Glutathione release from cultured brain cells: Multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells

Abstract

To investigate the release of glutathione (GSH) from brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells derived from rat brain were studied. During incubation of astroglial cultures, GSH accumulated in the medium with a rate of 3.1 +/- 0.6 nmol x h(-1) x mg protein(-1). In contrast, only marginal amounts of extracellular GSH were detectable in the media of the other brain cell cultures investigated. The mechanism of GSH release from astroglial cells, as yet, has not been reported. Multidrug resistance protein 1 (Mrp1), a transport protein known to mediate cellular export of glutathione disulfide and glutathione conjugates, is expressed in astroglial cultures. Inhibitors of Mrp1 were used to test for a function of this transporter in mediating GSH release from astroglial cells. The presence of the competitive Mrp1 inhibitor MK571 at a concentration of 50 microM inhibited the rate of GSH release by 63%. In contrast, the low concentration of 1 microM of MK571 increased the rate of GSH release by 83%. This bimodal concentration-dependent effect of MK571 is in accord with literature data for the effects of Mrp1 substrates on GSH release from cells. In addition, the presence of cyclosporin A (10 microM) reduced the GSH release rate significantly and completely blocked the stimulating effect of 1 microM MK571 on the release of GSH from astroglial cells. In conclusion, the data presented are a strong indication that Mrp1 participates in the release of GSH from astroglial cells.

Two-photon Imaging of Glutathione Levels in Intact Brain Indicates Enhanced Redox Buffering in Developing Neurons and Cells at the Cerebrospinal Fluid and Blood-Brain Interface

“…In contrast, in astrocyte cell culture at 37°C the conjugate was largely effluxed from cells. Astrocyte efflux could be inhibited by treatment with MK571, a blocker of multidrug resistance proteins (Mrps) (13,21,22). In brain slice experiments at room temperature, we observed no significant effect of MK571 (50 M) inhibition of Mrps on MCB fluorescence ( p Ͼ 0.05 from five separate slice experiments).…”

Multidrug Resistance-Associated Proteins: Expression and Function in the Central Nervous System

“…Addition of 20 M MK571 to astrocyte cultures decreases GSSG efflux by 50%, suggesting the involvement of Mrp1 and/or another Mrp isoform. Efflux of reduced GSH by astrocytes is also inhibited by MK571 at concentrations Ͼ10 M (Hirrlinger et al, 2002c). Given these results, Mrp1 may play an essential role in maintaining GSH concentrations and redox balance of astrocytes during oxidative stress (Hirrlinger et al, 2001(Hirrlinger et al, , 2002c.…”

“…Given these results, Mrp1 may play an essential role in maintaining GSH concentrations and redox balance of astrocytes during oxidative stress (Hirrlinger et al, 2001(Hirrlinger et al, , 2002c. Incomplete inhibition of GSH and GSSG efflux from primary astrocyte cultures in the presence of high concentrations of MK571 (50 M) suggests that other transporters (e.g., Mrp4) might also contribute to thiol removal (Hirrlinger et al, 2002c).…”

Coordinate Regulation of Glutathione Biosynthesis and Release by Nrf2-Expressing Glia Potently Protects Neurons from Oxidative Stress

“…Of particular interest is the induction of many genes associated with the biosynthesis, use, and export of GSH, including the following: (1) uptake of cystine at the cell surface via the xCT cystine/glutamate antiporter, shown by RT-PCR (see Fig. 9) (Bannai, 1986;Sato et al, 1999); (2) synthesis of gGluCys by the rate-limiting enzyme for GSH synthesis ␥-GCS; (3) incorporation of Gly to gGluCys to make the complete GSH tripeptide by GSH synthetase; (4) use of GSH by various GSTs and glutathione reductase; (5) possible efflux of GSH via the multidrug resistance protein (MRP1), a mechanism previously described for astrocyte GSH release (Hirrlinger et al, , 2002); and (6) possible extracellular cleavage of GSH by ␥-glutamyl transpeptidase (␥GT). …”