CYSTIC-FIBROSIS TRANSMEMBRANE REGULATOR-INDEPENDENT RELEASE OF ATP - ITS IMPLICATIONS FOR THE REGULATION OF P2Y(2) RECEPTORS IN AIRWAY EPITHELIA

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AM P-dependent Cl- channel that is defective in CF cells. It has been hyp othesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-i ndependent Ca2+-activated Cl- conductance is regulated by the P2Y(2) r eceptor. Thus, ATP may function as an autocrine signaling factor promo ting Cl- secretion in normal but not CF epithelia if ATP release is de fective. We have tested for CFTR-dependent ATP release using four inde pendent detection systems. First, a luciferase assay detected no diffe rences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A ma rked accumulation of extracellular ATP resulted from mechanical stimul ation effected by a medium displacement. Second, high pressure liquid chromatography analysis of H-3-labeled species released from [H-3]aden ine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells, Mechanical stimulation of HNE cells again result ed in enhanced accumulation of extracellular [H-3]ATP and [H-3]ADP, Th ird, 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-stimul ated 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) recepto r were used as a reporter system for detection of ATP via P2Y(2) recep tor-promoted formation of [H-3]inositol phosphates, Basal [H-3]inosito l phosphate accumulation was of the same magnitude in control and CFTR -transduced cells, and no change was observed following addition of fo rskolin and isoproterenol, In both cell types, mechanical stimulation resulted in hexokinase-attenuable [H-3]inositol phosphate formation. I n summary, our data suggest that ATP release may be triggered by mecha nical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.