Gastroenterology

Gastroenterology

Volume 118, Issue 2, February 2000, Pages 395-403
Gastroenterology

Liver, Pancreas, and Biliary Tract
Functional interactions between oxidative stress, membrane Na+ permeability, and cell volume in rat hepatoma cells,☆☆

https://doi.org/10.1016/S0016-5085(00)70222-8Get rights and content

Abstract

Background & Aims: Oxidative stress leads to a rapid initial loss of liver cell volume, but the adaptive mechanisms that serve to restore volume have not been defined. This study aimed to evaluate the functional interactions between oxidative stress, cell volume recovery, and membrane ion permeability. Methods: In HTC rat hepatoma cells, oxidative stress was produced by exposure to H2O2 or D-alanine plus D-amino acid oxidase (40 U/mL). Results: Oxidative stress resulted in a rapid decrease in relative cell volume to 0.85 ± 0.06. This was followed by an ~100-fold increase in membrane cation permeability and partial volume recovery to 0.97 ± 0.05 of original values. The volume-sensitive conductance was permeable to Na+ ≃ K+ >> Tris+, and whole-cell current density at −80 mV increased from −1.2 pA/pF at 10−,5 mol/L H2O2 to −95.1 pA/pF at 10−,2 mol/L H2O2. The effects of H2O2 were completely inhibited by dialysis of the cell interior with reduced glutathione, and were markedly enhanced by inhibition of glutathione synthase. Conclusions: These findings support the presence of dynamic functional interactions between cell volume, oxidative stress, and membrane Na+ permeability. Stress-induced Na+ influx may represent a beneficial adaptive response that partially restores cell volume over short periods, but sustained cation influx could contribute to the increase in intracellular [Na+] and [Ca2+] associated with cell injury and necrosis.

GASTROENTEROLOGY 2000;118:395-403

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).

. Effect of oxidative stress on cell volume. (A) The effect of oxidative stress on cell area was assessed in cells superfused with D-alanine–containing buffer. Cells

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.).

    ☆☆

    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.

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