Chronic hypoxia and pH homeostasis in pulmonary myocytes

描述（由申请人提供）：许多肺部疾病长期暴露于氧分压降低，导致肺动脉高压，预后显著恶化。 这一过程的细胞机制仍然知之甚少。 慢性缺氧时肺动脉平滑肌细胞（PASMC）收缩和生长的调节可能与细胞内pH（pHi）的改变有关。 碱性pHi引起PASMC收缩，并且是PASMC响应于生长因子而增殖所必需的。 此外，Na+/H+交换（NHE）的拮抗剂抑制缺氧性肺动脉高压的发展。 我们的数据表明，暴露于CH导致PASMCs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;然而，影响NHE表达的因素尚不清楚。 缺氧诱导因子1（HIF-1）是一种转录因子，通过调节基因诱导介导多种缺氧适应性反应。 我们已经开发了一种小鼠模型的缺氧性肺动脉高压，并发现，部分缺陷的HIF-1的α亚基的转基因小鼠表现出降低肺动脉高压的反应CH。此外，CH诱导的碱化和激活的NHE活性减少在PASMCs从这些小鼠。 HIF-1对NHE表达的调节尚未得到证实，但由于NHE 1的启动子含有推定的HIF-1结合位点，因此是可能的。 除了碱性pHi对PASMC收缩和生长的直接影响之外，pHi还可以通过控制[Ca 2 +]i来调节血管口径，因为由于NHE活性增加而导致的细胞内Na+增加可以通过Na+/Ca 2+交换改变Ca 2+挤出。 我们已经表明，静息[Ca 2 +]i也升高PASMCs从CH小鼠，这种反应是依赖于HIF-1。 基于这些考虑，我们假设在CH期间，低氧诱导HIF-1激活NHE 1转录，导致NHE 1蛋白表达增加和Na+/H+交换增加。 NHE活性的增加导致pHi的碱性偏移，这通过调节PASMC细胞内Ca 2+浓度、收缩和生长而导致肺动脉压升高。 我们将测试这一假设使用的技术组合，包括等距张力记录在动脉段，分子生物学，电生理和显微荧光技术。

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.