REGULATION OF CHOLANGIOCYTE CL SECRETION BY PHOSPHATIDYLINOSITOL 3-KINASE

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149 Intrahepatic bile duct epithelial cells (cholangiocytes) have a major influence on the volume and composition of bile through absorption and secretion of fluid and electrolytes (e.g. Cl-). Recent studies indicate that increases in cholangiocyte cell volume stimulate i) ATP efflux, ii) autocrine stimulation of purinergic (P2) receptors, and iii) Cl- channel opening. Therefore, cell volume may be an important determinant of cholangiocyte secretion. The cellular signals involved in these processes, however, have not been identified. Aim: The purpose of these studies was to determine if PI 3-kinase (PI3K), a newly described phosphoinositide kinase, is involved in volume-activated Cl- secretion in cholangiocytes. Methods: In Mz-Cha-1 cells, a human cholangiocarcinoma cell line, membrane Cl- permeability was assessed by whole-cell patch clamp techniques, ATP efflux by a luciferin-luciferase assay(reported as arbitrary light units (ALU), and cell size by Coulter-Multisizer. Wortmannin (Wort, 50 nM), LY294002 (LY, 10 μM), and antibodies to the 110 kD catalytic subunit of PI3K (5 μg/ml) were used as specific, but unrelated, inhibitors of PI3K. Results: Exposure of cells to hypotonic buffer (20% less NaCl) rapidly increased cell volume (120 ± 1% of basal size), which was followed by recovery toward basal values (107± 1%) by 30 minutes. In the presence of Wort, however, volume recovery was inhibited (114 ± 2% at 30 minutes, n=5, p<.001). In parallel studies, increases in cell volume also activated currents carried by Cl- ions (-45.9 ± 6.5 pA/pF). Volume-sensitive current activation was also inhibited by Wort (-6.1 ± 2.5 pA/pF, n=7, p<.001), LY (-9.5 ± 4.8, n=5, p<.005) and intracellular dialysis(by inclusion in the patch pipette) with antibodies to the 110 kD catalytic subunit of PI3K (-2.9 ± 0.4, n=5, p<.005). To evaluate the site of action of PI3K in volume-stimulated Cl- channel activation, the effects on ATP release and the response to exogenous ATP were assessed. Hypotonic perfusate increased cellular ATP efflux (52.5 ± 4.6 ALU) and this response was partially inhibited by both Wort (38.1 ± 4.1 ALU) and LY(35.7 ± 5.1 ALU), (n=5 for each, p<.05). In the presence of Wort, exposure to exogenous ATP (10 μM) still activated Cl- currents(-25.5 ± 7.3 pA/pF, n=4, p<.01). Conclusion: These findings indicate that PI3K regulates cellular release of ATP, and the resulting increase in P2 receptor agonists leads to substantial increases in membrane Cl- permeability. Thus, PI3K represents a novel signaling pathway, coupling changes in cell volume with ATP efflux and membrane Cl- permeability. Further elucidation of the signaling pathways involved may provide novel strategies for pharmacologic approaches to increase bile flow in cholestatic liver diseases.