Volume-Sensitive Anion Channels Mediate Osmosensitive Glutathione Release from Rat Thymocytes

Abstract

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 PGSH/PCl 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.

Mechanical stretch exacerbates the cell death in SH-SY5Y cells exposed to paraquat: mitochondrial dysfunction and oxidative stress

“…These results suggest that lower levels of stretch may trigger ROS production in the cytosol, as well to deplete intracellular GSH. It is also possible that stretch might be affecting GSH synthesis and metabolism or that GSH depletion might also be mediated by stretch activated efflux pathways (Franco and Cidlowski, 2012, Sabirov et al, 2013). …”

Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel

“…For patch-clamping, the cells were cultured in suspension under mild stirring during 3-5 h. All animal experiments were conducted in accordance with the ARRIVE guidelines and approved by the Bioethics Committee of the Institute of Biophysics and Biochemistry. The isolation of thymic lymphocytes (thymocytes) was performed as described previously (Kurbannazarova et al, 2003(Kurbannazarova et al, , 2008(Kurbannazarova et al, , 2011Sabirov et al, 2013). Briefly, the 6-8 weeks old rats, kept in vivarium on an average diet, were anaesthetized with halothane or diethyl ether and painlessly euthanized by cervical dislocation.…”

“…In order to test this possibility, we employed the immature thymic lymphocytes which possess fully functional volume regulation machinery (Arrazola et al, 1993;Kurbannazarova et al, 2003;Soler et al, 1993). We have previously shown that thymocytes express the VSOR channels with the same biophysical and pharmacological profile as other cell types, and that VSOR blockers completely abolish RVD in these cells (Kurbannazarova et al, 2011;Sabirov et al, 2013). Therefore, we supposed that tannin preparations might affect the volume regulation in thymocytes.…”

Volume-regulated anion channel—a frenemy within the brain

“…The size of the pore was estimated based on the fit of the relative permeabilities of inorganic anions with known radii, or derived from electrophysiological recordings in the presence of pore permeating or blocking sulfonic-calix arenes and polyethylene glycol molecules with various diameters [51, 52, 200]. The VRAC pore may be suitable for conducting molecules as large as ATP [24, 85] and the tripeptide glutathione [178]. This channel poorly discriminates between various anions and neutral molecules due to the weak charge interactions of transported molecules with the amino acid moieties lining its vestibule.…”