1Medicine and 2Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303
Submitted 17 November 2003 ; accepted in final form 9 February 2004
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ABSTRACT |
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thiazine dyes; ascorbic acid; ferricyanide; phenylarsine oxide; oxidant stress; redox cycling
A cell surface site for MB+ reduction also brings into question the long-held notion that MB+ is directly reduced within cells by NADPH (29, 36). Clearly, NADPH generated in the pentose phosphate cycle is required for erythrocyte reduction of MB+, because cells of patients with glucose 6-phosphate dehydrogenase deficiency have impaired ability to take up the dye (36) and to carry out MB+-dependent methemoglobin reduction (43). This could be explained if NADPH or reducing equivalents derived from NADPH served as the cofactor for a transmembrane thiazine dye reductase that reduces MB+ on the cell surface. However, the substrate specificity of this activity has not been determined.
MBH that has entered or has been generated within cells can be oxidized by molecular oxygen (9, 25), but its major fate in cells is to reduce Fe3+ in prosthetic groups of numerous cellular proteins and enzymes (25). In erythrocytes, the major acceptor for electrons from MBH is Fe3+ in (met)hemoglobin (37), whereas in other cells, MBH donates electrons to heme groups of cytochrome c (25), guanylate cyclase (8), and nitric oxide synthase (24). Although MB+ accumulates in certain as yet unidentified organelles in endothelial cells (27), whether MB+ or MBH accumulates in erythrocytes is unclear. Although Sass et al. (36) could detect little MB+ in diluted suspensions of erythrocytes treated with MB+, blue staining of erythrocytes has been proposed as the basis for a clinical test for glucose 6-phosphate dehydrogenase deficiency (32).
Because the mechanisms of uptake or reduction of MB+ are relevant to its clinical use as well as in providing insight into cellular metabolism redox dyes, we studied the effects of MB+ in human erythrocytes. We report that, as in other cell types, there is extracellular reduction of MB+ and that MB+ accumulates as MB+ and MBH in erythrocytes as a function of its extracellular concentration. At MB+ concentrations >5 µM, MB+ generates an oxidant stress in the cells that consumes ascorbic acid. The transmembrane reduction of extracellular MB+ seems to be mediated by a protein, because it is sensitive to inhibition by thiol reagents and enzyme digestion in erythrocyte ghosts devoid of cytoplasm.
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MATERIALS AND METHODS |
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Preparation of erythrocytes and erythrocyte ghosts. Erythrocytes were prepared from blood obtained by venipuncture from healthy volunteers. The cells were washed three times in 10 volumes of PBS that contained 140 mM NaCl and 12.5 mM Na2PO4, pH 7.4. The buffy coat of white cells was removed with each wash. Where indicated, cells at a 5% packed cell volume were loaded with ascorbate by incubation for 15 min at 37°C with 0.2 mM freshly prepared dehydroascorbic acid (DHA). Leaky or clear erythrocyte ghosts were prepared from intact cells as previously described (40).
Assay of ferricyanide reduction. Reduction of extracellular ferricyanide to ferrocyanide was measured in aliquots of cell supernatants by the ortho-phenanthroline method of Avron and Shavit (2). Corrections were made as necessary in a paired sample for the absorbance generated in the presence of cells by the same concentration of MB+ without ferricyanide. At 20 µM MB+, this value was <20% of that observed in the presence of ferricyanide. Results are expressed relative to the intracellular water space, which was taken as 70% of the packed cell volume (31).
Uptake of MB+. The uptake and/or reduction of MB+ by erythrocytes was measured as the decrease in absorbance of cell supernatants after removal of cells by microfuging at 13,600 g for 1 min. Absorbance of MB+ was measured at 610 nm, and the MB+ concentration was calculated by using a measured extinction coefficient of 8.8 mM1·cm1 in PBS.
The intracellular content of MB+ and MBH was assessed as follows. Cells at a 10% packed cell volume were loaded with 40 µM MB+ in PBS containing 5 mM D-glucose for 30 min at 37°C. The cells were then pelleted in the cold in a microfuge, and the supernatant was then removed for measurement of extracellular MBH and MB+. The former was determined on the basis of the absorbance at 610 nm, and the latter was calculated as the difference between total dye concentration (determined after addition of 2 mM ferricyanide) and MB+ alone. The cells were then hemolyzed with 10 volumes of deionized water that contained either no additives or 2 mM ferricyanide. The hemolysate was ultrafiltered through a Centricon YM-10 membrane (Amicon, Beverly, MA) by performing centrifugation at 3°C for 10 min at 5,000 g. The concentration of MB+ was calculated on the basis of the absorbance at 610 nm. Dye that was bound to cell protein or lipid was measured as follows. The remaining hemolysate was treated with 0.1 ml of metaphosphoric acid, diluted 10-fold with 100 mM sodium phosphate (pH 8.0), and microfuged. The concentration of MB+ was determined on the basis of the absorbance at 610 nm. Each sample obtained from cells treated with MB+ was corrected for absorbance in a paired sample not exposed to MB+ that had or had not been treated with ferricyanide, as appropriate. This correction was <15% of the reading in MB+-treated cells.
Measurement of the ascorbate free radical by electron paramagnetic resonance spectroscopy. Time-dependent changes in ascorbate free radical (AFR) concentration were measured by electron paramagnetic resonance spectroscopy as previously described (21).
Assay of radiolabeled glucose metabolism in human erythrocytes. Glucose metabolism through the pentose phosphate pathway was measured as the generation of 14CO2 from D-[1-14C]glucose, as previously described in erythrocytes (6).
Other assays. Intracellular concentrations of glutathione (GSH) were measured according to the method of Hissin and Hilf (11), as previously described (22). The recycling method of Zerez et al. (44) was used to measure pyridine nucleotide concentrations in 2-ml lysates of erythrocytes, except that a concentration of 1 mM (rather than 2 mM) phenazine ethosulfate was used, as recommended by Wagner and Scott (42). Intracellular ascorbic acid was measured as previously described (18) by performing HPLC with electrochemical detection (23), except that tetrapentylammonium bromide was used as an ion pair reagent. Intracellular concentrations of GSH, pyridine nucleotides, and ascorbate were expressed relative to the intracellular water space as noted in Assay of ferricyanide reduction.
Data analysis. Results are shown as means ± SE. Differences between treatments were analyzed with SigmaStat 2.0 software (Jandel Scientific, San Rafael, CA). Comparisons of more than two treatments were made by using either one- or two-way ANOVA with post hoc testing by the Dunnett test. P < 0.05 was considered significant.
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RESULTS |
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Correlation of MB+ uptake with ferricyanide reduction.
The reduction and uptake of MB+ were measured as the decrease in its concentration in the incubation medium with time. In the presence of D-glucose, the MB+ concentration outside the cells gradually decreased during 30 min of incubation (Fig. 2A, ). This could be due either to reduction of MB+ to the colorless MBH outside the cells or to uptake of MB+ or MBH into the cells, or both. When D-glucose was omitted from the incubation, the extracellular MB+ concentration did not change (Fig. 2A,
). This finding is in agreement with the decrease in MB+-stimulated ferricyanide reduction in the absence of D-glucose, shown in Fig. 1. These results confirm those shown in Fig. 1B in that reducing equivalents from glucose metabolism are necessary for sustained reduction of MB+.
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The question of whether the dye accumulated in the cells as MB+ or as MBH was addressed by measuring MB+ in erythrocyte hemolysates, as shown in Table 1. After 30 min of incubation of 10% packed cells with 38 µM MB+, the cells took up and/or reduced 71 ± 6% of the dye. One-half of the intracellular dye was free, and one-half was bound to proteins and/or lipids in the hemolysate. The fact that acid treatment released the dye suggests that binding was ionic, but it was not possible to determine the redox state of the bound dye. The cells concentrated free MB+ 14-fold, free MBH 4.3-fold, and bound dye 18-fold. These findings confirm a marked accumulation of both forms of the dye against a concentration gradient.
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PAO was first used to assess the extent to which MB+-dependent ferricyanide reduction might be mediated by the transmembrane ferricyanide reductase activity that uses intracellular ascorbate as a source of electrons (31). To allow direct comparison, results were expressed as fractions of ascorbate- or MB+-stimulated activity, corrected for basal ferricyanide reduction (Fig. 6). At a 20 µM concentration, MB+ increased ferricyanide reduction 2.6-fold compared with treatment of cells with ferricyanide alone, from 6.0 ± 0.5 to 15.9 ± 1.5 µmol·ml cells1·30 min1. MB+-stimulated ferricyanide reduction decreased significantly at 10 µM PAO and was decreased maximally by >90% at 50 µM PAO. Preloading the cells with 0.5 mM DHA to yield intracellular ascorbate concentrations of 1.52 mM (20) increased ferricyanide reduction 4.8-fold, from 4.6 ± 1.0 to 22 ± 3.8 µmol·ml cells1·30 min1. Ascorbate-stimulated ferricyanide reduction was significantly decreased with 20 µM PAO, but it was inhibited by only 27% with 100 µM PAO. This contrasts with nearly complete inhibition of MB+-stimulated ferricyanide reduction and suggests that the two activities are not mediated by the same protein.
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Involvement of a membrane protein in MB+ reduction.
Erythrocyte ghosts lacking hemoglobin and other cytosolic components were prepared and incubated with increasing concentrations of MB+ in the presence of NADH or NADPH as electron donors. Because of the low absorbance of MB+ and MBH at 340 nm (results not shown), it was possible to follow NAD(P)H oxidation at this wavelength in the presence of MB+. Rates of NADH and NADPH oxidation were linear for 3 min (results not shown) and increased with increasing MB+ concentrations (Fig. 8A). The relative increases in NADH and NADPH oxidation rates were similar, although the rate for NADPH appeared to level off. For both nucleotides, the rates were corrected for direct oxidation of NADH and NADPH by MB+, which were linear and 22 ± 4% and 16 ± 3% of the total rate at 10 µM MB+, respectively. These results show a saturable increase in NAD(P)H oxidation that is best explained by the activity of a membrane-bound enzyme. To further investigate this protein, we studied its sensitivity to trypsin and to PAO.
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DISCUSSION |
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Redox cycling of MB+ is readily quantified as MB+-stimulated ferricyanide reduction. At an initial extracellular concentration of 10 µM, correcting for basal rates of ferricyanide reduction and considering that 1 mole of MBH reduces 2 moles of ferricyanide, erythrocytes at a 5% packed cell volume reduce
88 nmol of MB+ for 30 min in the absence of D-glucose and 225 nmol in the presence of D-glucose (Fig. 1B). The latter is >20 times the amount of MB+ in the system. This is likely the reason that MBH is also strongly concentrated against a gradient in the cells. Paradoxically, it is likely the high capacity of the cells to reduce MB+ and generate MBH, not the direct effects of MB+ itself, that causes excess oxidant stress. That both NADH and NADPH are severely depleted by low extracellular MB+ concentrations (Fig. 3) demonstrates their crucial role in MB+ reduction. In addition, our results point to contributions by both ascorbate and GSH in MB+ recycling (Fig. 4). Although MB+ can be reduced directly by both ascorbate (Fig. 5A; see Ref. 35) and GSH (13), the glucose dependence and depletion of both at higher MB+ concentrations may also reflect ascorbate scavenging of intracellular ROS, with subsequent GSH-dependent reduction of ascorbate (Fig. 4).
Whereas activation of the pentose phosphate cycle by NADP+ generated by NADPH oxidation is clearly required to sustain MB+ reduction (5, 38), a role for NADH has been suggested only indirectly in studies using glycolytic inhibitors in erythrocytes (37) and in pulmonary artery endothelial cells (26). Our results, showing nearly complete oxidation of NADH by low concentrations of MB+ (Fig. 3B), indicate that NADH generated in glycolysis also plays a role. The differential sensitivity to oxidation of NAD(P)H compared with ascorbate and GSH by MB+ in glucose-utilizing erythrocytes suggests, in fact, that low concentrations of MB+ might be a good tool with which to selectively deplete the pyridine nucleotides.
The present findings are in accord with the results in endothelial cells pointing to a transmembrane thiazine dye reductase as the initial mechanism for MB+ reduction to MBH, which then diffuses into the cells. That this activity is mediated by a protein is indicated by its sensitivity to inhibition by thiol reagents. This may in part reflect the dependence of a crucial protein thiol on GSH or on the cell redox state, because DEM should have some specificity for GSH alone. The results with PAO also suggest that there are sensitive vicinal thiols on the protein, especially because PAO inhibited MB+-dependent ferricyanide reduction more strongly than it decreased GSH. The specificity of PAO is further indicated by its lack of effect on NADPH or on pentose phosphate cycle activity at concentrations effective for inhibition of MB+-stimulated ferricyanide reduction (Fig. 7). PAO strongly inhibited MB+-dependent NADH oxidation in erythrocyte ghosts (Fig. 8C), which supports the notion that the ghost activity might correspond to reduction of MB+ by cells.
The identity of the putative transmembrane thiazine dye reductase remains to be established. Merker et al. (28) concluded that because it was not activated by intracellular ascorbate in endothelial cells, it did not correspond to the transmembrane ascorbate-dependent ferricyanide oxidoreductase. We provide additional evidence that the two activities are mediated by different proteins, with the observation that MB+ reductase is much more sensitive than the ascorbate-dependent activity to inhibition by PAO (Fig. 6). It is possible that the thiazine dye reductase corresponds to the MB+ reductase activity that we were able to measure in erythrocyte ghosts. The latter likely reflects the activity of an enzyme, because it was saturable with MB+ and inhibited by both trypsin and PAO. Inhibition by trypsin in ghosts but not in intact cells suggests that the protein is mostly exposed on the cytosolic membrane face. Because either NADH or NADPH was a substrate for the ghost activity, the observed depletion of NAD(P)H in intact cells by low concentrations of extracellular MB+ (Fig. 3) suggests that pyridine nucleotides are substrates for the initial extracellular reduction of MB+. It is possible to compare the rates of ghost NAD(P)H-dependent MB+ reductase with those of ferricyanide reduction in cells, because the latter reflect only rates of MB+ reduced at the cell surface. At 10 µM MB+, ghosts oxidized 10 nmol·mg protein)1·min1 of NADH or NADPH (Fig. 8A). Considering that 1 ml of packed erythrocytes yields
3.5 mg of ghost protein, this would correspond to a rate of 35 nmol·ml packed red cells1·min1. Because 2 moles of ferricyanide reduces 1 mole of NAD(P)H, the rate of MB+-dependent NAD(P)H oxidation induced by ferricyanide in intact cells would be
79 nmol·mg protein1·min1 from the 10-min point shown in Fig. 1A. Thus the ghost activity accounts for roughly one-half of the rate of ferricyanide reduction in intact cells. This seems to be a reasonable concordance because the MB+ reductase activity might not be fully active in ghosts compared with intact cells. Whereas the evidence that the ghost activity and that measured in cells reflects activity of the same protein is only circumstantial at this point, erythrocyte ghosts provide a system in which to test this possibility. Furthermore, the presence of sensitive thiols on the protein will be useful in its purification, reconstitution, and ultimate identification.
In conclusion, we have confirmed the presence of a trans-plasma membrane thiazine dye reductase in erythrocytes that reduces extracellular MB+ to MBH, which then is taken up by the cells and undergoes redox cycling. The latter generates an oxidant stress that also depletes low-molecular-weight antioxidants. MB+ reductase activity is sensitive to inhibition by thiol reagents in intact cells, suggesting that it is a protein. This activity may correspond to MB+-dependent NAD(P)H reductase activity in erythrocyte ghosts, which would aid in its identification.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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