THE LIPID PRODUCTS OF PHOSPHOINOSITIDE 3-KINASE REGULATE CHOLANGIOCYTE ATP AND CHLORIDE TRANSPORT

3 ATP stimulates Cl- secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. In other cell types, increases in cell volume are a potent stimulus for ATP release, however the origin and regulation of biliary ATP is unknown. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. Methods: Studies were performed in Mz-Cha-1 human cholangiocarcinoma cells and normal rat cholangiocyte (NRC) monolayers. PI 3-kinase (PI3K) activity was assessed utilizing an assay which measures effector, Akt, phosphorylation; membrane Cl- permeability by patch clamp techniques; and ATP efflux by luminometry (reported as arbitrary light units (ALU)). Wortmannin (50nM) and LY294002 (10µM) were used as specific inhibitors of PI3K. Results: increases in cell volume (hypotonic exposure, 20% less NaCl) were followed by increases in: PI3K activity (20-50% increase by 10 min.), ATP release (increase of 0.73±07 ALU), and membrane Cl- permeability (increase of 41.9±6.5 pA/pF). PI3K signaling plays a regulatory role since ATP release was inhibited by wortmannin (0.19±01 ALU) or LY294002 (.12±.01 ALU); and since wortmannin also inhibited volume-sensitive current activation (2.1±2.5 pA/pF). Activated PI3K phosphorylates the D3 position of the inositol ring forming distinct lipid products which function as secondary messengers. To determine if Cl- secretion is mediated by these products, the synthetic lipids (PI-3-P, PI-3,4-P2, and PI-3,4,5-P3, 10 µM each) were delivered to the cell interior by inclusion in the patch pipette. Currents were activated by intracellular dialysis with PI-3,4-P2 (11.7±4.8 pA/pF) and PI-3,4,5-P3 (7.9±2.5 pA/pF), but not by PI-3-P (2.8±1.5 pA/pF) or control lipid, PIP-2 (1.4±0.3 pA/pF). PI-induced currents were analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase, and the generation of lipid messengers, modulates cholangiocyte ATP release, Cl- secretion, and hence, bile formation. Further elucidation of the signaling pathways involved may provide novel strategies for stimulation of bile flow in cholestatic liver diseases through effects on PI3K and ATP release.