The injurious effects of hypercapnia on the alveolar epithelium

描述(申请人提供)：慢性阻塞性肺疾病患者和机械通气患者的“容许性高碳酸血症”患者的二氧化碳分压显著升高。我们推测，高碳酸血症通过特异性下调肺泡上皮Na，K-ATPase和减少肺泡液清除而导致肺泡上皮功能障碍。这一应用的重点是确定短期(30-60分钟)高二氧化碳是否通过抑制Na，K-ATPase，促进其内吞从质膜进入细胞内，从而通过涉及JNK和ERK激酶以及蛋白激酶C(PKC)信号分子的特定途径，对肺泡液的重吸收造成可逆性损害。我们还建议确定长期(5天和7天)高碳酸血症是否通过泛素/蛋白小体途径使肺泡上皮细胞对通气性肺损伤敏感，并且不仅引起内吞作用，而且引起Na，K-ATPase的降解。感知高碳酸血症的细胞信号在很大程度上是未知的，因此我们将利用线虫基因组已被完整定位的优势，在线虫中进行实验，研究可能参与感知和响应高二氧化碳的潜在基因。因此，我们将通过四个相互关联的目标来研究高二氧化碳对肺泡上皮和线虫的影响：在特定目标1中，我们建议确定高二氧化碳是否降低了正常和受损肺的肺泡液重吸收，以及这些影响是由于高二氧化碳还是相关的酸中毒所致；在特定目标2中，我们将确定高二氧化碳促进肺泡上皮细胞Na，K-ATPase内吞的信号通路；在特定目标3中，我们将确定高二氧化碳是否通过磷酸化和泛素化抑制Na，K-ATPase活性，从而导致Na内吞和降解。K-ATPase蛋白和特定目标4，我们将确定二氧化碳水平升高对线虫发育、运动和繁殖的影响，并建立一系列基因突变株，这些基因突变调节细胞对二氧化碳水平升高的感知和反应。针对每个具体目标进行了实验，初步结果支持了该方案的可行性。这些研究的完成将为高碳酸血症对肺泡上皮细胞的影响提供新的信息，特别是关于肺泡上皮屏障功能障碍的机制，这可能对高碳酸血症患者的治疗具有重要意义。

DESCRIPTION (provided by applicant): Patients with COPD and mechanically ventilated patients with "permissive hypercapnia" can have significant elevation of pCO2. We hypothesize that hypercapnia causes dysfunction of the alveolar epithelium by specifically downregulating the alveolar epithelial Na,K-ATPase and decreasing alveolar fluid clearance. The focus of this application is to determine whether short term (30-60 min) hypercapnia impairs reversibly alveolar fluid reabsorption by inhibiting the Na,K-ATPase and promoting its endocytosis from the plasma membrane into intracellular compartments via specific pathways involving JNK and ERK kinases and protein kinase C (PKC) signaling molecules. We also propose to determine whether long term (5 and 7 days) hypercapnia sensitizes the alveolar epithelium to ventilation induced lung injury and causes not only endocytosis but degradation of the Na,K-ATPase via the ubiquitin/proteosome pathway. The cellular signals that sense hypercapnia are largely unknown, therefore we will take advantage that the genome of C. Elegans has been completely mapped and conduct experiments in C. elegans to study potential genes that may participate in the sensing and response to high pCO2. As such, we will study the effects of hypercapnia on the alveolar epithelium and C. elegans via four interrelated aims: in Specific Aim 1 we propose to determine whether hypercapnia decreases alveolar fluid reabsorption in normal and injured lungs and whether these effects are due to high pCO2 or the associated acidosis, in Specific Aim 2 we will determine the signaling pathways by which hypercapnia promotes alveolar epithelial Na,K-ATPase endocytosis in alveolar epithelial cells , in Specific Aim 3 we will determine whether hypercapnia inhibits Na,K-ATPase activity via phosphorylation and ubiquitination leading to the endocytosis and degradation of Na,K-ATPase proteins and in Specific Aim 4 we will determine the effect of elevated levels of CO2 on C. elegans development, motility and fertility and establish a collection of lines mutated in genes that regulate the sensing and cellular response to elevated levels of CO2. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility of this proposal. Completion of the proposed studies will provide novel information on the effects of hypercapnia on the alveolar epithelium, specifically as it pertains to mechanisms of alveolar epithelial barrier dysfunction which may be of importance for the treatment of hypercapnic patients.