Chronic hypoxia and pH homeostasis in pulmonary myocytes

描述（由申请人提供）：许多肺部疾病患者长期暴露于低氧压力下，导致肺动脉高压，显著恶化预后。这一过程背后的细胞机制仍然知之甚少。慢性缺氧（CH）时，细胞内pH （pHi）的改变可能有助于调节肺动脉平滑肌细胞（PASMC）的收缩和生长。碱性pHi引起PASMC收缩，是PASMC响应生长因子增殖的必要条件。此外，Na+/H+交换拮抗剂（NHE）可抑制低氧性肺动脉高压的发展。我们的数据表明，暴露于CH会导致pasmc的碱化和NHE活性的增加。NHE活性的增加可能是由于交换剂表达的变化；但影响NHE表达的因素尚不清楚。缺氧诱导因子1 （hypoxia -inducible factor 1, HIF-1）是一种通过调控基因诱导介导多种缺氧适应性反应的转录因子。我们建立了一个小鼠缺氧肺动脉高压模型，发现部分缺乏HIF-1 α亚基的转基因小鼠对CH的反应表现出肺动脉高压的降低。此外，CH诱导的PASMCs中的碱化和NHE活性的激活也减少了。HIF-1对NHE表达的调控尚未得到证实，但这是可能的，因为NHE1的启动子含有一个假定的HIF-1结合位点。除了对PASMC收缩和生长的直接影响外，pHi还可以通过控制[Ca2+]i来调节血管直径，因为由于NHE活性增加而增加的细胞内Na+可以通过Na+/Ca2+交换改变Ca2+挤出。我们已经证明静息[Ca2+]i在CH小鼠的pasmc中也升高，并且这种反应依赖于HIF-I。基于这些考虑，我们假设在CH过程中，缺氧诱导HIF-1激活NHE1转录，导致NHE1蛋白表达增加，Na+/H+交换增加。NHE活性的增加引起pHi的碱性转移，这通过调节PASMC细胞内Ca2+浓度、收缩和生长来促进肺动脉压的升高。我们将使用一系列技术来检验这一假设，包括动脉段的等长张力记录、分子生物学、电生理学和微荧光技术。

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.