EXTRACELLAR NUCLEOTIDE SIGNALING IN CYSTIC FIBROSIS

囊性纤维化（CF）是一种外分泌上皮疾病。不仅在CF上皮细胞中观察到缺乏环amp激活的氯离子（C1-）通道活性，而且其他上皮C1-和Na+通道，上皮囊泡运输，和上皮免疫功能也在CF中观察到。PI最近表明，在基础或等渗和低渗条件下，CF上皮顶端膜的ATP释放减少，细胞外ATP信号可能在CF中丢失。CF上皮中没有基础或刺激的ATP释放，我们假设CF上皮功能障碍的发病机制可能在于ATP信号传导缺陷和CF上皮细胞体积调节缺陷。观察到气道表面液体中的离子不平衡；我们假设这反映了由于自分泌ATP控制上皮细胞体积调节的固有缺陷而导致的渗透不平衡。因此，这个完全重构的建议集中在一个简洁的中心假设上：ATP的释放和信号传导对于细胞体积调节的自分泌控制至关重要。这一假设产生了两个目的：(1)验证RVD需要ATP释放、ATP受体和质膜中CFTR表达的假设；(2)验证ATP释放是通过运输和胞外驱动机制发生的假设。为了实现这些目标，将亲代CF上皮细胞与短暂或稳定补充野生型CFTR的CF细胞进行比较。细胞体积调节试验、细胞释放ATP的生物发光检测试验、ATP全细胞电流和膜电容的膜片钳电生理试验以及胞吐试验的协同组合将用于解决每个目标。这些试验将确定ATP释放机制，这些机制对CFTR表达的依赖性，以及参与自分泌ATP控制细胞体积调节的嘌呤能受体。我们相信，关于自分泌ATP释放、信号传导和细胞体积调节的细胞和分子机制将会发现新的结果。

Cystic fibrosis (CF) is a disease of exocrine epithelia. Not only is a lack of cyclic AMP-activated chloride (C1-) channel activity observed in CF epithelial cells, but dysregulation of other epithelial C1- and Na+ channels, epithelial vesicle trafficking, and epithelial immune function are also observed in CF. The PI has shown recently that there is a loss of ATP release across the apical membrane of CF epithelia under basal or isotonic and hypotonic conditions and that extracellular ATP signaling may be lost in CF. Without basal or stimulated ATP release in CF epithelia, we hypothesize that the pathogenesis of CF epithelial dysfunction may lie in defective ATP signaling and defective cell volume regulation by the CF epithelia. An ionic imbalance in the airway surface liquid has been observed; we hypothesize that this reflects an osmotic imbalance due to an inherent defect in autocrine ATP control of epithelial cell volume regulation. As such, this completely re-constructed proposal focuses on one concise, central hypothesis: ATP release and signaling is essential for autocrine control of cell volume regulation. Two aims derive from this hypothesis: (1) Test the hypothesis that RVD requires ATP release, ATP receptors, and CFTR expression in the plasma membrane, and (2) Test the hypothesis that ATP release occurs via transport- and exocytotic-driven mechanisms. To fulfill these Aims, parental CF epithelial cells will be compared to CF cells complemented transiently or stably with wild-type CFTR. A synergistic combination of cell volume regulation assays, bioluminescence detection assays of ATP released from cells, patch-clamp electrophysiological assays of ATP whole cell current and membrane capacitance, and exocytosis assays will be used to address each Aim. These assays will identify the ATP release mechanisms, the dependence of these mechanisms upon CFTR expression, and the purinergic receptors involved in autocrine ATP control of cell volume regulation. We are confident that novel results will be found concerning the cellular and molecular mechanisms of autocrine ATP release, signaling, and regulation of cell volume.