Watt W C, Lazarowski E R, Boucher R C
Cystic Fibrosis Research and Treatment Center, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA.
J Biol Chem. 1998 May 29;273(22):14053-8. doi: 10.1074/jbc.273.22.14053.
The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.
囊性纤维化（CF）跨膜调节因子（CFTR）是一种环磷酸腺苷（cAMP）依赖性氯离子通道，在CF细胞中存在缺陷。据推测，CFTR具有ATP释放功能，可控制气道表面ATP浓度。在气道上皮细胞中，不依赖CFTR的钙激活氯离子电导受P2Y2受体调节。因此，如果ATP释放存在缺陷，ATP可能作为自分泌信号因子促进正常上皮而非CF上皮中的氯离子分泌。我们使用四种独立检测系统对依赖CFTR的ATP释放进行了检测。首先，荧光素酶测定未检测到对照与cAMP刺激的原代正常人鼻上皮（HNE）细胞培养基中ATP浓度的差异。培养基置换引起的机械刺激导致细胞外ATP显著积累。其次，对[3H]腺嘌呤加载的HNE细胞释放的3H标记物质进行高压液相色谱分析，结果显示基础状态与cAMP刺激的细胞之间无差异。对HNE细胞的机械刺激再次导致细胞外[3H]ATP和[3H]ADP积累增加。第三，当通过核苷二磷酸激酶催化的[α-33P]dADP磷酸化来测量ATP浓度时，在对照和cAMP刺激的HNE细胞以及野生型和CF小鼠的鼻上皮细胞培养基中观察到等量的[33P]dATP形成。两种细胞类型中机械刺激引起的[33P]dATP形成相似。第四，稳定表达人P2Y2受体的1321N1细胞用作报告系统，通过P2Y2受体促进的[3H]肌醇磷酸形成来检测ATP。对照细胞和转导CFTR的细胞中基础[3H]肌醇磷酸积累量相同，添加福斯可林和异丙肾上腺素后未观察到变化。在两种细胞类型中，机械刺激均导致己糖激酶可减弱的[3H]肌醇磷酸形成。总之，我们的数据表明，ATP释放可能由细胞表面的机械刺激触发。未发现支持CFTR在ATP释放中起作用的证据。
