Serous Cell Secretion and Cystic Fibrosis Lung Disease

描述（由申请人提供）：拟议的研究是计划的一部分，旨在确定囊性纤维化（CF）患者患有慢性气道感染的原因。一般的假设是，有缺陷的气道粘膜下腺体产生的粘液水化不足，损害粘液清除和抗微生物和抗炎化合物的生物利用度。在以前的工作中，我们发现CF腺体对升高cAMP的药物的反应存在严重缺陷。为了从这些观察中取得进展，我们提出了四个具体目标。目标1：确定局部（内在）神经元，特别是含有VIP的神经元，如何直接和通过与胆碱能输入的相互作用控制腺体分泌。目标二：从单细胞水平研究浆液腺泡、粘液小管、集合管和纤毛管四个腺室的分泌机制。使用微分干涉对比（DIC）时间推移数字成像，我们光学部分隔离，功能腺体从控制和CF主题量化细胞反应。我们将研究四个隔室中的每一个如何响应于在对照溶液（Krebs-碳酸氢盐缓冲液）或其中离子取代或转运抑制剂用于解剖腺体内的转运过程的溶液的存在下增加[cAMP]i、[Ca 2 +]i或两者的试剂。我们还将阐明浆液和粘液细胞分泌液体和蛋白质的机制：我们的成像方法使我们能够量化单个胞吐事件。目标3：通过使用注射到腺腔中的荧光指示剂（BCECF）的比率成像来确定4个腺隔室中的腔粘液的pH值。目标4：采用膜片钳技术部分分离腺浆细胞和粘液细胞，为反驳CFTR只存在于浆细胞顶端阴离子通道而粘液细胞不存在的假说提供证据。这些目标的成功完成将阐明腺体的贡献，产生约95%的气道粘液，气道先天防御的粘液清除和化学屏障组件。

DESCRIPTION (provided by applicant): The proposed research is part of program that seeks to determine why people with cystic fibrosis (CF) have chronic airway infections. The general hypothesis is that defective airway submucosal glands produce mucus that is under-hydrated, compromising mucus clearance and the bio-availability of antimicrobial and anti-inflammatory compounds. In previous work we showed a profound defect in the response of CF glands to agents that elevate cAMP. To progress from those observations we propose 4 specific aims. Aim 1: To determine how local (intrinsic) neurons, especially those containing VIP, control gland secretion directly and via interaction with cholinergic input. Aim 2: To study secretory mechanisms at the single cell level within four anatomical compartments of the glands: the serous acini, mucous tubules, collecting duct and ciliated duct. Cellular responses are quantified using differential interference contrast (DIC) time-lapse digital imaging with which we optically section isolated, functioning glands from control and CF subjects. We will study how each of the four compartments respond to agents that increase [cAMP]i, [Ca2+]i or both, in the presence of control solution (Krebs-bicarbonate buffer) or solutions in which ion substitutions or transport inhibitors are used to dissect the transport processes within the glands. We will also clarify mechanisms for fluid and protein secretion by serous and mucous cells: our imaging methods allow us to quantify individual exocytotic events. Aim 3: To determine the pH of luminal mucus within the 4 gland compartments by using ratiometric imaging of fluorescent indicators (BCECF) injected into the gland lumen. Aim 4: To patch clamp partially dissociated gland serous and mucous cells, to obtain evidence for against the hypothesis that CFTR in the only apical anion channel in serous cells and is absent from mucous cells. Successful completion of these aims will clarify the contribution of glands, which produce approximately 95% of airway mucus, to the mucus clearance and chemical shield components of airway innate defenses.