Serous Cell Secretion and Cystic Fibrosis Lung Disease

描述（由申请人提供）：拟议的研究是项目的一部分，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产生的粘液水分不足，损害了粘液的清除和抗菌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的试剂的反应存在深刻缺陷。为了从这些观察中取得进展，我们提出了4个具体目标。目的1：确定局部（内在）神经元，特别是那些含有VIP的神经元，如何通过与胆碱能输入的相互作用直接控制腺体分泌。目的2：研究腺的浆液腺泡、粘液小管、集管和纤毛管四个解剖腔内的单细胞分泌机制。细胞反应是用微分干涉对比（DIC）延时数字成像来量化的，我们用光学切片从对照组和CF受试者中分离出功能正常的腺体。我们将研究在对照溶液（krebs -碳酸氢盐缓冲液）或使用离子取代或运输抑制剂来解剖腺体内运输过程的溶液中，四个腔室中的每一个如何对增加[cAMP]i， [Ca2+]i或两者同时增加的药物做出反应。我们还将阐明浆液和黏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外事件。目的3：利用注入腺体管腔的荧光指示剂（BCECF）比例成像法测定4个腺体腔内粘液的pH值。目的4：对部分游离的腺体浆液和黏液细胞进行膜片钳夹，以获得证据来反驳CFTR在浆液细胞中唯一的根尖阴离子通道中而在黏液细胞中不存在的假设。这些目标的成功完成将阐明腺体的作用，它产生大约95%的气道粘液，对气道先天防御的粘液清除和化学屏蔽成分的贡献。

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.