Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

Department of Environmental Medicine, Box EHSC, University of Rochester School of Medicine, 575 Elmwood Avenue, Rochester, NY 14642, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 07/2008; 1778(10):2413-20. DOI: 10.1016/j.bbamem.2008.06.011
Source: PubMed


The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.

Download full-text


Available from: Rosemarie Marchan, Oct 13, 2014
  • Source
    • "Cellular release of GSH is required for operation of the gamma-glutamyl cycle, in which GSH conjugated to extracellular amino acids plays a key role in amino acid uptake and metabolism (Meister and Powers, 1978; Viña et al., 1989) and protection against oxidative damage (Vargas and Johnson, 2009). Members of the VRAC family of channels and MDR transporters have been implicated in GSH release (Marchan et al., 2008; Sabirov et al., 2013). Flux of GSH through Cx26 hemichannels to the extracellular environment, driven by its 1,000-fold concentration gradient, may have important physiological implications in the inner ear, liver, and skin. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC. © 2015 Tong et al.
    Full-text · Article · Aug 2015 · The Journal of General Physiology
  • Source
    • "First is the family of multidrug resistance-associated proteins (ABCC/MRP), which are implicated in high constitutive GSH efflux in the liver [19], [20], [68] and brain astrocytes [21], [69]. MRP1-overexpression studies with BHK [46] and HEK293 [50] cells as well as siRNA-mediated MRP1 gene silencing in Jurkat cells [54] supported the notion of MRP-mediated apoptotic GSH release. In our present study, however, an MRP inhibitor, probenecid, produced stimulation rather than inhibition of the swelling-induced GSH release from rat thymocytes arguing against the contribution of this pathway in immature thymic lymphocytes. "
    [Show abstract] [Hide abstract]
    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 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.
    Full-text · Article · Jan 2013 · PLoS ONE
  • Source
    • "One of the mechanisms whereby cells maintain their redox status is by maintaining the GSH/GSSG ratio. The transporters involved in GSH release remain largely unknown, however, some studies describe involvement of MRPs in the transport of GSH and GSSG [15], [30], [35], [36], MRP1 is expressed in all mammalian cell types and is well characterized [9], [37]. Our data demonstrate that MRP1 is an effective transporter of GSH/GSSG in RPE cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.
    Full-text · Article · Mar 2012 · PLoS ONE
Show more