Mechanisms Controlling Airway Surface Liquid pH

项目摘要/摘要 呼吸道感染是许多疾病发病和死亡的常见原因，包括囊性疾病 纤维化(CF)。尽管目前有多种治疗方法，但超过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，CFTR是一种阴离子 频道。这种阴离子通道的缺陷是如何导致呼吸道感染的尚不清楚。为了更好地了解CF 肺病，我们生产了CF零的猪。Cf猪表现出宿主防御缺陷，与人类相似。我们发现 Cf猪有异常酸性的呼吸道表面液体(ASL)，这是一层薄薄的液体衬里在肺上皮细胞。 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中是由于酸性的ASL pH，部分是由于功能失调的CFTR，它通常会分泌HCO3-。在……里面 除了CFTR，一些离子通道和转运体被假设参与了HCO3- 分泌和H+分泌。然而，哪些离子通道和转运体是影响ASL pH的主要因素 不清楚。如果能够确定优势通路，最有效的药物治疗将增加 ASL的pH值和推测可以减少肺部感染。要做到这一点，我们建议 以下是具体目标： 1)确定分泌HCO3-进入ASL的通道和转运体 2)确定H+分泌到ASL的主要机制。 为了确定哪些HCO3-分泌和哪些H+分泌蛋白质是最重要的，我们将使用小分子 干扰RNA(SiRNA)选择性地敲除特定的mRNAs和药物以抑制离子 渠道和传输者。我们将测量抑制这些蛋白对呼吸道ASL pH的影响 上皮细胞。我们将使用三种技术来量化这种影响：PH值敏感的荧光团、Ussing小室 电生理学和酸碱度。通过将pH敏感的荧光团分散到ASL中，我们可以测量 对ASL pH的直接影响。在Ussing Chambers，我们将通过电压钳制培养来测量生电 HCO3-由药理刺激产生的转运。为了解释电中性离子的传输，我们将使用 测定H+或HCO3-的分泌速率。我们希望通过这些技术，我们 将确定哪些蛋白质对ASL的HCO3-分泌和H+分泌贡献最大。然后我们将测试 通过测定细菌杀灭和ASL粘度，研究了体外培养条件下对CF寄主防御缺陷的影响。 了解哪些蛋白质是ASL pH的主要贡献者，是迈向我们长期目标的第一步 开发提高ASL pH值的药物。利用我们接触到的CF猪，我们可以在体内测试药物，然后 开发它们作为人类的治疗方法。这些药物不仅对慢性萎缩性胃炎有重大影响，而且对 其他疾病，如COPD，也表现为酸性ASL和细菌感染。这个项目是一个直接的 应用了解生理机制以更好地促进人类健康，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.