Mechanisms Controlling Airway Surface Liquid pH

项目总结/摘要 呼吸道感染是许多疾病发病和死亡的常见原因， 纤维化（CF）。尽管目前的治疗方法，超过90%的CF患者死于呼吸道感染。CF 是由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起的， 频道这种阴离子通道的缺陷如何导致呼吸道感染尚不清楚。更好地理解CF 肺病时，我们生产CF无效猪。CF猪表现出与人类相似的宿主防御缺陷。我们发现 CF猪具有异常酸性的气道表面液体（ASL），即肺上皮细胞内衬的薄层液体。 提高CF猪ASL的pH值可挽救宿主防御缺陷。因此，我们假设感染 在CF是由于酸性ASL的pH值，部分，由功能失调的CFTR，它通常分泌HCO 3-。在 除了CFTR，许多离子通道和转运蛋白被假设参与HCO 3- 分泌和H+分泌。然而，哪些离子通道和转运蛋白主要对ASL pH起作用， 不清楚如果能够确定主要途径，则增加 ASL的pH值和可能减少肺部感染可以开发。为了实现这一目标，我们建议 具体目标如下： 1)确定将HCO 3-分泌到ASL中的通道和转运蛋白 2)确定H+分泌到ASL中的主要机制。 为了确定哪种HCO 3-分泌蛋白和哪种H+分泌蛋白是最重要的，我们将使用小的 选择性敲低特定mRNA的干扰RNA（siRNA）和抑制离子的药理学试剂 渠道和运输。我们将测量抑制这些蛋白对气道中ASL pH的影响 上皮细胞我们将使用三种技术量化这种效应：pH敏感荧光团， 电生理学和pH值通过将pH敏感荧光团分散到ASL中，我们可以测量 直接影响ASL pH值。在Ussing Chambers，我们将电压钳培养来测量产电 HCO 3-药物刺激产生的转运。为了解释电中性离子传输，我们将使用 pH值恒定至pH钳并测量H+或HCO 3-分泌速率。我们希望通过这些技术， 将确定哪些蛋白质对HCO 3-分泌和H+分泌到ASL中贡献最大。然后我们将测试 通过测定细菌杀灭率和ASL粘度，研究了CF宿主体外防御缺陷的影响。 了解哪些蛋白质是ASL pH的主要贡献者，是实现我们长期目标的第一步， 开发药物来增加ASL pH值。利用我们对CF猪的访问，我们可以在体内测试药物，然后 将其发展为人类的疗法。这些药物不仅会对CF产生重大影响， 其他疾病，如COPD，也显示酸性ASL和细菌感染。该项目是一个直接 应用了解生理机制以改善人类健康，这是NHLBI的使命。

Project Summary/Abstract Respiratory infections are a common cause of morbidity and mortality in many diseases including Cystic Fibrosis (CF). In spite of current therapies more than 90% of people with CF die from respiratory infections. CF is caused by mutations in the gene for cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. How defects in this anion channel led to respiratory infections is unclear. To better understand CF lung disease, we produced CF null pigs. CF pigs show host defense defects, similar to humans. We found that CF pigs have an abnormally acidic airway surface liquid (ASL), the thin layer of fluid lining the lung epithelia. Increasing the pH of the ASL in CF pigs rescues the host defense defect. Thus, we hypothesize that infections in CF are due to an acidic ASL pH, in part, by a dysfunctional CFTR, which normally secretes HCO3-. In addition to CFTR, a number of ion channels and transporters have been hypothesized to be involved in HCO3- secretion and H+ secretion. However, which ion channels and transporters primarily contribute to ASL pH is unclear. If the dominant pathways can be identified, the most efficacious pharmacological therapy to increase ASL pH and presumably decrease lung infections can be developed. To accomplish this, we propose the following specific aims: 1) Determine the channels and transporters that secrete HCO3- into the ASL 2) Determine the primary mechanism of H+ secretion into the ASL. To determine which HCO3--secreting and which H+-secreting proteins are most important, we will use small interfering RNA (siRNA) to selectively knock down specific mRNAs and pharmacologic agents to inhibit ion channels and transporters. We will measure the effect of inhibiting these proteins on ASL pH in airway epithelia. We will quantify this effect using three techniques: pH sensitive fluorophores, Ussing chamber electrophysiology, and pH stat. By dispersing a pH sensitive fluorophore into the ASL, we can measure the effects of on ASL pH directly. In Ussing Chambers, we will voltage-clamp cultures to measure electrogenic HCO3- transport generated by pharmacologic stimulus. To account for electro-neutral ion transport, we will use pH stat to pH-clamp and measure rates of H+ or HCO3- secretion. We expect that with these techniques, we will identify which proteins contribute most to HCO3- secretion and H+ secretion into the ASL. We will then test the effects on CF host defense defects in vitro by measuring bacterial killing and ASL viscosity. Knowing which proteins are the major contributors to ASL pH, is the first step towards our long-term goal of developing drugs to increase ASL pH. Using our access to CF pigs, we can test the drugs in vivo, and then develop them as therapies for humans. These drugs would have a major impact for not only CF, but also for other diseases like COPD, which also show acidic ASL and bacterial infections. This project is a direct application of understanding a physiology mechanism to better human health, the mission of NHLBI.