H AND HC03 TRANSPORT BY AIRWAY EPITHELIA IN CF

H 和 HC03 CF 中的气道上皮运输

• 批准号: 6476236
• 负责人: TERRY E MACHEN
• 金额: $ 22.66万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476236
• 关键词: Golgi apparatus; acid base balance; bicarbonates; chloride channels; cystic fibrosis; electrophysiology; fluorescence microscopy; human tissue; hydrogen transport; ion transport; membrane permeability; mucus; respiratory epithelium; secretion; sodium channel; tight junctions; tissue /cell culture; western blottings

DESCRIPTION (Applicant's Abstract): Acid - base transport by airway epithelial cells likely play key roles in cystic fibrosis (CF) due to effects on mucus hydration and rheology, ciliary transport, bacterial binding and anti-microbial factors. Airway surface liquid (ASL) appears to be slightly acidic (pH 6.9), and acidity increases in CF. Cells in the submucosal glands secrete mucus, and secretion of (primarily) HCO3 and (some) Cl along with Na through tight junctions (to maintain electroneutrality) and fluid sweeps the mucus out of the glands. Permeability of tight junctions between submucosal gland cells is critical for the glands' ability to secrete ions. In contrast, surface cells seem to have tight junctions that are impermeant to ions, and this assures that these cells operate only to absorb Na (through ENaC), Cl (through CFTR) and water. According to these proposals, the absence of CFTR in CF will cause: (i) decreased HCO3, Na and water secretion by submucosal gland cells (leading to mucus clogging); (ii) increased acidity of ASL (due to reduced HCO3 secretion); (iii) thicker and stickier mucus (due to reversal of fluid secretion to absorption and increased acidity of ASL). According to these hypotheses, it is crucial to determine mechanisms of H and HCO3 transport across the membranes and tight junctions of WT and CF submucosal gland and surface epithelial cells, including the potential role of mucus secretion to the acid-base balance of the ASL. The specific aims of this proposal are: (i) Use fluorescence microscopic imaging, immunomicroscopy and western blots to identify mechanisms for HCO3 and H transport across apical and basolateral membranes of submucosal gland and surface cells of WT and CF airway monolayers. (ii) Using genetic targeting of pH-sensitive dyes and imaging microscopy, measure pH (and regulation) in mucus granules and at the lumenal aspect of WT and CF airway epithelia. (iii) Using isotopic methods, measure secretion of mucus and of constitutive and regulated Golgi-derived vesicles and correlate to H/HCO3 transport. (iv) Using electrophysiological, isotopic flux and immunomicroscopic methods, determine the permeability/conductance properties of the tight junctions.

描述（申请人摘要）：气道上皮的酸碱转运 由于对粘液的影响，细胞可能在囊性纤维化（CF）中发挥关键作用 水合和流变学、纤毛运输、细菌结合和抗菌 因素。气道表面液体 (ASL) 呈弱酸性 (pH 6.9)， CF 中酸度增加。粘膜下腺细胞分泌粘液， (主要) HCO3 和 (一些) Cl 以及 Na 通过紧密分泌 连接处（以保持电中性）和液体将粘液扫出 腺体。粘膜下腺细胞之间紧密连接的渗透性为 对于腺体分泌离子的能力至关重要。相比之下，表面细胞 似乎具有离子不可渗透的紧密连接，这确保了 这些细胞仅吸收Na（通过ENaC）、Cl（通过CFTR）和 水。根据这些提案，CF 中 CFTR 的缺失将导致：(i) 粘膜下腺细胞的 HCO3、Na 和水分泌减少（导致 粘液堵塞）； (ii) ASL 酸度增加（由于 HCO3 分泌减少）； (iii) 粘液变厚和粘稠（由于液体分泌逆转） ASL 的吸收和酸度增加）。根据这些假设， 对于确定 H 和 HCO3 跨膜运输机制至关重要 以及 WT 和 CF 粘膜下腺与表面上皮细胞的紧密连接， 包括粘液分泌对酸碱平衡的潜在作用 美国手语。该提案的具体目标是： (i) 使用荧光显微镜 成像、免疫显微镜和蛋白质印迹来确定 HCO3 和 H 穿过粘膜下腺的顶膜和基底外侧膜的转运 WT 和 CF 气道单层的表面细胞。 (ii) 使用基因靶向 pH 敏感染料和成像显微镜，测量粘液中的 pH（和调节） 颗粒以及 WT 和 CF 气道上皮的管腔方面。 (三) 使用 同位素方法，测量粘液以及组成型和调节型的分泌 高尔基体衍生的囊泡与 H/HCO3 运输相关。 (四) 使用 电生理学、同位素通量和免疫显微镜方法，确定 紧密连接的磁导率/电导特性。