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Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells¹

THERESE SÖDERDAHL^*, MARI ENOKSSON^,, MATHIAS LUNDBERG, ARNE HOLMGREN, OLE P. OTTERSEN, STEN ORRENIUS, GEORGE BOLCSFOLDI^|| and IAN A. COTGREAVE^*2

^* Division of Biochemical Toxicology;
Division of Toxicology, Institute of Environmental Medicine;
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm Sweden;
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Blindern, 0317 Oslo, Norway; and
^|| Department of Genetic Toxicology, AstraZeneca R and D Södertälje, Safety Assessment, S-15185 Södertälje, Sweden

²Correspondence: Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, S-17177, Stockholm, Sweden. E-mail: Ian.Cotgreave{at}I mm.ki.se

SPECIFIC AIMS

Many studies have alluded to the bulk levels of glutathione and oxidized glutathione as glutathione disulfide and protein-glutathione mixed disulfides in intact cells. A variety of intracellular glutathione pools have been postulated by use of broken cell preparations. These include the cytosolic pool, a separate pool in mitochondria, and a nuclear pool whose composition has been a matter of debate. So far, however, such compartmentalization has not been visualized in intact cells. The primary aim of the work was to combine the specificity of reaction of a polyclonal anti-glutathione antibody with the power of resolution of laser confocal microscopy and fluorescence-activated cell sorting (FACS) to illuminate the 3-dimensional compartmentalization of reduced glutathione (GSH) in cultured cells. The possibility of visualizing S-glutathionylation of cellular protein formed during intracellular oxidative stress was to be explored. Variations in these parameters within populations of cells and organelles and with respect to the cell cycle were the subject of study.

PRINCIPAL FINDINGS

Cytosolic and nuclear concentrations of glutathione lie near equilibrium and a cytosolic concentration gradient in glutathione exists in intact cells. Topological analysis of the cellular GSH content of a variety of cell lines immunochemically stained with an anti-GSH antibody by standard fluorescence microscopy revealed a discontinuous pattern across the cell, with the nucleus appearing as a "black hole". However, confocal microscopic analysis of the cells showed that the peripheral cytosolic and nuclear contents of GSH are close to equilibrium at a point halfway through the z-plane of A549 cells. Comparisons of images taken from serial z-sections of the cells display a gradient of staining within the peripheral cytosol, suggesting that concentrations of the tripeptide rise transiently to a maximum toward the basolateral surface of the cells (Fig. 1 ).

View larger version (61K): [in this window] [in a new window]   Figure 1. Laser confocal imaging of cells stained with an anti-GSH antibody. A549 cells were fixed, stained, and imaged using laser confocal microscopy. The images are obtained in 0.7 µm cuts of the z-plane, starting from the apex of the cell (top left image), moving right toward the basolateral surface (bottom right-hand image).

The mitochondrial compartment is confirmed as possessing the highest GSH concentration in intact cells
Fluorescence microscopic analysis of A549 and other cells stained with the anti-GSH antibody revealed intense punctate staining, diffuse in the perinuclear region of the cell. This colocalized well with staining for the mitochondrial enzyme marker cytochrome oxidase. Inspection of the laser confocal images of A549 cells in Fig. 1 show this intense perinuclear punctate staining, whose fluorescence intensity across the z-plane, halfway between the apex of the cell and the basolateral surface, was more than twice that of the peripheral cytosol and nucleus at this level. Taken together, these data indicate that individual mitochondria constitute the area of the cell displaying the highest glutathione concentrations within the intact cell.

Protein-glutathione mixed disulfides are regionally located during diamide-induced oxidative stress
Immunocytochemical staining of cells fixed and washed free of soluble glutathione species, then stained with the anti-GSH antibody, reveals a discontinuous staining pattern in cells treated with the thiol oxidant diamide under conditions known to promote S-glutathionylation of cellular proteins. The regions of most intense staining include membrane blebs, the nuclear area, and areas of punctate staining in the perinuclear region.

Levels of glutathione and protein-glutathione mixed disulfides vary considerably within cell and isolated mitochondrial populations and with the cell cycle
The combination of immunocytochemical staining and FACS can be applied to determine individual levels of GSH and protein-GSH mixed disulfides within cell populations and populations of subcellular organelles such as mitochondria. Suspension cultures of Jurkat T lymphocytes exhibit heterogeneity in their cellular content of GSH. Cell cycle-associated variations in GSH content were noted, rising steadily from G phase through S phase, reaching a maximum at G2/M. GSH content of isolated mitochondria of similar granularity varies considerably.

CONCLUSIONS

Glutathione is the major low molecular weight thiol-containing compound found in mammalian cells. The tripeptide possesses a variety of important biological functions related to its nucleophilic, redox-active thiol group ranging from partaking in conjugation reactions with reactive electrophilic metabolites of drugs and foreign chemicals, to dictating reductive, antioxidative reactions with reactive O- and N-centered reactive metabolites, and finally being involved in protein regulation by reversible S-glutathionylation of protein cysteines. Our knowledge of the intracellular complement of the glutathione redox couple is limited largely to bulk assays of the tripeptide, yielding an average concentration in an average of cells. Based largely on broken cell preparations, however, some evidence demonstrated the existence of separate cytosolic and mitochondrial pools of GSH in cells. Even so, direct visualization of the compartmentalization of intracellular GSH throughout the volume of the cell is lacking.

In this work we use a polyclonal anti-GSH antibody to illuminate the cellular glutathione compartmentalization in intact cells using fluorescence microscopy, laser confocal microscopy, and FACS analysis. Confocal analysis of stained A549 cells reveals that the nuclear region of the cell (Fig. 1) , which appears deficient in GSH in a variety of cell types using conventional microscopy, is actually close to equilibrium with the peripheral cytosol in A549 cells at a z-plane cut halfway through the cell. This contradicts previous data obtained with mercury orange staining and dynamic staining with various fluorescent cosubstrates for glutathione conjugation, which have demonstrated nuclear:cytosolic ratios ranging from 3:1 to 0.5:1. Our data thus seem to suggest that the nuclear pool of GSH in intact cells is maintained by diffusion from the cytosol, although we cannot exclude that import into the nucleus might be energy dependent.

Conventional fluorescence viewing of cells stained with anti-GSH antibody revealed a punctate staining pattern in the perinuclear region of the cytosol. This clearly colocalized with immunocytochemical staining for cytochrome oxidase. The intense mitochondrial staining against the peripheral cytosol and nucleus suggested that mitochondrial GSH levels are superior to those in other areas of surrounding compartments of the cell. This was strengthened when confocal images of A549 cells stained with the anti-GSH antibody were carefully inspected. They reveal intense perinuclear staining, beginning at the apex of the cell and working down in the z-plane. Again, intense punctate staining was particularly evident in z-plane images obtained closer to the median of the cell, indicating a higher concentration of mitochondrial GSH with respect to surrounding compartments, at least at these levels. Taken together, the data demonstrate that previous estimates of mitochondrial GSH levels obtained from broken cell mitochondrial preparations may have underestimated the mitochondrial GSH content in intact cells. It may be argued that this discrepancy is largely due to an artifactual loss of GSH, perhaps from the intermitochondrial space, during isolation of the organelle. This might be further investigated in ultrastructural studies using immunogold staining with the GSH-specific antibody. However, in the present work, FACS studies with isolated mitochondria show considerable heterogeneity in the level of GSH staining within the isolate. The demonstration of high levels of GSH associated with the mitochondria of intact cells would be logical in terms of the importance of this organelle as a major site of oxygen consumption and reactive oxygen metabolite production in aerobic cells. Conversely, it may be that most attempts to study the potential role of GSH in controlling redox-dependent events, such as the regulation of mitochondrial membrane potential and events leading to release of cytochrome c in apoptosis, are being routinely performed in organelles with severely compromised thiol homeostasis.

It has generally been assumed that the cytosolic compartment of the cell is a water-filled continuum, in which small solutes are dissipated uniformly throughout the phase. Inspection of the z-plane images of GSH levels in the cytosol, particularly closer to the cell periphery, indicates the presence of localized concentration gradients in GSH within the cytosol. This is most evident in the z-plane of the imaging, where staining intensity transiently rises to a maximum as the images progress from the upper quadrant of the cell to the lower quadrant (Fig. 1) . Glutathione is a highly charged molecule and theoretically can form aggregates in the aqueous phase of the cytosolic gel. Such self-association or loose electrostatic association with other cellular components, particularly cytoskeletal constituents, may underlie regional difference in GSH concentration in these cells.

Disturbance in intracellular redox potential are usually accompanied by a shift in the reduced:oxidized GSH state in the cell. Having demonstrated intracellular compartmentalization of GSH in intact cells with the antibody labeling technique, we probed the possible application of the technique to determine oxidized GSH species in the cell. As the antibody is not able to distinguish between GSH and its oxidized form, GSSG, we were limited in this effort to attempts to visualize GSH bound to protein thiols in a mixed disulfide (PrSSG). Rapid fixation of control A549 cells with methanol:acetone (1:1), followed by washing with PBS, resulted in removal of all soluble low molecular weight glutathione species. This was confirmed with a conventional HPLC assay of cellular GSH. When cells were treated with diamide, a residual of immunoreactivity remained within the cells, representing PrSSG, which is known to be formed during diamide metabolism in these. To our knowledge, this is the first time these species have been directly visualized in cells. Once again, the images reveal a regional distribution of the antibody staining, indicating compartmentalization. One feature immediately apparent is the intense staining associated with plasma membrane blebs. This may be speculated to be due to the accumulation of oxidized cytoskeletal elements, such as actin, in the blebs. Similarly intense perinuclear staining was visualized, as was nuclear staining, indicating considerable accumulation of protein-GSH mixed disulfides.

When this procedure was applied to suspensions of Jurkat T lymphocytes and control and diamide-treated cells were analyzed by FACS, the data revealed population differences in the control GSH content of the cells and in the protein-GSH mixed disulfide levels of the cells formed in response to diamide metabolism. The variation in basal GSH levels of the cells may be related to the position of individual cells in the cell cycle and may dictate the variations in mixed disulfide levels formed in the cells in response to diamide. Indeed, using double labeling with propidium iodide and the anti-GSH antibody, we demonstrated cell cycle-associated variations in GSH content rising steadily from G phase, through S phase, and reaching a maximum at G2/M (Fig. 2) . These FACS data reveal the suitability of the antibody labeling technique for rapid determination of GSH redox balance in populations of cells which may potential comprise of many distinct cell types, such as peripheral blood lymphocytes. Finally, we could demonstrate population variation in the GSH content of isolated mitochondria or similar granularity using FACS. These data can be interpreted in terms of either selective loss during isolation or variation in GSH content with maturity of the organelle and/or their intracellular origins. Thus, caution should be applied in interpreting results made on the role of GSH in controlling biological and toxicological processes in nonsynchronous cultures and in isolated mitochondrial preparations

View larger version (26K): [in this window] [in a new window]   Figure 2. Schematic representation of application of the anti-GSH antibody to the quantitation of GSH and protein-GSH mixed disulfides in cells and organelles. The relative sizes of GSH and PrSSG represent the relative levels of these species at each level of biological organization studied.

In conclusion, the methods described allow us to illuminate more closely the organization of intracellular GSH in intact cells, the variation in levels of GSH in homogeneous and heterogeneous cells and organelle populations, and the disposition of GSH in such systems with respect to reversible S-glutathionylation of protein. Application of the techniques may therefore add new dimensions to our understanding of the biochemistry of this important cellular mediator (Fig. 2 ).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0259fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0259fje

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