Chronic hypoxia and pH homeostasis in pulmonary myocytes

描述(由申请人提供)：长期暴露在氧分压降低的环境中，会导致许多肺部疾病，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。细胞内pH(Phi)的变化可能参与了慢性低氧(CH)时对肺动脉平滑肌细胞(PASMC)收缩和生长的调节。碱性pHi引起PASMC收缩，是PASMC对生长因子的反应中增殖所必需的。此外，Na/H交换(NHE)拮抗剂可抑制缺氧性肺动脉高压的形成。我们的数据表明，暴露于CH会导致PASMCs的碱化和NHE活性的增加。这种NHE活性的增加可能是由于交换子的表达发生了变化；然而，影响NHE表达的因素尚不清楚。低氧诱导因子1(HIF-1)是一种转录因子，通过基因诱导调节介导多种对低氧的适应性反应。我们已经建立了缺氧性肺动脉高压的小鼠模型，并发现部分缺乏HIF-1α亚基的转基因小鼠表现出对CH的反应降低了肺动脉高压。此外，在这些小鼠的PASMCs中，CH诱导的碱化和NHE活性的激活也减少。HIF-1对NHE表达的调控尚未被证实，但由于NHE1的启动子包含一个假定的HIF-1结合位点，因此NHE1的表达是可能的。除了碱性Phi对PASMC收缩和生长的直接影响外，Phi还可能通过控制[Ca~(2+)]i来调节血管管径，因为NHE活性增加导致的细胞内Na增加可能通过Na/Ca~(2+)交换改变Ca~(2+)排出。我们已经证明，在CH小鼠的PASMC中，静息[Ca~(2+)]i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，缺氧诱导的HIF-1激活了NHE1的转录，导致NHE1蛋白表达增加，Na/H交换增加。NHE活性的增加导致PHI的碱性漂移，从而通过调节PASMC细胞内钙浓度、收缩和生长而导致肺动脉压的升高。我们将使用多种技术组合来验证这一假设，包括动脉节段的等长张力记录、分子生物学、电生理和微荧光技术。

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.