EXTRACELLAR NUCLEOTIDE SIGNALING IN CYSTIC FIBROSIS

囊性纤维化是一种外分泌上皮病。不仅在CF上皮细胞中观察到环状AMP激活的氯(C_1-)通道活性的缺乏，而且在CF中还观察到其他上皮C_1~-和Na~+通道的失调、上皮泡的转运和上皮免疫功能的异常。PI最近表明，在基础或等渗和低渗条件下，CF上皮细胞的顶膜上存在ATP释放的丢失，并且在CF中细胞外的ATP信号可能丢失。如果CF上皮细胞没有基础或刺激的ATP释放，我们推测，CF上皮功能障碍的发病机制可能在于CF上皮细胞的ATP信号转导和细胞体积调节缺陷。已经观察到呼吸道表面液体中的离子失衡；我们假设这反映了渗透失衡，这是由于自分泌ATP控制上皮细胞体积调节的固有缺陷所致。因此，这个完全重建的建议集中在一个简洁的中心假设：ATP释放和信号是细胞体积调节的自分泌控制所必需的。这一假说有两个目的：(1)检验RVD需要质膜上的ATP释放、ATP受体和CFTR表达的假说；(2)检验通过转运和胞吐驱动的机制释放ATP的假说。为了实现这些目标，将亲本的CF上皮细胞与暂时或稳定地补充野生型CFTR的CF细胞进行比较。细胞体积调节试验、细胞释放三磷酸腺苷的生物发光检测试验、膜片钳全细胞电流和膜电容的电生理试验以及胞吐试验的协同作用将被用来解决每个目标。这些检测将确定ATP的释放机制，这些机制对CFTR表达的依赖，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，关于自分泌三磷酸腺苷释放、信号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.