title: Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia.
abstract: 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.
authors.affiliation: Cystic Fibrosis Research and Treatment Center, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA.
authors.forename: W C
authors.initials: WC
authors.lastname: Watt
authors.forename: E R
authors.initials: ER
authors.lastname: Lazarowski
authors.forename: R C
authors.initials: RC
authors.lastname: Boucher
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D051379:Mice; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D014132:Trachea / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: C505032:CFTR protein, human; D018048:Receptors, Purinergic P2; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate