Liver, Pancreas, and Biliary TractFunctional interactions between oxidative stress, membrane Na+ permeability, and cell volume in rat hepatoma cells☆,☆☆
Section snippets
Cell isolation and culture
All studies were performed using HTC cells from rat hepatoma. These cells exhibit volume-regulatory mechanisms similar to primary cells but provide a more stable and reproducible model for electrophysiological recording.19 Cells were maintained in culture at 37°C in 5% CO2 in HCO3−-containing modified minimal essential medium as described previously.19 Human PMNs were isolated from peripheral blood by density centrifugation and were provided by J. M. McCord, Ph.D., as described previously.20
Patch clamp recordings
Effect of oxidative stress on cell volume
To estimate the time course of short-term exposure to oxidants on cell volume, the CSA of single cells was measured during basal conditions and after exposure to either H2O2 (10−3 mol/L) or D-alanine plus DAO. As shown in Figure 1, oxidant exposure caused a rapid initial decrease in relative CSA to 0.56 ± 0.07 (1 minute; P < 0.001).
Discussion
Liver cells are subject to wide changes in solute transport and metabolism between the fed and fasted states. Under basal conditions, membrane Na+ permeability is low. However, exposure to certain hormones (e.g., vasopressin) or physiological decreases in cell volume stimulate Na+ influx as a result of opening of nonselective cation channels in the plasma membrane. The present studies of a model liver cell line support the presence of dynamic functional interactions between oxidative stress,
Acknowledgements
The authors thank Joe M. McCord, Ph.D. (Webb Waring Institute, University of Colorado Health Sciences Center, Denver, Colorado) for helpful discussions and strategies for experimental design.
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Supported in part by DFG grants SCHL 380/1-1 and SCHL 380/2-1 (to T.S.); Cystic Fibrosis Foundation (to A.P.F.); and National Institutes of Health grants DK43278 and DK46082 and Waterman Foundation (to J.G.F.).
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Address requests for reprints to: J. Greg Fitz, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Campus Box B-158, Denver, Colorado 80262. e-mail: [email protected]; fax: (303) 315-5711.