Chronic hypoxia and pulmonary vascular smooth muscle

慢性缺氧与肺血管平滑肌

• 批准号: 6789297
• 负责人: Larissa A. Shimoda
• 金额: $ 28.61万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-08 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6789297
• 关键词: actin binding protein; calcium ion; cell proliferation; cell type; endothelin; hypoxia inducible factor 1; immunocytochemistry; intracellular transport; laboratory rat; lung ischemia /hypoxia; muscle cells; muscle contraction; myosins; northern blottings; phosphorylation; potassium channel; pulmonary hypertension; sodium ion; vascular smooth muscle; voltage /patch clamp; voltage gated channel; western blottings

DESCRIPTION (provided by applicant): Prolonged exposure to decreased oxygen tension, as occurs with many pulmonary diseases, results in pulmonary hypertension, significantly worsening prognosis. The mechanism underlying the pathogenesis of this process remains unknown. Pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be caused by elevated intracellular Ca2+ concentration ([Ca2+]i). In PASMCs, depolarization is observed with CH, fueling speculation that [Ca2+]i is increased due to activation of voltage-gated Ca2+ channels or enhanced Na+/Ca2+ exchange. The endothelium-derived constricting factor, endothelin-1 (ET-1), may contribute to the pathogenesis of CHPH, since ET-1 increases [Ca2+]i. Following exposure to CH, the ET-1-induced rise in [Ca2+]i is reduced but contraction is maintained, suggesting activation of Ca2+-independent contractile pathways. This may be due to ET-1-induced activation of tyrosine kinases (TK). Hypoxic induction of ET-1 occurs via activation of the transcription factor, HIF-1, in systemic endothelium and in mice partially deficient for HIF-1, CH-induced pulmonary hypertension is markedly reduced. Therefore, we hypothesize induction of HIF-1 is an initiating step in the development of pulmonary hypertension, leading to elevated ET-1 levels. ET-1 then diffuses to PASMCs, activating three contractile mechanisms. First, ET-1 decreases voltage-gated K+ channel expression, leading to depolarization-driven activation of Na+/Ca2+ exchange and elevation of resting [Ca2+]i. Second, ET-1 causes TK-mediated Ca2+ influx through L-type Ca2+ channels. Both of these mechanisms increase phosphorylation of myosin light chains (MLCs). Finally, ET-1 causes changes in Ca2+-sensitivity of the contractile apparatus via TK-mediated regulation of actin binding proteins. This final step allows actin to interact with the phosphorylated MLCs generated in steps 1 and 2, and results in contraction. To test these hypotheses, we will use a combination of techniques in our model of hypoxic pulmonary hypertension, including isometric tension recording in arterial segments, Northern and Western blot analysis, whole-cell patch-clamp and microfluorescence measurements, to accomplish the following Specific Aims: 1) confirm that HIF-1 regulates hypoxic induction of ET-1 in the pulmonary vasculature and identify the cell type(s) involved; 2) determine the mechanisms responsible for the CH-induced increase in resting [Ca2+]i and 3) determine the mechanisms by which ET-1 causes contraction during CH.

描述（由申请人提供）： 长期暴露于氧气张力降低，如许多肺部疾病，导致肺动脉高压，预后明显恶化。该过程发病机理的基础机制仍然未知。与慢性缺氧（CH）相关的肺动脉平滑肌细胞（PASMC）收缩可能是由细胞内Ca2+浓度升高引起的（[Ca2+] I）。在PASMC中，通过CH观察到去极化，加油猜测[Ca2+] I因电压门控Ca2+通道的激活或增强的Na+/Ca2+交换而增加。内皮衍生的收缩因子内皮素-1（ET-1）可能有助于CHPH的发病机理，因为ET-1增加[Ca2+] i。暴露于CH后，ET-1诱导的[Ca2+] I中的上升减少但保持收缩，表明CA2+非依赖性收缩途径的激活。这可能是由于ET-1诱导的酪氨酸激酶（TK）的激活所致。 ET-1的低氧诱导是通过在系统性内皮中的转录因子HIF-1的激活和HIF-1部分缺陷的小鼠中发生的，CH诱导的肺动脉高压明显降低。因此，我们假设HIF-1的诱导是肺动脉高压发展的启动步骤，导致ET-1水平升高。 ET-1然后扩散到PASMC，激活三种收缩机制。首先，ET-1降低了电压门控的K+通道表达，从而导致Na+/Ca2+交换的去极化驱动的激活以及静息[Ca2+] i的升高。其次，ET-1引起TK介导的Ca2+流入L型Ca2+通道。这两种机制都会增加肌球蛋白光链（MLC）的磷酸化。最后，ET-1通过TK介导的肌动蛋白结合蛋白的调节而导致CA2+ - 敏感性的变化。最后一步允许肌动蛋白与步骤1和2中产生的磷酸化的MLC相互作用，并导致收缩。 To test these hypotheses, we will use a combination of techniques in our model of hypoxic pulmonary hypertension, including isometric tension recording in arterial segments, Northern and Western blot analysis, whole-cell patch-clamp and microfluorescence measurements, to accomplish the following Specific Aims: 1) confirm that HIF-1 regulates hypoxic induction of ET-1 in the pulmonary vasculature and identify the cell type(s) 涉及; 2）确定负责CH诱导的静止增加的机制[Ca2+] i和3）确定ET-1在CH期间引起收缩的机制。