Chronic hypoxia and pH homeostasis in pulmonary myocytes

肺肌细胞的慢性缺氧和 pH 稳态

• 批准号: 6765150
• 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: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765150
• 关键词: acidity /alkalinity; binding sites; calcium ion; cell proliferation; charge coupled device camera; electrophysiology; enzyme linked immunosorbent assay; genetically modified animals; growth factor; hypoxia; hypoxia inducible factor 1; immunocytochemistry; laboratory mouse; muscle contraction; northern blottings; polymerase chain reaction; pulmonary artery; pulmonary hypertension; sodium hydrogen exchanger; vascular smooth muscle; western blottings

DESCRIPTION (provided by applicant): Prolonged exposure to decreased oxygen tension occurs with many pulmonary diseases, resulting in pulmonary hypertension, significantly worsening prognosis. The cellular mechanisms underlying this process remain poorly understood. Alterations in intracellular pH (pHi) may contribute to regulation of pulmonary arterial smooth muscle cell (PASMC) contraction and growth during chronic hypoxia (CH). Alkaline pHi causes PASMC contraction and is necessary for PASMC proliferation in response to growth factors. Moreover, antagonists of Na+/H+ exchange (NHE) inhibit development of hypoxic pulmonary hypertension. Our data indicate that exposure to CH results in alkalinization of PASMCs and increased NHE activity. This increase in NHE activity could be due to a change in the expression of the exchanger; however, the factors influencing NHE expression are not clear. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates numerous adaptive responses to hypoxia through the regulation of gene induction. We have developed a mouse model of hypoxic pulmonary hypertension, and have found that transgenic mice with partial deficiency for the alpha subunit of HIF-1 exhibit reduced pulmonary hypertension in response to CH. Moreover, the CH-induced alkalinization and activation of NHE activity is reduced in PASMCs from these mice. Regulation of NHE expression by HIF-1 has not been demonstrated, but is possible since the promoter of NHE1 contains a putative HIF-1 binding site. In addition to direct effects of alkaline pHi on PASMC contraction and growth, pHi may also regulate vascular caliber through control of [Ca2+]i, as an increase in intracellular Na+ due to increased NHE activity could alter Ca2+ extrusion through Na+/Ca2+ exchange. We have shown that resting [Ca2+]i is also elevated in PASMCs from CH mice, and that this response was dependent on HIF-I. Based on these considerations, we hypothesize that during CH, hypoxic induction of HIF-1 activates NHE1 transcription, resulting in an increase in NHE1 protein expression and increased Na+/H+ exchange. The increase in NHE activity causes an alkaline shift in pHi, which contributes to the elevation in pulmonary artery pressure by modulating PASMC intracellular Ca2+ concentration, contraction, and growth. We will test this hypothesis using a combination of techniques, including isometric tension recording in arterial segments, molecular biological, electrophysiological and micro fluorescence techniques.

描述（由申请人提供）：长时间暴露于氧气张力降低的情况下发生了许多肺部疾病，导致肺动脉高压，预后明显恶化。 该过程基础的细胞机制仍然鲜为人知。 细胞内pH（PHI）的改变可能有助于调节肺动脉平滑肌细胞（PASMC）收缩和慢性缺氧期间的生长。 碱性PHI会导致PASMC收缩，这对于响应生长因子而言是PASMC增殖所必需的。 此外，Na+/H+交换（NHE）的拮抗剂抑制了低氧肺动脉高压的发展。 我们的数据表明，接触CH会导致PASMC的碱化和NHE活性增加。 NHE活性的增加可能是由于交换器表达的变化。但是，影响NHE表达的因素尚不清楚。 低氧诱导因子1（HIF-1）是转录因子，通过调节基因诱导来介导许多对缺氧的适应性反应。 我们已经开发了低氧肺动脉高压的小鼠模型，并发现HIF-1α亚基部分缺乏的转基因小鼠响应于CH，表现出较低的肺动脉高压。 此外，在这些小鼠的PASMC中，CH诱导的NHE活性的碱化和激活降低。 HIF-1对NHE表达的调节尚未得到证明，但由于NHE1的启动子包含假定的HIF-1结合位点，因此可能是可能的。 除了碱性PHI对PASMC收缩和生长的直接影响外，PHI还可以通过控制[Ca2+] I的控制来调节血管口径，因为由于NHE活性的增加，细胞内Na+的增加可能会通过Na+/Ca2+交换来改变Ca2+挤出。 我们已经表明，在CH小鼠的PASMC中，静止[Ca2+] I也升高，并且这种响应取决于HIF-I。 基于这些考虑，我们假设在CH期间，HIF-1的低氧诱导激活NHE1转录，从而导致NHE1蛋白表达的增加并增加Na+/H+交换。 NHE活性的增加导致PHI的碱性转移，这通过调节PASMC细胞内Ca2+浓度，收缩和生长来导致肺动脉压力的升高。 我们将使用技术的组合来检验该假设，包括在动脉片段，分子生物学，电生理和微荧光技术中记录等距张力。