Volume-sensitive purinergic signaling in human hepatocytes

Background/Aims: Purinergic signaling potentially contributes to many liver functions. Therefore, the purpose of these studies was to characterize adenosine 5′-triphosphate (ATP) release from human hepatocytes, and to determine the role of extracellular ATP in the autocrine regulation of Cl− permeability and cell volume homeostasis. Methods: Release of ATP (luciferase-luciferin assay), Cl− currents (whole-cell patch clamp), and cell volume (Coulter Multisizer) were measured in human hepatocytes within 12 h of isolation. Results: Hepatocyte swelling increased bioluminescence from basal values of 11.21±0.45 to 178.29±44.49 and 492.15±89.41 arbitrary light units following 20 and 40% buffer dilutions, respectively (p<0.001), representing an increase in extracellular ATP from 10 to >300 nM. Whole-cell Cl− currents activated during exposure to hypotonic buffer (15% less mosmol, 126.34±36.49 pA/pF) and ATP (10 μM, 71.92±15.48 pA/pF) exhibited outward rectification, time-dependent inactivation at depolarizing potentials, and sensitivity to the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Removal of extracellular ATP (apyrase) prevented volume-sensitive current activation. Exposure to hypotonic buffer (30% less mosmol) increased mean relative volume to 1.092±0.004 by 2.5 min, and volume recovery (1.019±0.002 by 30 min) was abolished by NPPB, apyrase, and the P2 receptor antagonist suramin. Conclusions: These findings indicate that human hepatocytes exhibit constitutive and volume-dependent ATP release, which is a critical determinant of membrane Cl− permeability and cell volume regulation. ATP release may represent an extracellular signaling pathway that couples the cellular hydration state to important hepatic functions.