Volume-sensitive anion channels mediate osmosensitive glutathione release from rat thymocytes.

Glutathione (GSH) is a negatively charged tripeptide, which is a major determinant of the cellular redox state and defense against oxidative stress. It is assembled inside and degraded outside the cells and is released under various physiological and pathophysiological conditions. The GSH release mechanism is poorly understood at present. In our experiments, freshly isolated rat thymocytes were found to release GSH under normal isotonic conditions at a low rate of 0.82±0.07 attomol/cell/min and that was greatly enhanced under hypoosomotic stimulation to reach a level of 6.1±0.4 attomol/cell/min. The swelling-induced GSH release was proportional to the cell density in the suspension and was temperature-dependent with relatively low activation energy of 5.4±0.6 kcal/mol indicating a predominant diffusion mechanism of GSH translocation. The osmosensitive release of GSH was significantly inhibited by blockers of volume-sensitive outwardly rectifying (VSOR) anion channel, DCPIB and phloretin. In patch-clamp experiments, osmotic swelling activated large anionic conductance with the VSOR channel phenotype. Anion replacement studies suggested that the thymic VSOR anion channel is permeable to GSH(-) with the permeability ratio P(GSH)/P(Cl) of 0.32 for influx and 0.10 for efflux of GSH. The osmosensitive GSH release was trans-stimulated by SLCO/OATP substrates, probenecid, taurocholic acid and estrone sulfate, and inhibited by an SLC22A/OAT blocker, p-aminohippuric acid (PAH). The inhibition by PAH was additive to the effect of DCPIB or phloretin implying that PAH and DCPIB/phloretin affected separate pathways. We suggest that the VSOR anion channel constitutes a major part of the γ-glutamyl cycle in thymocytes and, in cooperation with OATP-like and OAT-like transporters, provides a pathway for the GSH efflux from osmotically swollen cells

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

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

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

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