum; vascular smooth muscle; vasomotion; voltage /patch clamp; western blottings

Chronic hypoxia, as occurs in many pulmonary diseases, results in vascular myocyte proliferation and pulmonary hypertension. Pulmonary arterial smooth muscle cells (PASMCs) from animals of chronic hypoxia are associated with an elevated [Ca2+]i and altered reactivity to agonists, suggesting alterations in Ca2+ homeostasis intrinsic to PASMCs. It has been postulated that chronic hypoxia suppresses KV channel expression, leading to membrane depolarization, activation of L-type Ca2+ channels, and increase in [Ca2+]i. However, some studies found that inhibition of L-type Ca2+ channels was ineffective in reducing the elevated [Ca2+]i and vasomotor tone, suggesting additional Ca2+ pathway(s) may be involved. Recently, we have identified multiple TRPC channel isoforms (TRP1, TRP3, and TRP6) in rat intra-lobar PASMCs. Semi-quantitative RT-PCR analyses show that TRPC1 and TRPC6 mRNA level were increased in chronic hypoxic PASMCs; functional studies showed significant increase in store- and receptor-operated Ca2+ entry. In addition, we found that local Ca2+ release transients, "Ca2+ sparks", have very robust interactions with IP3-receptors and TRPCs. Both spontaneous and agonist-evoked Ca2+ spark activities were altered in chronic hypoxic PASMCs. Based on these findings, we hypothesis that multiple Ca2+ influx and release pathways are altered by chronic hypoxia; the increase in cation entry via TRPCs, and alterations in SR Ca2+ release processes play major roles in the increase in basal [Ca2+]i and vasomotor tone, and the alterations in vascular reactivity. To test these hypotheses, we will apply a combination of state-of-the-art techniques including whole-cell patch clamp, laser-scanning confocal microscopy, UV-pulse laser flash photolysis, microarray analysis, antisense gene knockout and isolated microvessels to examine (i) changes in gene-expressions of various Ca2+ transporters, and the associated changes in vascular reactivity, (ii) changes in ryanodine- and IP3-receptors dependent Ca2+ release, (iii) alterations in store-operated and receptor-operated Ca2+ entries, and (iv) specific TRPC subtypes responsible for the elevated basal [Ca2+]i in PASMCs and vasomotor tone in pulmonary arteries of chronic hypoxic rats. This project will provide unique information on the subcellular Ca2+ signaling and homeostasis in pulmonary vasculatures in chronic hypoxia-induce pulmonary hypertension.

正如许多肺部疾病中发生的那样，慢性缺氧会导致血管肌细胞增殖和肺动脉高压。慢性缺氧动物的肺动脉平滑肌细胞 (PASMC) 与 [Ca2+]i 升高和对激动剂的反应性改变相关，表明 PASMC 固有的 Ca2+ 稳态发生改变。据推测，慢性缺氧抑制 KV 通道表达，导致膜去极化、L 型 Ca2+ 通道激活和 [Ca2+]i 增加。然而，一些研究发现，抑制 L 型 Ca2+ 通道对于降低升高的 [Ca2+]i 和血管舒缩张力无效，这表明可能涉及其他 Ca2+ 途径。最近，我们在大鼠脑叶内 PASMC 中发现了多种 TRPC 通道亚型（TRP1、TRP3 和 TRP6）。半定量RT-PCR分析表明，慢性缺氧PASMCs中TRPC1和TRPC6 mRNA水平增加；功能研究表明，钙库和受体操纵的 Ca2+ 进入显着增加。此外，我们发现局部 Ca2+ 释放瞬态（“Ca2+ 火花”）与 IP3 受体和 TRPC 具有非常强大的相互作用。慢性缺氧 PASMC 中自发和激动剂诱发的 Ca2+ 火花活性均发生改变。基于这些发现，我们假设慢性缺氧会改变多种 Ca2+ 流入和释放途径。通过 TRPC 进入的阳离子增加以及 SR Ca2+ 释放过程的改变在基础 [Ca2+]i 和血管舒缩张力的增加以及血管反应性的改变中起主要作用。为了检验这些假设，我们将应用最先进的技术组合，包括全细胞膜片钳、激光扫描共聚焦显微镜、紫外脉冲激光闪光光解、微阵列分析、反义基因敲除和分离的微血管来检查(i) 各种 Ca2+ 转运蛋白基因表达的变化，以及血管反应性的相关变化，(ii) 兰尼碱和 IP3 受体依赖性 Ca2+ 释放的变化， (iii) 钙池操纵和受体操纵的 Ca2+ 条目的改变，以及 (iv) 特定 TRPC 亚型导致 PASMC 中基础 [Ca2+]i 升高和慢性缺氧大鼠肺动脉血管舒缩张力升高。该项目将提供有关慢性缺氧引起的肺动脉高压的肺血管中的亚细胞 Ca2+ 信号传导和稳态的独特信息。