Volume 118, Issue 2, Pages 395-403 (February 2000) Functional interactions between oxidative stress, membrane Na+ permeability, and cell volume in rat hepatoma cells  Thorsten Schlenker*,‡, Andrew P. Feranchak§, Lukas Schwake‡, Wolfgang Stremmel‡, Richard M. Roman*, J.Gregory Fitz*  Gastroenterology  Volume 118, Issue 2, Pages 395-403 (February 2000) DOI: 10.1016/S0016-5085(00)70222-8 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 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 were placed on the stage of an inverted microscope and viewed on a video monitor. Changes in CSA were recorded after exposure to DAO (added at t = 0) to stimulate local generation of ROS. Data are presented as mean ± SE (n = 4) of relative values over time, where the value at t = 0 minute equals 1. (B) The effect of hypertonic exposure was assessed in an analogous manner. Initially cells were exposed to hypertonic buffer containing 50 mmol/L sucrose in which Na+ was replaced by Tris+. After 7.5 minutes, the perfusate was switched to an Na+-containing buffer with the same sucrose concentration. There was no recovery of CSA in the absence of Na+. In the presence of Na+, CSA showed partial recovery despite the continued presence of sucrose (n = 7). Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of cell volume and oxidative stress on membrane Na+ permeability. Whole-cell currents were recorded from a holding potential of −40 mV and at test potentials of 0 and −80 mV. (A) In the presence of Na+, decreases in cell volume caused by hypertonic exposure (addition of 50 mmol/L sucrose) activated large inward currents at −80 mV. Currents were eliminated when bath Na+ was replaced with Tris+ (0 Na+). (B) Similar currents were activated by enzymatic generation of ROS in the absence of an osmolar challenge (isotonic buffer containing 1 mmol/L D-alanine [DAA] + 40 mU/mL DAO). Currents were eliminated when bath Na+ was replaced with Tris+ (0 Na+). (C) Current-voltage relations from the cell shown in B. Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Current-voltage relationships for ROS-activated currents. Whole-cell current-voltage relationships were measured after exposure to ROS generated by D-alanine + DAO. Extracellular buffers contained either Na+ (○), K+ (▾), or Tris+ (▿) as the major cation. •, Basal. Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Concentration-dependent activation of currents. (A) Using the standard solutions described in Materials and Methods, maximal current density at a potential of −80 mV was measured at different bath H2O2 concentrations (n = 3–15 for each point). Currents activated by oxidative stress were fully reversible on return to control conditions. (B) Preincubation with BSO (1 mmol/L, •) to cause glutathione depletion caused a marked increase in the sensitivity to H2O2. In control cells, H2O2 failed to activate currents at concentrations of <10−5 mol/L (○). Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of intracellular glutathione on the current response to oxidative stress. Maximal current density produced by exposure to H2O2 (10−2 mol/L, left) and enzymatic generation of ROS (right) were measured in the absence (2, n = 4) vs. presence of glutathione (10 mmol/L, ▨, n = 4). Glutathione was delivered to the cell interior by inclusion in the pipette solution and substantially inhibited the current response to oxidative stress. Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 ROS release by PMNs activates Na+ conductance. HTC cells were coincubated with PMNs. Currents were small during basal conditions. (A) Release of ROS from PMNs stimulated by addition of 1 nmol/L PMA resulted in activation of inward currents. (B) Average maximal current density at −80 mV was measured in cells coincubated with PMNs under basal conditions and after stimulation of ROS release by PMA (control). Preincubation of HTC cells with BSO to inhibit glutathione synthase caused a small increase in the response to ROS. Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 ROS release by PMNs activates Na+ conductance. HTC cells were coincubated with PMNs. Currents were small during basal conditions. (A) Release of ROS from PMNs stimulated by addition of 1 nmol/L PMA resulted in activation of inward currents. (B) Average maximal current density at −80 mV was measured in cells coincubated with PMNs under basal conditions and after stimulation of ROS release by PMA (control). Preincubation of HTC cells with BSO to inhibit glutathione synthase caused a small increase in the response to ROS. Gastroenterology 2000 118, 395-403DOI: (10.1016/S0016-5085(00)70222-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions