Local Calcium Signaling in Pulmonary Arteries

肺动脉中的局部钙信号传导

• 批准号: 6824048
• 负责人: JAMES SK SHAM
• 金额: $ 32.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6824048
• 关键词: antisense nucleic acid; calcium channel; calcium flux; confocal scanning microscopy; endothelin; flash photolysis; laboratory rat; membrane potentials; microfilaments; neuromuscular transmission; potassium channel; pulmonary artery; vascular smooth muscle; vasoconstriction; vasomotion; voltage /patch clamp

EXCEED THE SPACE PROVIDED. Local Ca2+ release transients, or "Ca2+ sparks", originated from ryanodine receptors on sarcoplasmic receptors have been identified in cardiac, skeletal, and smooth muscles. In systemic vascular smooth muscles, they have been implicated as a feedback modulator of membrane potential by increasing local [Ca2+], activating nearby Ca2+-activated K+ (KCa) channels, leading to membrane hyperpolarization and vasodilation. Recently, we have identified and characterized Ca2+ sparks in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). In contrast to systemic vascular smooth muscle, activation of Ca2+ spark in PASMCs causes membrane depolarization. Endothelin-1 (ET-1), a major mediator of acute and chronic hypoxia induced pulmonary vasoconstriction, causes dramatic increase in Ca2+ spark frequency, which can be blocked by ET-A receptor antagonist, and non-selective cation channels. Inhibition of Ca2+ sparks with ryanodine significantly attenuated the ET-1 induced contraction of pulmonary arteries. Enhancement of Ca2+ spark by ET-1 is evident even at a threshold concentration (10-10 M), which at the best cause minimal contraction but is capable of potentiating hypoxic pulmonary vasoconstriction and contraction induced by other vasoconstrictor. Moreover, norepinephrine, which activates the PLC/PKC pathway similar to ET-1, causes the opposite effect of inhibiting Ca2+ sparks in PASMCs. Based on these novel findings, we propose that Ca2+ spark in PASMCs promote vasoconstriction, and vasoconstrictors modulate Ca2+ sparks through specific receptor dependent mechanisms. To test this hypotheses, we will apply a combination of state-of-the-art techniques including whole-cell patch clamp, laser-scanning confocal microscopy, and UV-pulse laser flash photolysis, RT-PCR, and gene-knockout with antisense oligonucleotide to examine if Ca2+ spark of PASMCs promotes vasoconstriction by (i) inducing membrane depolarization, (ii) providing Ca2+ for direct myofilament activation, and/or (iii) potentiating the contractile effects of other physiological stimuli. We will also characterize and identify signaling pathways for agonist- induced Ca2+ sparks activation, and determine the molecular identity and functional role of non-selective cation channels in Ca2+ spark actiivation. This project will provide important information on the unique control of pulmonary circulation by subcellular local Ca2+ signaling. PERFORMANCE SITE ========================================Section End===========================================

超过提供的空间。局部Ca2+释放瞬变或“ Ca2+火花”起源于肌浆受体上的ryanodine受体，在心脏，骨骼和光滑的肌肉中鉴定出来。在全身性血管平滑肌肉中，通过增加局部[Ca2+]，激活附近Ca2+活化的K+（KCA）通道，从而导致膜超极化和血管舒张，将它们与膜电位的反馈调节剂有关。最近，我们已经确定并表征了大鼠肺内动脉平滑肌细胞（PASMC）中的Ca2+火花。与全身血管平滑肌相反，PASMC中Ca2+火花的激活导致膜去极化。内皮素-1（ET-1）是急性和慢性缺氧的主要介质引起的肺血管收缩，会导致Ca2+火花频率的急剧增加，可以通过ET-A受体拮抗剂和非选择性阳离子通道阻止。 ryanodine对Ca2+火花的抑制显着减弱了ET-1诱导的肺动脉收缩。即使在阈值浓度（10-10 m）中，通过ET-1增强Ca2+火花也很明显，这最佳导致最小收缩，但能够增强其他血管收缩剂引起的低氧肺血管收缩和收缩。此外，激活与ET-1相似的PLC/PKC途径的去甲肾上腺素会导致抑制PASMC中Ca2+火花的相反作用。基于这些新的发现，我们提出PASMC中的Ca2+火花促进血管收缩，血管收缩器通过特定受体依赖机制调节Ca2+火花。为了检验这一假设，我们将采用最先进的技术组合，包括全细胞贴片夹，激光扫描的共聚焦显微镜和UV-Pulse激光闪光光解，RT-PCR和Gene-nockout和ant gene-nockout用反义核苷酸进行反义核苷酸，以检查pasmcys vasemctions vasbrane vasemctional vasextrion（Ii spasks sprandiation（II）（ii）（II）（ii spasks）（ii spasks sprance insoctions vasextription（II）（II）（II）（II）（II）（II）（II）（II）（II）。为直接肌丝激活提供Ca2+，和/或（iii）增强其他生理刺激的收缩作用。我们还将表征和识别激动剂诱导的Ca2+火花激活的信号传导途径，并确定非选择性阳离子通道在Ca2+火花作用中的分子身份和功能作用。该项目将通过亚细胞局部CA2+信号传导提供有关肺循环独特控制的重要信息。表演站点=============================================================================================