Glutamate and GSH permeability of the whole-cell currents activated by cell swelling in rat thymocytes.

<p>(A) Whole-cell current responses to ramp pulses from −70 to +70 mV before and after replacement of extracellular Cl⁻ with glutamate. The pipette solution was Cl⁻-rich (125 mM CsCl); the bath solution was normal Ringer (curve 1) or low-Cl⁻ Ringer solution in which 135 mM NaCl was replaced with 135 mM Na-glutamate (curve 2). (B) Whole-cell current responses to ramp pulses from −70 to +70 mV before and after replacement of extracellular Cl⁻ with GSH. The pipette solution was Cl⁻-rich (125 mM CsCl); the bath solution was the normal Ringer (curve 1) or low-Cl⁻ Ringer solution in which 135 mM NaCl was replaced with 135 mM Na-GSH (curve 2). (C) Instantaneous whole-cell I–V relationships in response to step pulses in 20 mV increments. The pipette solution was Cl⁻-rich (curve 1: 125 mM CsCl) or GSH⁻-rich (curve 2: 100 mM Cs-GSH plus 25 mM CsCl). The bath solution was the normal Ringer solution. Averaged data of 6 experiments performed in each configuration are plotted.</p

Osmosensitive release of GSH from rat thymocytes.

<p>(A) Dependence of the GSH concentration in the extracellular fluid on the number of cells in suspension under control isotonic (290 mosmol/kg-H₂O) conditions (filled symbols) and upon hypotonic (147 mosmol/kg-H₂O) stimulation (open symbols). The cells were incubated 10 min at 25^oC. (B) Time course of GSH release from rat thymocytes under control isotonic conditions (filled symbols) and upon hypotonic (147 mosmol/kg-H₂O) stimulation (open symbols). The suspension containing 1 x 10⁸ cells/ml was incubated at 25^oC for the indicated time. (C) Osmolality dependence of GSH release from rat thymocytes. The suspension containing 1 x 10⁸ cells/ml was incubated at 25^oC for 20 min in solutions with various tonicity. *Significantly different from the isotonic control values at P<0.05.</p

Whole-cell macroscopic currents activated by cell swelling in rat thymocytes.

<p>(A) Time course of whole-cell current activation in response to cell swelling. Currents were elicited by application of alternating pulses from 0 to ±25 mV (every 5 s). (B) Representative traces of current responses recorded at the steady-state in (A) marked with # symbol. The holding potential was 0 mV; after a pre-pulse to −100 mV (500 ms), currents were elicited by application of step pulses (1000 ms) from −100 to +100 mV in 20-mV increments. (C) Instantaneous I-V relationship measured at the beginning of test pulses from recordings similar to those shown in (B). (D) Effects of VSOR channel blockers, phloretin (200 µM) and DCPIB (10 µM), on the whole-cell macroscopic currents activated by cell swelling in rat thymocytes. Open and filled bars represent the fractional currents measured at +25 mV and −25 mV, respectively. *Significantly different from the control values at P<0.05.</p

Effects of ambient temperature on the osmosensitive release of GSH from rat thymocytes.

<p>(A) The suspension containing 1×10⁸ cells/ml was incubated at indicated temperature for 10 min. All values measured in hypoosmotic conditions were significantly different from the isotonic control values at P<0.05. (B) The data from (A) are presented as an Arrhenius plot. The solid lines are linear fits with the activation energies given in the text.</p

Effects of organic anion transporter substrates and inhibitors on the swelling-induced GSH release from rat thymocytes.

<p>Stimulating effects of the SLCO/OATP substrates, probenecid (A), taurocholic acid (B) and estrone sulfate (C), and a suppressing effect of the inhibitor of SLC23A/OAT transporter, <i>p</i>-aminohippuric acid (PAH), alone and in combination with VSOR blockers, phloretin (200 µM) (D) and DCPIB (20 µM) (F) on the net GSH release from rat thymocytes upon hypotonic stimulation are shown. The suspension containing 1×10⁸ cells/ml was incubated for 20 min at 25 °C in the hypoosmotic (147 mosmol/kg-H₂O) conditions in the absence of any drug (control) or presence of the above-indicated drugs used at the concentration of 500 µM (A), 2 mM (B and C) and 1 mM (D and F). In order to minimize day-to-day variations, the results shown in (D) and (F) were obtained within one experiment performed in one day. *Significantly different from the control values at P<0.05. ^#Significantly different from phloretin (D) or DCPIB (F) alone at P<0.05.</p

Hypothetical pathways that contribute to the GSH release from swollen thymocytes.

<p>The scheme depicts three main pathways of the osmotic swelling-induced GSH release revealed in the present study: VSOR anion channel (sensitive to DCPIB and phloretin), SLC22A/OAT-like transporter (sensitive to PAH), and SLCO/OATP-like transporter (stimulated by the substrates such as probenecid, taurocholate and estrone sulfate). Significant contribution of the MRP protein (sensitive to MK571 and probenecid) was not detected in the present study. The grey box denotes other possible routes for GSH exit from swollen thymocytes.</p

Effects of anion channel blockers on the GSH release from rat thymocytes upon hypotonic stimulation.

<p>The suspension containing 1×10⁸ cells/ml was incubated for 20 min at 25 °C in the hypoosmotic (147 mosmol/kg-H₂O) conditions in the absence of any drug (control) or presence of 200 µM SITS (A), 200 µM NPPB (B), 50 µM Gd³⁺ (C), 200 µM phloretin (D), 20 µM DCPIB (E) and 200 µM glibenclamide (F). *Significantly different from the control values at P<0.05.</p