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: 6706704
• 负责人: JAMES SK SHAM
• 金额: $ 39.29万
• 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/6706704
• 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.

如许多肺部疾病中，慢性缺氧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升高有关，并且对激动剂的反应性改变了，这表明Ca2+稳态的改变了PASMC的固有性。据推测，慢性缺氧会抑制KV通道的表达，从而导致膜去极化，L型Ca2+通道的激活以及[Ca2+] i的增加。然而，一些研究发现，抑制L型Ca2+通道在降低升高的[Ca2+] I和血管肌张力张张的方面无效，这表明可能涉及其他CA2+途径。最近，我们已经确定了大鼠内部PASMC中多个TRPC通道同工型（TRP1，TRP3和TRP6）。半定量RT-PCR分析表明，在慢性低氧PASMC中，TRPC1和TRPC6 mRNA水平升高。功能研究表明，储存和受体操的Ca2+进入。此外，我们发现局部Ca2+释放瞬变“ Ca2+ Sparks”与IP3受体和TRPC具有非常强大的相互作用。在慢性低氧PASMC中，自发和激动剂引起的CA2+火花活性都改变了。基于这些发现，我们假设慢性缺氧改变了多个CA2+涌入和释放途径。通过TRPCS进入阳离子的增加以及SR Ca2+释放过程的变化在基础[Ca2+] I和血管舒缩张张的增加以及血管反应性的变化中起着重要作用。 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-和ip3受体依赖于Ca2+释放，（iii）在商店操作和受体操作的Ca2+ entries中的变化，以及（iv）特定的TRPC亚型，负责在PASMCS和血管中升高的基础[Ca2+] I，在肺部肺部的PASMCS和血管肌张力中，肺部炎性过敏性过氧性氧气透明度过高。该项目将提供有关慢性缺氧 - 诱导肺动脉高压的肺血管中亚细胞Ca2+信号传导和稳态的独特信息。