TY - JOUR
T1 - Volume-sensitive purinergic signaling in human hepatocytes
AU - Feranchak, Andrew P.
AU - Fitz, J. Gregory
AU - Roman, Richard M.
N1 - Funding Information:
We would like to acknowledge Erik Schwiebert, Ph.D. (University of Alabama at Birmingham) for his gracious held and advice in setting up the luciferase assay, and Dennis Guenette (UCHSC) for preparation of human hepatocytes. This work was supported by the Cystic Fibrosis Clinical Fellowship Grant, The Children's Hospital Research Institute Professional Development Award, National Institute of Diabetes and Kidney Diseases Grants DK-46082, DK-43278, and K08 DK 02539-01 and by the Waterman Family Fund for Liver Research.
PY - 2000/8
Y1 - 2000/8
N2 - 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.
AB - 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.
KW - Cl channel
KW - Hepatocyte
KW - Luminescence
KW - P2 receptor
KW - Purinergic
KW - Volume
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U2 - 10.1016/S0168-8278(00)80357-8
DO - 10.1016/S0168-8278(00)80357-8
M3 - Article
C2 - 10952234
AN - SCOPUS:0033913902
SN - 0168-8278
VL - 33
SP - 174
EP - 182
JO - Journal of Hepatology
JF - Journal of Hepatology
IS - 2
ER -