Cystic fibrosis transmembrane regulator-independent release of ATP -: Its implications for the regulation of P2Y2 receptors in airway epithelia

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 P2Y(2) 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 H-3-labeled species released from [H-3]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 [H-3]ATP and [H-3]ADP, Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-P-33]dADP, equivalent formation of [P-33]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 [P-33]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y(2) receptor were used as a reporter system for detection of ATP via P2Y(2) receptor-promoted formation of [H-3]inositol phosphates, Basal [H-3]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 [H-3]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.