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Enzyme-Dependent Ascorbate Recycling in Human Erythrocytes: Role of Thioredoxin Reductase
Free Radical Biology & Medicine, Vol. 25, No. 2, pp. 221–228, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00060-4
Original Contribution ENZYME-DEPENDENT ASCORBATE RECYCLING IN HUMAN ERYTHROCYTES: ROLE OF THIOREDOXIN REDUCTASE SHALU MENDIRATTA, ZHI-CHAO QU,
and JAMES
M. MAY
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA (Received 25 November 1997; Revised 20 January 1998; Accepted 27 February 1998)
Abstract—Human erythrocytes efficiently reduce dehydroascorbic acid (DHA) to ascorbate, which helps to maintain the ascorbate content of blood. Whereas erythrocyte DHA reduction is thought to occur primarily through a direct chemical reaction with GSH, this work addresses the role of enzyme-mediated DHA reduction by these cells. The ability of intact erythrocytes to recycle DHA to ascorbate, estimated as DHA-dependent ferricyanide reduction, was decreased in parallel with GSH depletion by glutathione-S-transferase substrates. In contrast, the sulfhydryl reagent phenylarsine oxide inhibited DHA reduction to a much greater extent than it decreased GSH in intact cells. DHA reduction in excess of that due to a direct chemical reaction with GSH was also observed in freshly prepared hemolysates. Hemolysates likewise showed NADPH-dependent reduction of DHA that appeared due to thioredoxin reductase, because this activity was inhibited 68% by 10 mM aurothioglucose, doubled by 5 mM E. coli thioredoxin, and had an apparent K m for DHA (1.5 mM) similar to that of purified thioredoxin reductase. Additionally, aurothioglucose-sensitive, NADPH-dependent DHA reductase activity was decreased 80% in hemolysates prepared from phenylarsine oxide-treated cells. GSHdependent DHA reduction in hemolysates was more than 10-fold that of NADPH-dependent reduction. Nonetheless, the ability of phenylarsine oxide to decrease DHA reduction in intact cells with little effect on GSH suggests that enzymes, such as thioredoxin reductase, may contribute more to this activity than previously considered. © 1998 Elsevier Science Inc. Keywords—Dehydroascorbate reductase, Oxidant stress, Glutathione, Phenylarsine oxide, Free radical
INTRODUCTION
nonenzymatic reaction has been reported to be the only mechanism of GSH-dependent DHA reduction in human erythrocyte hemolysates.5 More recently, however, a DHA reductase activity has been purified from human erythrocytes that likely corresponds to the thioltransferase glutaredoxin.6,7 Thioredoxin reductase (TR) has also been purified from erythrocytes,8 and we have shown that mammalian TR can reduce DHA to ascorbate.9 These observations raise the question of whether one or both of these enzymes contribute to DHA reduction in the erythrocyte. The ability of the intact erythrocyte to recycle DHA to ascorbate can be measured as the appearance of ascorbate within cells,4 or indirectly as DHA-dependent reduction of extracellular ferricyanide.10 –12 Ferricyanide does not enter the cells,13 but can oxidize intracellular ascorbate, presumably through a transmembrane oxidoreductase enzyme.11,12 The appearance of extracellular ferrocyanide then provides an integrated measure of the capacity of the cells to regenerate ascorbate from
Humans lack the ability to synthesize vitamin C, or ascorbic acid. To maintain cellular stores of the vitamin, it must be regenerated or recycled from its oxidized forms. Ascorbate recycling is especially important in blood, which may be exposed to oxidant stresses in the vascular bed in areas of inflammation or atherosclerosis.1 A one-electron oxidation of ascorbate results in the monodehydroascorbyl free radical, which can then either donate another electron to form dehydroascorbic acid (DHA) or disproportionate to reform ascorbate and DHA.2 The erythrocyte has a substantial capacity to recycle DHA back to ascorbate through direct reaction with GSH in a two-electron reduction that does not involve the monodehydroascorbyl free radical.3,4 This Address correspondence to: J. M. May, 736 Medical Research Building II, Vanderbilt University School of Medicine, Nashville, TN 37232-6303; Tel: (615) 936-1653; Fax: (615) 936-1667; E-Mail: [email protected]. 221
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DHA. Because ferricyanide reacts directly with ascorbate,10 in cell lysates ascorbate regeneration from DHA must be measured as the appearance of ascorbate. Using these approaches, we investigated the ability of erythrocytes and erythrocyte hemolysates to reduce DHA to ascorbate. We found that whereas GSH-dependent DHA reduction is prominent in erythrocytes, not all GSHdependent reduction occurs by direct chemical reaction. There is also an enzymatic component of DHA reduction that has the features of TR. EXPERIMENTAL PROCEDURES
Materials Analytical reagents, including ascorbate, DHA, 1-chloro-2,4-dinitrobenzene (CDNB), phenylarsine oxide (PAO), phorone (diisopropylene acetone), and E. coli thioredoxin were from Sigma Chemical Co. (St. Louis, MO). Aldrich Chemical Co. (Milwaukee, WI) supplied the dehydroascorbate and tridecylamine. CDNB, PAO, and phorone were dissolved in dimethyl sulfoxide before addition to cells, with maximal dimethyl sulfoxide concentrations of 0.5% (w/v) or less. TR, purified from rat liver,14 was a generous gift from Drs. Raymond F. Burk and Kristina E. Hill.
Separation was accomplished on a 10 3 1 cm Waters RadialPak C18 column (300 mm, 4 m), with a 4-mm guard column of the same packing material. Peaks were detected using an ESA Model 5010 analytical cell with the detecting electrode set at 10.4 volts and a Model 5100A detector. At a flow rate of 1 ml/min ascorbate was eluted at 7.5– 8.5 min, and the detection limit was 10 pmol/sample. Erythrocyte GSH content was measured using the fluorometric method of Hissen and Hilf.17 The a-tocopherol content of erythrocytes was determined by HPLC,18 following cell lysis and a-tocopherol extraction by the method of Lang et al.19 The concentrations of these antioxidants within cells were expressed relative to a milliliter of erythrocyte cytoplasm, which was taken as 70% of the packed cell volume.10 Measurement of ferricyanide reduction The ability of erythrocytes to reduce ferricyanide was measured as previously described.20 Briefly, erythrocytes at a 5% hematocrit were incubated for 30 min at 37°C in PBS that contained 5 mM D-glucose and 1 mM potassium ferricyanide. The cells were pelleted in a microfuge, and duplicate aliquots of the supernatant were taken for assay of ferricyanide, using o-phenanthroline for detection.21 Results are expressed relative to erythrocyte cytoplasmic volume, as for the antioxidants.
Cell preparation Blood was obtained from normal volunteers by venipuncture and anticoagulated with heparin. Erythrocytes were washed three times in 5 vol of phosphate-buffered saline (PBS), which consisted of deionized water that contained 12.5 mM Na2HPO4 and 140 mM NaCl, pH 7.4. The buffy coat of leukocytes was carefully removed from above the cell pellet with each wash. The cells were prepared to the final hematocrit as noted for incubations. Assays of ascorbate, GSH, and a-tocopherol. The concentration of ascorbate in cells or hemolysates was measured by HPLC with electrochemical detection. Cells at a 25% hematocrit were lysed by a single freeze– thaw cycle in dry ice–acetone, and allowed to thaw on ice. The resulting hemolysate was ultrafiltered in a Centricon-10 filter apparatus (Amicon, Inc., Beverly, MA) as described by Iheanacho et al.15 Following a suitable dilution, ascorbate was measured in the clear ultrafiltrate with the ion-pairing method of Pachla and Kissinger16 using tridecylamine as the ion-pair reagent. Modifications to the method included use of 30% methanol in the mobile phase and preoxidation of the mobile phase before sample injection using an ESA Model 5020 guard cell set at 10.5 volts (ESA, Inc., Chelmsford, MA).
Preparation of erythrocyte hemolysates for in vitro incubations Erythrocytes at a 25% hematocrit in PBS were lysed by freezing in dry ice–acetone, and thawing on ice. Prepared in this manner, the hemolysate GSH content was the same as that found in cells lysed directly in the GSH assay (results not shown). Aliquots of the hemolysates were incubated in the specified volume of PBS with additives as noted for 10 min at the indicated temperature. The hemolysates were ultrafiltered and assayed for ascorbate. The amount of ascorbate generated is expressed as a concentration in the incubation volume, normalized to the original erythrocyte cytosolic volume. Data analysis Results are shown as mean 6 SEM for the indicated number of experiments. Statistical analysis was carried out using paired t-tests for single comparisons, and analysis of variance for multiple comparisons with appropriate post hoc tests, using the statistical software package Sigmastat 2.0 (Jandel Scientific, San Rafael, CA). Curve fitting was performed using the graphics analysis program Fig.P (Biosoft, Cambridge, U.K.).
Erythrocyte ascorbate recycling
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RESULTS
The contribution of GSH to the ability of intact erythrocytes to reduce DHA to ascorbate was first assessed by depleting cells of GSH. Phorone was used to selectively deplete cellular GSH in a reaction catalyzed by glutathione-S-transferase.22 Erythrocytes were preincubated with increasing concentrations of phorone, washed by centrifugation, and treated with 100 mM DHA to test their ability to convert DHA to ascorbate. The latter was measured indirectly as the ability of the cells to reduce extracellular ferricyanide. In the experiments of Fig. 1, loading with 100 mM DHA alone had no effect on intracellular GSH (1.6 6 0.2 mM in control, and 1.7 6 0.2 mM in DHA-loaded cells, n 5 9), but it more than tripled the ability of cells to reduce ferricyanide, from 2.2 6 0.3 to 8.1 6 1.0 mmol z (ml erythrocytes)21 z (30 min)21 (n 5 9). Under these conditions, ferricyanide does not affect cellular GSH.4 To allow direct comparisons between the different assays, the data in Figs. 1 and 2 are shown relative to the DHA-treated controls. Pretreatment with phorone caused a linear decrease in the cellular content of GSH to 76% of control, with a slope significantly less than zero by linear regression (r 5 2.98, p , .001). Phorone decreased ferricyanide reduction in a monoexponential fashion, although not significantly greater than it decreased GSH (Fig. 1A). The relatively small effects of phorone differ from those observed with CDNB, another agent that depletes GSH by glutathione-S-transferase-dependent conjugation.23 Preincubation of cells with increasing concentrations of CDNB, followed by DHA loading, caused monoexponential decreases in cellular GSH, ascorbate, and DHA-dependent ferricyanide reduction, as shown by the fitted lines (Fig. 1B). The half-maximal effect of CDNB was about 65 mM for each inhibition. Membrane a-tocopherol was unaffected, even at the highest dose of CDNB (Fig. 1B). At the concentrations used in the studies of Fig. 1B, CDNB did not directly destroy or react with ascorbate, as assessed by in vitro HPLC analysis (results not shown). As with phorone, these results suggest that ascorbate recycling requires GSH. The results of primary GSH depletion can be contrasted with those observed with PAO, an agent that reacts with vicinal sulfhydryl groups.24 Pretreatment of cells with up to 100 mM PAO, followed by DHA-loading under conditions similar to those of Fig. 1, resulted in only a 17% decrease in the cellular GSH concentration, but a 69% decrease in ability of DHA-loaded cells to reduce ferricyanide (Fig. 2). Both inhibitions were incomplete, leaving residual uninhibited activity. Although not shown, the portion of ferricyanide reduction that was insensitive to PAO corresponded exactly to DHA-independent reduction, measured in parallel incubations. De-
Fig. 1. Erythrocyte responses to GSH depletion by glutathione-Stransferase substrates. Erythrocytes at a 6% hematocrit were incubated at 37°C in PBS that contained 5 mM D-glucose and the indicated concentration of phorone (A) or CDNB (B). At 15 min, DHA was added to an initial concentration of 100 mM, and incubations were continued for an additional 15 min. The cells were pelleted in a microfuge and washed three times in 10 vol of PBS. Aliquots of the washed cells were taken for assay of GSH (circles), ascorbate (squares), a-tocopherol (diamonds), and for their ability to reduce extracellular ferricyanide (triangles), as detailed under Experimental Procedures. Results are shown as a fraction of control from three experiments with phorone, and six experiments with CDNB. Initial values in A were: GSH, 1.7 6 0.4 mM; ferricyanide reduction, 5.3 6 1.6 mmol z (ml erythrocytes)21 z (30 min)21. Initial values in panel B were: GSH, 1.7 6 0.2 mM; ascorbate, 22 6 3 mM; ferricyanide reduction, 9.3 6 0.8 mmol z (ml erythrocytes)21 z (30 min)21, and a-tocopherol, 3.6 6 0.2 nmol z (ml erythrocytes)21.
spite only a small effect on the cellular GSH content, PAO markedly decreased the ability of cells to reduce added DHA. The decrease in DHA-stimulated ferricyanide reduction caused by preincubation with PAO paralleled the decrease in the ability of erythrocytes to recycle DHA to ascorbate, as shown in Fig. 3. In these experiments, cells were pretreated with 100 mM PAO, followed by loading with increasing concentrations of DHA. The ability of erythrocytes to reduce DHA to ascorbate was markedly impaired over
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Fig. 2. Inhibition of erythrocyte DHA reduction by increasing concentrations of PAO. Erythrocytes at a 6% hematocrit were incubated at 37°C in PBS that contained 5 mM D-glucose and the indicated concentration of PAO. At 20 min, DHA was added to an initial concentration of 100 mM and the incubation was continued for an additional 20 min. The cells were pelleted in a microfuge and washed three times in 10 vol of PBS. Aliquots of the washed cells were taken for assay of GSH (circles), and for their ability to reduce extracellular ferricyanide (triangles), as detailed under Experimental Procedures. Results are expressed as a fraction of control values from at least six experiments for each assay, with the solid lines indicating fits to a monoexponential model with residual.
the range of DHA concentrations tested (Fig. 3A), as was the ability of PAO-treated cells to reduce ferricyanide (Fig. 3B). These results could not be explained by direct reaction of PAO with ascorbate, because an eightfold molar excess of PAO did not affect ascorbate concentrations when incubated in vitro (results not shown). Further, PAO up to 100 mM did not significantly decrease 3-O-methylglucose efflux from erythrocytes, making it unlikely that PAO inhibited DHA uptake on the glucose transporter, which could have contributed to the observed decrease in DHA recycling to ascorbate. The effect of PAO to inhibit ascorbate recycling more severely than it decreased intracellular GSH concentrations suggests that PAO decreases DHA recycling by one or more mechanisms in addition to reacting with intracellular GSH. GSH-dependent DHA reduction was studied next in erythrocyte hemolysates. Use of hemolysates removes the membrane barrier and allows direct access of DHA and inhibitors to cytoplasmic substrates and enzymes. Erythrocyte hemolysates were initially dialyzed overnight to remove GSH and enzyme cofactors, such as NADPH. Whereas these dialysates supported reduction of DHA by added GSH, there was no activity in excess of that seen in control incubations with GSH and DHA
Fig. 3. Inhibition by PAO of erythrocyte recycling of DHA to ascorbate. Erythrocytes were incubated under the conditions described in the legend to Fig. 2 without (open symbols) or with (closed symbols) 100 mM PAO. After 20 min of incubation, DHA was added to the indicated initial concentration and incubation was continued for another 20 min. Following three centrifugation washes, aliquots of the cells were taken for assay of ascorbate, and for their ability to reduce extracellular ferricyanide. (A) This shows intracellular ascorbate concentrations (squares); (B) Shows ferricyanide reduction as mmol z (ml erythrocytes)21 z (30 min)21 (triangles) from three experiments.
alone (results not shown). On the other hand, incubation of freshly prepared lysates with 4 mM GSH and increasing concentrations of DHA did result in ascorbate generation in excess of that attributable to a direct chemical reduction (Fig. 4). In the absence of added GSH, the rate of DHA reduction was about 10% of that observed with 4 mM GSH, reflecting dilution of intracellular GSH in the lysates. The rate of chemical reduction of DHA by 4 mM GSH rose linearly with increases in added DHA (Fig. 4). Subtraction of the rate ascorbate generation in the chemical reaction from the rate in the presence of hemolysate at each DHA concentration resulted in a progressively increasing plot, which showed no tendency to saturate over the range of DHA concentrations used. The rate of DHA reduction in excess of the chemical reaction, calculated relative to the volume of original cell cytoplasm and as a function of the mM DHA concentration, was 23.1 nmol z (ml cells)21 z min21 (r 5 .99).
Erythrocyte ascorbate recycling
Fig. 4. GSH-dependent DHA reduction in erythrocyte hemolysates. Erythrocyte hemolysates were prepared as described under Materials and Methods. The freshly prepared hemolysate was incubated at room temperature in 1 vol of PBS that contained 4 mM GSH and the indicated initial concentration of DHA. At 10 min, the hemolysate was ultrafiltered as described for the ascorbate assay, and the ascorbate concentration in the ultrafiltrate was measured (squares, solid line). The direct chemical reaction was also measured in the absence of cells (circles, solid line). Activity that could not be accounted for by direct reaction (triangles, dashed line) was obtained by subtracting the direct chemical reaction from the total in each of the four experiments performed. Data are from four experiments, fit to linear models with r . .99 for each fit.
To examine the possibility of NADPH-dependent DHA reduction, 400 mM NADPH was added to freshly prepared erythrocyte hemolysates. This resulted in a 23% increase in the rate of ascorbate generation compared to control ( p , .01, n 5 6 determinations). However, this small increase in NADPH-dependent reduction could have been due simply to direct reaction of DHA with NADPH or to the ability of NADPH to recycle GSSG to GSH through glutathione reductase. Increased specificity for TR was sought as follows. First, because TR is sensitive to inhibition by aurothioglucose,14 we incubated freshly prepared erythrocyte lysates with 10 mM aurothioglucose. As shown in Fig. 5, the rate of ascorbate generation, expressed relative to the original cytoplasmic volume, was inhibited 68% by aurothioglucose (first pair of bars). Further, addition of 5 mM E. coli thioredoxin almost doubled DHA reduction compared to control, and this activity was inhibited to the same level by aurothioglucose (second pair of bars). To correct for direct reduction of DHA by NADPH and for GSH-dependent reduction of DHA, we calculated aurothioglucose-sensitive DHA reductase activity (basal activity minus activity in the presence of aurothioglucose), and this was
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Fig. 5. NADPH-dependent DHA reduction in erythrocyte hemolysates. Hemolysates prepared as described under Materials and Methods were incubated at room temperature in PBS in the presence (open bars) or absence (hatched bars) of 10 mM aurothioglucose. After 5 min, NADPH and DHA were added to concentrations of 0.4 mM and 40 mM, respectively. At the same time, thioredoxin (TRx) was added to a final concentration of 5 mM to one pair of samples, but not to the other (Con). The incubations were continued another 10 min at room temperature, followed by ultrafiltration of the samples for assay of the ascorbate concentration. The third pair of bars, noted by (2)ATG, shows total activity minus activity observed with aurothioglucose in each experiment, with the double crosshatched bar indicating control, and the solid bar indicating thioredoxin-treated samples. Data are from five experiments, with an asterisk (*) indicating p , .05 compared to the adjacent bar.
more than doubled in the presence of thioredoxin (last pair of bars). This supports the hypothesis that TR contributed to the reduction of DHA in these hemolysates. Aurothioglucose-sensitive DHA reductase activity was also observed in overnight dialyzed hemolysates, but this was only 5% of the aurothioglucose-sensitive activity in fresh hemolysates (results not shown). To further study the role of TR in erythrocyte DHA reduction, two additional experiments were performed. In the first, DHA reductase activity was measured with increasing concentrations of DHA in the presence or absence of aurothioglucose. As shown in Fig. 6, aurothioglucose was a more effective inhibitor of hemolysate DHA reductase activity at low than at high DHA concentrations. Subtraction of rates of aurothioglucose-sensitive ascorbate regeneration from rates of total ascorbate regeneration resulted in a curvilinear plot that had a apparent K m of 1.5 mM when fit to a simple hyperbolic model. When the total activity was fit to an hyperbolic model with a nonsaturable component, the apparent kinetic parameters were similar, as noted in the legend to Fig. 6. This saturability of the aurothioglucose-depen-
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Fig. 6. DHA dose-dependence of gold-sensitive, NADPH-dependent DHA reductase activity. Cell lysates were prepared and incubated in 1.25 vol of PBS under the conditions described in the legend to Fig. 5. Following a 3-min preincubation with (squares) or without (circles) 10 mM aurothioglucose, NADPH was added to 0.4 mM, followed by the indicated concentration of DHA. After 10 min, the samples were ultrafiltered, and ascorbate concentrations were measured. The difference between the total and the aurothioglucose-sensitive ascorbate concentration was calculated at each point (triangles). The solid line is a linear fit to the gold-inhibited data (r 5 .99, p , .002), the dotted line is a fit to an hyperbolic model with a nonsaturable component of the total activity (K m 5 1.2 mM, V max 5 99 mM, nonsaturable 5 10% of V max, and r 5 .9998), and the dashed line is an hyperbolic fit without nonsaturable components to the difference between total and gold-inhibited activity (K m 5 1.5 mM, V max 5 100 mM, r 5 .997). Data are shown from four experiments.
dent activity provides additional support for the notion that it is mediated by an enzyme. In the second experiment, we returned to the use of PAO, which appeared to inhibit ascorbate regeneration in intact cells to an extent greater than could be accounted for by reaction with cellular GSH. As shown in Fig. 7, lysates that were prepared from cells that had been treated with PAO showed a 75% decrease in total (first pair of bars) and an 80% decrease in aurothioglucose-sensitive NADPH-dependent DHA reductase activity (last pair of bars). To ensure adequate ascorbate generation in PAO-treated samples, incubations were carried out at 37°C, which accounts for the increased rate of ascorbate generation in the control sample compared to that shown in Fig. 5. These effects of PAO on DHA reductase activity could not be accounted for by the 10% measured decrease in GSH in the cell lysates. Further, the DHA reductase activity of purified rat liver TR was very sensitive to PAO, and was inhibited half-maximally by preincubation with 21 mM PAO (Fig. 8). These data support the notion that inhibition of this enzyme in
Fig. 7. Inhibition of aurothioglucose-sensitive DHA reductase activity by pretreatment of cells with PAO. Erythrocytes were treated as described in the legend to Fig. 2 with 100 mM PAO, washed, and hemolyzed for assay of DHA reductase activity at 37°C in an equal volume of PBS as described under Materials and Methods. The hatched bars show the amount of ascorbate generated by control hemolysates, and the solid bars show ascorbate generated in hemolysates from cells treated with PAO. The first pair of bars (TOTAL) indicates DHA reduction in control hemolysates, the second pair of bars (ATG) indicates activity in the presence of 10 mM aurothioglucose, and the third pair of bars (TOT-ATG) indicates the difference between control and aurothioglucose-treated hemolysates for each of five experiments. An asterisk (*) indicates p , .05 compared to the adjacent control.
erythrocytes and their hemolysates was involved in the cellular response to PAO.
DISCUSSION
In contrast to nucleated cells, which concentrate ascorbate against a gradient,25 human erythrocytes have similar concentrations of ascorbate compared to plasma.26 Nonetheless, erythrocytes have a high capacity to recycle DHA to ascorbate. When presented with DHA, erythrocytes can rapidly remove DHA from the extracellular space,4 and accumulate ascorbate against a concentration gradient.20,27 Based on rates of DHA-dependent ferricyanide reduction, we have calculated that the cells can regenerate the ascorbate content of blood every 3 min.20 This ascorbate recycling depends on an adequate supply of reducing equivalents in the cells, which derive ultimately from glucose metabolism. The proximal electron donors for ascorbate recycling have not been established with certainty. GSH appears to be involved in erythrocytes, because DHA recycling to ascorbate in the absence of glucose depletes GSH,9 and since GSH depletion with diamide9 and with glutathione-S-transferase substrates (Figs. 1 and 2) impairs DHA-
Erythrocyte ascorbate recycling
Fig. 8. Inhibition of rat liver TR by PAO. Purified from rat liver TR (21 nM) was incubated in 50 mM Tris-HCl, pH 7.4, at room temperature with the indicated concentration of PAO. At 15 min, NADPH and DHA were added to 0.4 mM and 2 mM, respectively, and the incubation was continued for another 20 min. Nine volumes of ice-cold methanol 80% (v/v) containing 1 mM EDTA were added, the sample was microfuged for 2 min, and the ascorbate concentration in the supernatant was determined. The data from three experiments are expressed relative to the control value, and fit to a monoexponential model with residual (r 5 .9997, residual 3.6%).
dependent ferricyanide reduction. GSH can directly reduce DHA to ascorbate,28 and such nonenzymatic recycling is considered a prominent, if not the sole mechanism operative in erythrocytes and other cells.3 Thus, dialyzed erythrocyte hemolysates fail to support GSH-dependent DHA reduction at levels beyond that attributable to the nonenzymatic reaction.5 We obtained similar results with dialyzed hemolysates, but were able to show DHA reduction in excess of that attributable to the nonenzymatic reaction with added GSH in freshly prepared hemolysates (Fig. 4). This suggests that erythrocytes do have GSH-dependent DHA reductase activity that could account for as much as a third of the total activity observed. The enzymatic component is lost upon dialysis in oxygenated buffer. Oxidation of sensitive sulfhydryl groups on glutaredoxin6 would account for this observation. It was also possible to demonstrate NADPH-dependent DHA reductase activity in freshly prepared erythrocyte hemolysates. This activity was present in overnight-dialyzed hemolysates, albeit at markedly reduced levels, again suggesting oxidative loss of NADPH-dependent activity. Several considerations suggest that the NADPH-dependent reductase activity in hemolysates is due to TR. First, we have recently reported that purified rat liver TR has DHA reductase activity.9 Second, he-
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molysate DHA reductase activity was inhibited almost 70% by 10 mM aurothioglucose (Fig. 5), a known inhibitor of selenoenzymes.14,29 –31 The selenoenzyme glutathione peroxidase is inhibited only slightly by this concentration of aurothioglucose,14 whereas TR is inhibited by more than 80%.14 Third, 5 mM E. coli thioredoxin doubled aurothioglucose-sensitive DHA reductase activity in erythrocyte hemolysates (Fig. 5). The ability of purified rat liver TR to reduce DHA to ascorbate is tripled by addition of the same concentration of E. coli thioredoxin.9 Fourth, the aurothioglucose-sensitive component of hemolysate DHA reductase activity showed a saturable dependence on the concentration of DHA in the assay, with an apparent K m of 1.5 mM (Fig. 6). The latter is similar to that measured for purified TR.9 The results of treatment of erythrocytes with PAO also suggest that TR contributes significantly to DHA reduction in intact cells. PAO is an agent with specificity for vicinal or nearby sulfhydryl groups,24,32 which are required for catalysis in TR.33 PAO was a potent inhibitor of purified rat liver TR (Fig. 8). When incubated with intact erythrocytes, PAO inhibited the ability of cells to carry out DHA-dependent ferricyanide reduction to a much greater extent than it decreased the GSH content of cells (Figs. 2 and 3). These results suggest involvement of one or more sulfhydryl-containing enzymes, such as glutaredoxin or TR. Because treatment of intact cells with PAO inhibited aurothioglucose-sensitive DHA reductase activity by 80% in hemolysates in the presence of NADPH (Fig. 7), at least part of the effect of PAO in intact cells appears due to inhibition of TR. The extent to which enzymatic NADPH-dependent versus either enzymatic or nonenzymatic GSH-dependent DHA reduction predominates in intact erythrocytes is difficult to ascertain. In hemolysates, comparison of the normalized data in Figs. 4 and 6 at 1 mM DHA reveals that rates of GSH-dependent DHA reduction are more than 10-fold those of NADPH-dependent DHA reduction. This may not reflect the situation in the intact cell, however, as indicated by the effects of PAO discussed above. Attempts to selectively deplete cellular GSH using glutathione-S-transferase substrates, although suggestive of GSH-dependent reduction, did not provide a conclusive answer. Phorone at concentrations effective for depletion of GSH from liver and other tissues22,34 had only a small effect on erythrocyte GSH content, although it did decrease DHA-dependent ferricyanide reduction (Fig. 1A). CDNB was much more potent than phorone in decreasing both erythrocyte DHA recycling and GSH content (Fig. 1B), but CDNB is also an inhibitor of mammalian TR.35 Still, there is more evidence for GSH dependence of DHA reduction in erythrocytes than in HL-60 cells, in which the depletion of GSH has little effect on rates of DHA reduction.36 In dialysed rat liver
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hemolysates, we found both GSH- and NADPH-dependent DHA reductase activities.9 Mechanisms of DHA reduction may vary in different cells, and at least in erythrocytes and liver, also appear to be redundant.
19.
20. Acknowledgements—This work was supported by NIH Grant DK 50435, and by a Research Grant from the American Diabetes Association. The authors appreciate the gift of purified rat liver TR from Drs. Raymond Burk and Kristina Hill, and reading of the manuscript by Dr. Burk.
21. 22.
REFERENCES 1. Diaz, M. N.; Frei, B.; Vita, J. A.; Keaney, J. F., Jr. Mechanisms of disease—antioxidants and atherosclerotic heart disease. N. Engl. J. Med. 337:408 – 416; 1997. 2. Iyanagi, T.; Yamazaki, I.; Anan, K. F. One-electron oxidationreduction properties of ascorbic acid. Biochim. Biophys. Acta 806: 255–261; 1985. 3. Winkler, B. S.; Orselli, S. M.; Rex, T. S. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med. 17:333–349; 1994. 4. May, J. M.; Qu, Z. C.; Whitesell, R. R.; Cobb, C. E. Ascorbate recycling in human erythrocytes: role of GSH in reducing dehydroascorbate. Free Radic. Biol. Med. 20:543–551; 1996. 5. Basu, S.; Som, S.; Deb, S.; Mukherjee, D.; Chatterjee, I. B. Dehydroascorbic acid reduction in human erythrocytes. Biochem. Biophys. Res. Commun. 90:1335–1340; 1979. 6. Papov, V. V.; Gravina, S. A.; Mieyal, J. J.; Biemann, K. The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells. Protein Sci. 3:428 – 434; 1994. 7. Xu, D. P.; Washburn, M. P.; Sun, G. P.; Wells, W. W. Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochem. Biophys. Res. Commun. 221:117–121; 1996. 8. Cha, M. K.; Kim, I.-H. Thioredoxin-linked peroxidase from human red blood cell: evidence for the existence of thioredoxin and thioredoxin reductase in human red blood cell. Biochem. Biophys. Res. Commun. 217:900 –907; 1995. 9. May, J. M.; Mendiratta, S.; Hill, K. E.; Burk, R. F. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J. Biol. Chem. 272:22607–22610; 1997. 10. Orringer, E. P.; Roer, M. E. An ascorbate-mediated transmembrane-reducing system of the human erythrocyte. J. Clin. Invest. 63:53–58; 1979. 11. Schipfer, W.; Neophytou, B.; Trobisch, R.; Groiss, O.; Goldenberg, H. Reduction of extracellular potassium ferricyanide by transmembrane NADH:(acceptor) oxidoreductase of human erythrocytes. Int. J. Biochem. 17:819 – 823; 1985. 12. May, J. M.; Qu, Z.-C.; Whitesell, R. R. Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes. Biochim. Biophys. Acta 1238:127–136; 1995. ¨ ber den Mechanismus der 13. Sze´kely, M.; Ma´nyai, S.; Straub, F. B. U osmotischen Ha¨molyse. Acta Physiol. Acad. Sci. Hung. 3:571– 583; 1952. 14. Hill, K. E.; McCollum, G. W.; Boeglin, M. E.; Burk, R. F. Thioredoxin reductase activity is decreased by selenium deficiency. Biochem. Biophys. Res. Commun. 234:293–295; 1997. 15. Iheanacho, E. N.; Hunt, N. H.; Stocker, R. Vitamin C redox reactions in blood of normal and malaria-infected mice studied with isoascorbate as a nonisotopic marker. Free Radic. Biol. Med. 18:543–552; 1995. 16. Pachla, L. A.; Kissinger, P. T. Analysis of ascorbic acid by liquid chromatography with amperometric detection. Methods Enzymol. 62:15–24; 1979. 17. Hissin, P. J.; Hilf, R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74: 214 –226; 1976. 18. May, J. M.; Qu, Z. C.; Morrow, J. D. Interaction of ascorbate and a-tocopherol in resealed human erythrocyte ghosts—Transmem-
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brane electron transfer and protection from lipid peroxidation. J. Biol. Chem. 271:10577–10582; 1996. Lang, J. K.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal. Biochem. 157:106 – 116; 1986. May, J. M.; Qu, Z.-C.; Whitesell, R. R. Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes. Biochemistry 34:12721–12728; 1995. Avron, M.; Shavit, N. A sensitive and simple method for determination of ferrocyanide. Anal. Biochem. 6:549 –554; 1963. Burk, R. F.; Hill, K. E. Reduced glutathione release into rat plasma by extrahepatic tissues. Am. J. Physiol. (Gastrointest. Liver Physiol.) 269:G396 –G399; 1995. Awasthi, Y. C.; Garg, H. S.; Dao, D. D.; Partridge, C. A.; Srivastava, S. K. Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2, 4-dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on hemoglobin. Blood 58:733–738; 1981. Webb, J. L. Arsenicals. enzyme and metabolic inhibitors, vol. 3. New York: Academic Press, Inc.; 1966: 595–701. Rose, R. C. Transport of ascorbic acid and other water-soluble vitamins. Biochim. Biophys. Acta 947:335–366; 1988. Evans, R. M.; Currie, L.; Campbell, A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br. J. Nutr. 47:473– 482; 1982. Christine, L.; Thomson, G.; Iggo, B.; Brownie, A. C.; Stewart, C. P. The reduction of dehydroascorbic acid by human erythrocytes. Clin. Chim. Acta 1:557–569; 1956. Winkler, B. S. Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. Biochim. Biophys. Acta 1117:287–290; 1992. Chaudiere, J.; Tappel, A. L. Interaction of gold(I) with the active site of selenium-glutathione peroxidase. J. Inorg. Biochem. 20: 313–325; 1984. Beckman, J. K.; Greene, H. L. Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation. Lipids 23: 899 –903; 1988. Mercurio, S. D.; Combs, G. F., Jr. Drug-induced changes in selenium-dependent glutathione peroxidase activity in the chick. J. Nutr. 115:1459 –1470; 1985. Eagle, H.; Hogan, R. B.; Doak, G. O.; Steinman, H. G. The toxicity, treponemicidal activity, and potential therapeutic utility of substituted phenylarsenoxides. J. Pharmacol. Exp. Ther. 70:221– 227; 1940. Holmgren, A.; Bjo¨rnstedt, M. Thioredoxin and thioredoxin reductase. Methods Enzymol. 252:199 –208; 1995. Burk, R. F.; Hill, K. E.; Awad, J. A.; Morrow, J. D.; Lyons, P. R. Liver and kidney necrosis in selenium-deficient rats depleted of glutathione. Lab. Invest. 72:723–730; 1995. Arne´r, E. S.; Bjo¨rnstedt, M.; Holmgren, A. 1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity. J. Biol. Chem. 270:3479 –3482; 1995. Guaiquil, V. H.; Farber, C. M.; Golde, D. W.; Vera, J. C. Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione. J. Biol. Chem. 272:9915–9921; 1997. ABBREVIATIONS
CDNB—1-chloro-2,4-dinitrobenzene DHA— dehydroascorbic acid HPLC— high-performance liquid chromatography PBS—phosphate-buffered saline PAO—phenylarsine oxide TR—thioredoxin reductase
PII S0891-5849(98)00060-4
Original Contribution ENZYME-DEPENDENT ASCORBATE RECYCLING IN HUMAN ERYTHROCYTES: ROLE OF THIOREDOXIN REDUCTASE SHALU MENDIRATTA, ZHI-CHAO QU,
and JAMES
M. MAY
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA (Received 25 November 1997; Revised 20 January 1998; Accepted 27 February 1998)
Abstract—Human erythrocytes efficiently reduce dehydroascorbic acid (DHA) to ascorbate, which helps to maintain the ascorbate content of blood. Whereas erythrocyte DHA reduction is thought to occur primarily through a direct chemical reaction with GSH, this work addresses the role of enzyme-mediated DHA reduction by these cells. The ability of intact erythrocytes to recycle DHA to ascorbate, estimated as DHA-dependent ferricyanide reduction, was decreased in parallel with GSH depletion by glutathione-S-transferase substrates. In contrast, the sulfhydryl reagent phenylarsine oxide inhibited DHA reduction to a much greater extent than it decreased GSH in intact cells. DHA reduction in excess of that due to a direct chemical reaction with GSH was also observed in freshly prepared hemolysates. Hemolysates likewise showed NADPH-dependent reduction of DHA that appeared due to thioredoxin reductase, because this activity was inhibited 68% by 10 mM aurothioglucose, doubled by 5 mM E. coli thioredoxin, and had an apparent K m for DHA (1.5 mM) similar to that of purified thioredoxin reductase. Additionally, aurothioglucose-sensitive, NADPH-dependent DHA reductase activity was decreased 80% in hemolysates prepared from phenylarsine oxide-treated cells. GSHdependent DHA reduction in hemolysates was more than 10-fold that of NADPH-dependent reduction. Nonetheless, the ability of phenylarsine oxide to decrease DHA reduction in intact cells with little effect on GSH suggests that enzymes, such as thioredoxin reductase, may contribute more to this activity than previously considered. © 1998 Elsevier Science Inc. Keywords—Dehydroascorbate reductase, Oxidant stress, Glutathione, Phenylarsine oxide, Free radical
INTRODUCTION
nonenzymatic reaction has been reported to be the only mechanism of GSH-dependent DHA reduction in human erythrocyte hemolysates.5 More recently, however, a DHA reductase activity has been purified from human erythrocytes that likely corresponds to the thioltransferase glutaredoxin.6,7 Thioredoxin reductase (TR) has also been purified from erythrocytes,8 and we have shown that mammalian TR can reduce DHA to ascorbate.9 These observations raise the question of whether one or both of these enzymes contribute to DHA reduction in the erythrocyte. The ability of the intact erythrocyte to recycle DHA to ascorbate can be measured as the appearance of ascorbate within cells,4 or indirectly as DHA-dependent reduction of extracellular ferricyanide.10 –12 Ferricyanide does not enter the cells,13 but can oxidize intracellular ascorbate, presumably through a transmembrane oxidoreductase enzyme.11,12 The appearance of extracellular ferrocyanide then provides an integrated measure of the capacity of the cells to regenerate ascorbate from
Humans lack the ability to synthesize vitamin C, or ascorbic acid. To maintain cellular stores of the vitamin, it must be regenerated or recycled from its oxidized forms. Ascorbate recycling is especially important in blood, which may be exposed to oxidant stresses in the vascular bed in areas of inflammation or atherosclerosis.1 A one-electron oxidation of ascorbate results in the monodehydroascorbyl free radical, which can then either donate another electron to form dehydroascorbic acid (DHA) or disproportionate to reform ascorbate and DHA.2 The erythrocyte has a substantial capacity to recycle DHA back to ascorbate through direct reaction with GSH in a two-electron reduction that does not involve the monodehydroascorbyl free radical.3,4 This Address correspondence to: J. M. May, 736 Medical Research Building II, Vanderbilt University School of Medicine, Nashville, TN 37232-6303; Tel: (615) 936-1653; Fax: (615) 936-1667; E-Mail: [email protected]. 221
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DHA. Because ferricyanide reacts directly with ascorbate,10 in cell lysates ascorbate regeneration from DHA must be measured as the appearance of ascorbate. Using these approaches, we investigated the ability of erythrocytes and erythrocyte hemolysates to reduce DHA to ascorbate. We found that whereas GSH-dependent DHA reduction is prominent in erythrocytes, not all GSHdependent reduction occurs by direct chemical reaction. There is also an enzymatic component of DHA reduction that has the features of TR. EXPERIMENTAL PROCEDURES
Materials Analytical reagents, including ascorbate, DHA, 1-chloro-2,4-dinitrobenzene (CDNB), phenylarsine oxide (PAO), phorone (diisopropylene acetone), and E. coli thioredoxin were from Sigma Chemical Co. (St. Louis, MO). Aldrich Chemical Co. (Milwaukee, WI) supplied the dehydroascorbate and tridecylamine. CDNB, PAO, and phorone were dissolved in dimethyl sulfoxide before addition to cells, with maximal dimethyl sulfoxide concentrations of 0.5% (w/v) or less. TR, purified from rat liver,14 was a generous gift from Drs. Raymond F. Burk and Kristina E. Hill.
Separation was accomplished on a 10 3 1 cm Waters RadialPak C18 column (300 mm, 4 m), with a 4-mm guard column of the same packing material. Peaks were detected using an ESA Model 5010 analytical cell with the detecting electrode set at 10.4 volts and a Model 5100A detector. At a flow rate of 1 ml/min ascorbate was eluted at 7.5– 8.5 min, and the detection limit was 10 pmol/sample. Erythrocyte GSH content was measured using the fluorometric method of Hissen and Hilf.17 The a-tocopherol content of erythrocytes was determined by HPLC,18 following cell lysis and a-tocopherol extraction by the method of Lang et al.19 The concentrations of these antioxidants within cells were expressed relative to a milliliter of erythrocyte cytoplasm, which was taken as 70% of the packed cell volume.10 Measurement of ferricyanide reduction The ability of erythrocytes to reduce ferricyanide was measured as previously described.20 Briefly, erythrocytes at a 5% hematocrit were incubated for 30 min at 37°C in PBS that contained 5 mM D-glucose and 1 mM potassium ferricyanide. The cells were pelleted in a microfuge, and duplicate aliquots of the supernatant were taken for assay of ferricyanide, using o-phenanthroline for detection.21 Results are expressed relative to erythrocyte cytoplasmic volume, as for the antioxidants.
Cell preparation Blood was obtained from normal volunteers by venipuncture and anticoagulated with heparin. Erythrocytes were washed three times in 5 vol of phosphate-buffered saline (PBS), which consisted of deionized water that contained 12.5 mM Na2HPO4 and 140 mM NaCl, pH 7.4. The buffy coat of leukocytes was carefully removed from above the cell pellet with each wash. The cells were prepared to the final hematocrit as noted for incubations. Assays of ascorbate, GSH, and a-tocopherol. The concentration of ascorbate in cells or hemolysates was measured by HPLC with electrochemical detection. Cells at a 25% hematocrit were lysed by a single freeze– thaw cycle in dry ice–acetone, and allowed to thaw on ice. The resulting hemolysate was ultrafiltered in a Centricon-10 filter apparatus (Amicon, Inc., Beverly, MA) as described by Iheanacho et al.15 Following a suitable dilution, ascorbate was measured in the clear ultrafiltrate with the ion-pairing method of Pachla and Kissinger16 using tridecylamine as the ion-pair reagent. Modifications to the method included use of 30% methanol in the mobile phase and preoxidation of the mobile phase before sample injection using an ESA Model 5020 guard cell set at 10.5 volts (ESA, Inc., Chelmsford, MA).
Preparation of erythrocyte hemolysates for in vitro incubations Erythrocytes at a 25% hematocrit in PBS were lysed by freezing in dry ice–acetone, and thawing on ice. Prepared in this manner, the hemolysate GSH content was the same as that found in cells lysed directly in the GSH assay (results not shown). Aliquots of the hemolysates were incubated in the specified volume of PBS with additives as noted for 10 min at the indicated temperature. The hemolysates were ultrafiltered and assayed for ascorbate. The amount of ascorbate generated is expressed as a concentration in the incubation volume, normalized to the original erythrocyte cytosolic volume. Data analysis Results are shown as mean 6 SEM for the indicated number of experiments. Statistical analysis was carried out using paired t-tests for single comparisons, and analysis of variance for multiple comparisons with appropriate post hoc tests, using the statistical software package Sigmastat 2.0 (Jandel Scientific, San Rafael, CA). Curve fitting was performed using the graphics analysis program Fig.P (Biosoft, Cambridge, U.K.).
Erythrocyte ascorbate recycling
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RESULTS
The contribution of GSH to the ability of intact erythrocytes to reduce DHA to ascorbate was first assessed by depleting cells of GSH. Phorone was used to selectively deplete cellular GSH in a reaction catalyzed by glutathione-S-transferase.22 Erythrocytes were preincubated with increasing concentrations of phorone, washed by centrifugation, and treated with 100 mM DHA to test their ability to convert DHA to ascorbate. The latter was measured indirectly as the ability of the cells to reduce extracellular ferricyanide. In the experiments of Fig. 1, loading with 100 mM DHA alone had no effect on intracellular GSH (1.6 6 0.2 mM in control, and 1.7 6 0.2 mM in DHA-loaded cells, n 5 9), but it more than tripled the ability of cells to reduce ferricyanide, from 2.2 6 0.3 to 8.1 6 1.0 mmol z (ml erythrocytes)21 z (30 min)21 (n 5 9). Under these conditions, ferricyanide does not affect cellular GSH.4 To allow direct comparisons between the different assays, the data in Figs. 1 and 2 are shown relative to the DHA-treated controls. Pretreatment with phorone caused a linear decrease in the cellular content of GSH to 76% of control, with a slope significantly less than zero by linear regression (r 5 2.98, p , .001). Phorone decreased ferricyanide reduction in a monoexponential fashion, although not significantly greater than it decreased GSH (Fig. 1A). The relatively small effects of phorone differ from those observed with CDNB, another agent that depletes GSH by glutathione-S-transferase-dependent conjugation.23 Preincubation of cells with increasing concentrations of CDNB, followed by DHA loading, caused monoexponential decreases in cellular GSH, ascorbate, and DHA-dependent ferricyanide reduction, as shown by the fitted lines (Fig. 1B). The half-maximal effect of CDNB was about 65 mM for each inhibition. Membrane a-tocopherol was unaffected, even at the highest dose of CDNB (Fig. 1B). At the concentrations used in the studies of Fig. 1B, CDNB did not directly destroy or react with ascorbate, as assessed by in vitro HPLC analysis (results not shown). As with phorone, these results suggest that ascorbate recycling requires GSH. The results of primary GSH depletion can be contrasted with those observed with PAO, an agent that reacts with vicinal sulfhydryl groups.24 Pretreatment of cells with up to 100 mM PAO, followed by DHA-loading under conditions similar to those of Fig. 1, resulted in only a 17% decrease in the cellular GSH concentration, but a 69% decrease in ability of DHA-loaded cells to reduce ferricyanide (Fig. 2). Both inhibitions were incomplete, leaving residual uninhibited activity. Although not shown, the portion of ferricyanide reduction that was insensitive to PAO corresponded exactly to DHA-independent reduction, measured in parallel incubations. De-
Fig. 1. Erythrocyte responses to GSH depletion by glutathione-Stransferase substrates. Erythrocytes at a 6% hematocrit were incubated at 37°C in PBS that contained 5 mM D-glucose and the indicated concentration of phorone (A) or CDNB (B). At 15 min, DHA was added to an initial concentration of 100 mM, and incubations were continued for an additional 15 min. The cells were pelleted in a microfuge and washed three times in 10 vol of PBS. Aliquots of the washed cells were taken for assay of GSH (circles), ascorbate (squares), a-tocopherol (diamonds), and for their ability to reduce extracellular ferricyanide (triangles), as detailed under Experimental Procedures. Results are shown as a fraction of control from three experiments with phorone, and six experiments with CDNB. Initial values in A were: GSH, 1.7 6 0.4 mM; ferricyanide reduction, 5.3 6 1.6 mmol z (ml erythrocytes)21 z (30 min)21. Initial values in panel B were: GSH, 1.7 6 0.2 mM; ascorbate, 22 6 3 mM; ferricyanide reduction, 9.3 6 0.8 mmol z (ml erythrocytes)21 z (30 min)21, and a-tocopherol, 3.6 6 0.2 nmol z (ml erythrocytes)21.
spite only a small effect on the cellular GSH content, PAO markedly decreased the ability of cells to reduce added DHA. The decrease in DHA-stimulated ferricyanide reduction caused by preincubation with PAO paralleled the decrease in the ability of erythrocytes to recycle DHA to ascorbate, as shown in Fig. 3. In these experiments, cells were pretreated with 100 mM PAO, followed by loading with increasing concentrations of DHA. The ability of erythrocytes to reduce DHA to ascorbate was markedly impaired over
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Fig. 2. Inhibition of erythrocyte DHA reduction by increasing concentrations of PAO. Erythrocytes at a 6% hematocrit were incubated at 37°C in PBS that contained 5 mM D-glucose and the indicated concentration of PAO. At 20 min, DHA was added to an initial concentration of 100 mM and the incubation was continued for an additional 20 min. The cells were pelleted in a microfuge and washed three times in 10 vol of PBS. Aliquots of the washed cells were taken for assay of GSH (circles), and for their ability to reduce extracellular ferricyanide (triangles), as detailed under Experimental Procedures. Results are expressed as a fraction of control values from at least six experiments for each assay, with the solid lines indicating fits to a monoexponential model with residual.
the range of DHA concentrations tested (Fig. 3A), as was the ability of PAO-treated cells to reduce ferricyanide (Fig. 3B). These results could not be explained by direct reaction of PAO with ascorbate, because an eightfold molar excess of PAO did not affect ascorbate concentrations when incubated in vitro (results not shown). Further, PAO up to 100 mM did not significantly decrease 3-O-methylglucose efflux from erythrocytes, making it unlikely that PAO inhibited DHA uptake on the glucose transporter, which could have contributed to the observed decrease in DHA recycling to ascorbate. The effect of PAO to inhibit ascorbate recycling more severely than it decreased intracellular GSH concentrations suggests that PAO decreases DHA recycling by one or more mechanisms in addition to reacting with intracellular GSH. GSH-dependent DHA reduction was studied next in erythrocyte hemolysates. Use of hemolysates removes the membrane barrier and allows direct access of DHA and inhibitors to cytoplasmic substrates and enzymes. Erythrocyte hemolysates were initially dialyzed overnight to remove GSH and enzyme cofactors, such as NADPH. Whereas these dialysates supported reduction of DHA by added GSH, there was no activity in excess of that seen in control incubations with GSH and DHA
Fig. 3. Inhibition by PAO of erythrocyte recycling of DHA to ascorbate. Erythrocytes were incubated under the conditions described in the legend to Fig. 2 without (open symbols) or with (closed symbols) 100 mM PAO. After 20 min of incubation, DHA was added to the indicated initial concentration and incubation was continued for another 20 min. Following three centrifugation washes, aliquots of the cells were taken for assay of ascorbate, and for their ability to reduce extracellular ferricyanide. (A) This shows intracellular ascorbate concentrations (squares); (B) Shows ferricyanide reduction as mmol z (ml erythrocytes)21 z (30 min)21 (triangles) from three experiments.
alone (results not shown). On the other hand, incubation of freshly prepared lysates with 4 mM GSH and increasing concentrations of DHA did result in ascorbate generation in excess of that attributable to a direct chemical reduction (Fig. 4). In the absence of added GSH, the rate of DHA reduction was about 10% of that observed with 4 mM GSH, reflecting dilution of intracellular GSH in the lysates. The rate of chemical reduction of DHA by 4 mM GSH rose linearly with increases in added DHA (Fig. 4). Subtraction of the rate ascorbate generation in the chemical reaction from the rate in the presence of hemolysate at each DHA concentration resulted in a progressively increasing plot, which showed no tendency to saturate over the range of DHA concentrations used. The rate of DHA reduction in excess of the chemical reaction, calculated relative to the volume of original cell cytoplasm and as a function of the mM DHA concentration, was 23.1 nmol z (ml cells)21 z min21 (r 5 .99).
Erythrocyte ascorbate recycling
Fig. 4. GSH-dependent DHA reduction in erythrocyte hemolysates. Erythrocyte hemolysates were prepared as described under Materials and Methods. The freshly prepared hemolysate was incubated at room temperature in 1 vol of PBS that contained 4 mM GSH and the indicated initial concentration of DHA. At 10 min, the hemolysate was ultrafiltered as described for the ascorbate assay, and the ascorbate concentration in the ultrafiltrate was measured (squares, solid line). The direct chemical reaction was also measured in the absence of cells (circles, solid line). Activity that could not be accounted for by direct reaction (triangles, dashed line) was obtained by subtracting the direct chemical reaction from the total in each of the four experiments performed. Data are from four experiments, fit to linear models with r . .99 for each fit.
To examine the possibility of NADPH-dependent DHA reduction, 400 mM NADPH was added to freshly prepared erythrocyte hemolysates. This resulted in a 23% increase in the rate of ascorbate generation compared to control ( p , .01, n 5 6 determinations). However, this small increase in NADPH-dependent reduction could have been due simply to direct reaction of DHA with NADPH or to the ability of NADPH to recycle GSSG to GSH through glutathione reductase. Increased specificity for TR was sought as follows. First, because TR is sensitive to inhibition by aurothioglucose,14 we incubated freshly prepared erythrocyte lysates with 10 mM aurothioglucose. As shown in Fig. 5, the rate of ascorbate generation, expressed relative to the original cytoplasmic volume, was inhibited 68% by aurothioglucose (first pair of bars). Further, addition of 5 mM E. coli thioredoxin almost doubled DHA reduction compared to control, and this activity was inhibited to the same level by aurothioglucose (second pair of bars). To correct for direct reduction of DHA by NADPH and for GSH-dependent reduction of DHA, we calculated aurothioglucose-sensitive DHA reductase activity (basal activity minus activity in the presence of aurothioglucose), and this was
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Fig. 5. NADPH-dependent DHA reduction in erythrocyte hemolysates. Hemolysates prepared as described under Materials and Methods were incubated at room temperature in PBS in the presence (open bars) or absence (hatched bars) of 10 mM aurothioglucose. After 5 min, NADPH and DHA were added to concentrations of 0.4 mM and 40 mM, respectively. At the same time, thioredoxin (TRx) was added to a final concentration of 5 mM to one pair of samples, but not to the other (Con). The incubations were continued another 10 min at room temperature, followed by ultrafiltration of the samples for assay of the ascorbate concentration. The third pair of bars, noted by (2)ATG, shows total activity minus activity observed with aurothioglucose in each experiment, with the double crosshatched bar indicating control, and the solid bar indicating thioredoxin-treated samples. Data are from five experiments, with an asterisk (*) indicating p , .05 compared to the adjacent bar.
more than doubled in the presence of thioredoxin (last pair of bars). This supports the hypothesis that TR contributed to the reduction of DHA in these hemolysates. Aurothioglucose-sensitive DHA reductase activity was also observed in overnight dialyzed hemolysates, but this was only 5% of the aurothioglucose-sensitive activity in fresh hemolysates (results not shown). To further study the role of TR in erythrocyte DHA reduction, two additional experiments were performed. In the first, DHA reductase activity was measured with increasing concentrations of DHA in the presence or absence of aurothioglucose. As shown in Fig. 6, aurothioglucose was a more effective inhibitor of hemolysate DHA reductase activity at low than at high DHA concentrations. Subtraction of rates of aurothioglucose-sensitive ascorbate regeneration from rates of total ascorbate regeneration resulted in a curvilinear plot that had a apparent K m of 1.5 mM when fit to a simple hyperbolic model. When the total activity was fit to an hyperbolic model with a nonsaturable component, the apparent kinetic parameters were similar, as noted in the legend to Fig. 6. This saturability of the aurothioglucose-depen-
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Fig. 6. DHA dose-dependence of gold-sensitive, NADPH-dependent DHA reductase activity. Cell lysates were prepared and incubated in 1.25 vol of PBS under the conditions described in the legend to Fig. 5. Following a 3-min preincubation with (squares) or without (circles) 10 mM aurothioglucose, NADPH was added to 0.4 mM, followed by the indicated concentration of DHA. After 10 min, the samples were ultrafiltered, and ascorbate concentrations were measured. The difference between the total and the aurothioglucose-sensitive ascorbate concentration was calculated at each point (triangles). The solid line is a linear fit to the gold-inhibited data (r 5 .99, p , .002), the dotted line is a fit to an hyperbolic model with a nonsaturable component of the total activity (K m 5 1.2 mM, V max 5 99 mM, nonsaturable 5 10% of V max, and r 5 .9998), and the dashed line is an hyperbolic fit without nonsaturable components to the difference between total and gold-inhibited activity (K m 5 1.5 mM, V max 5 100 mM, r 5 .997). Data are shown from four experiments.
dent activity provides additional support for the notion that it is mediated by an enzyme. In the second experiment, we returned to the use of PAO, which appeared to inhibit ascorbate regeneration in intact cells to an extent greater than could be accounted for by reaction with cellular GSH. As shown in Fig. 7, lysates that were prepared from cells that had been treated with PAO showed a 75% decrease in total (first pair of bars) and an 80% decrease in aurothioglucose-sensitive NADPH-dependent DHA reductase activity (last pair of bars). To ensure adequate ascorbate generation in PAO-treated samples, incubations were carried out at 37°C, which accounts for the increased rate of ascorbate generation in the control sample compared to that shown in Fig. 5. These effects of PAO on DHA reductase activity could not be accounted for by the 10% measured decrease in GSH in the cell lysates. Further, the DHA reductase activity of purified rat liver TR was very sensitive to PAO, and was inhibited half-maximally by preincubation with 21 mM PAO (Fig. 8). These data support the notion that inhibition of this enzyme in
Fig. 7. Inhibition of aurothioglucose-sensitive DHA reductase activity by pretreatment of cells with PAO. Erythrocytes were treated as described in the legend to Fig. 2 with 100 mM PAO, washed, and hemolyzed for assay of DHA reductase activity at 37°C in an equal volume of PBS as described under Materials and Methods. The hatched bars show the amount of ascorbate generated by control hemolysates, and the solid bars show ascorbate generated in hemolysates from cells treated with PAO. The first pair of bars (TOTAL) indicates DHA reduction in control hemolysates, the second pair of bars (ATG) indicates activity in the presence of 10 mM aurothioglucose, and the third pair of bars (TOT-ATG) indicates the difference between control and aurothioglucose-treated hemolysates for each of five experiments. An asterisk (*) indicates p , .05 compared to the adjacent control.
erythrocytes and their hemolysates was involved in the cellular response to PAO.
DISCUSSION
In contrast to nucleated cells, which concentrate ascorbate against a gradient,25 human erythrocytes have similar concentrations of ascorbate compared to plasma.26 Nonetheless, erythrocytes have a high capacity to recycle DHA to ascorbate. When presented with DHA, erythrocytes can rapidly remove DHA from the extracellular space,4 and accumulate ascorbate against a concentration gradient.20,27 Based on rates of DHA-dependent ferricyanide reduction, we have calculated that the cells can regenerate the ascorbate content of blood every 3 min.20 This ascorbate recycling depends on an adequate supply of reducing equivalents in the cells, which derive ultimately from glucose metabolism. The proximal electron donors for ascorbate recycling have not been established with certainty. GSH appears to be involved in erythrocytes, because DHA recycling to ascorbate in the absence of glucose depletes GSH,9 and since GSH depletion with diamide9 and with glutathione-S-transferase substrates (Figs. 1 and 2) impairs DHA-
Erythrocyte ascorbate recycling
Fig. 8. Inhibition of rat liver TR by PAO. Purified from rat liver TR (21 nM) was incubated in 50 mM Tris-HCl, pH 7.4, at room temperature with the indicated concentration of PAO. At 15 min, NADPH and DHA were added to 0.4 mM and 2 mM, respectively, and the incubation was continued for another 20 min. Nine volumes of ice-cold methanol 80% (v/v) containing 1 mM EDTA were added, the sample was microfuged for 2 min, and the ascorbate concentration in the supernatant was determined. The data from three experiments are expressed relative to the control value, and fit to a monoexponential model with residual (r 5 .9997, residual 3.6%).
dependent ferricyanide reduction. GSH can directly reduce DHA to ascorbate,28 and such nonenzymatic recycling is considered a prominent, if not the sole mechanism operative in erythrocytes and other cells.3 Thus, dialyzed erythrocyte hemolysates fail to support GSH-dependent DHA reduction at levels beyond that attributable to the nonenzymatic reaction.5 We obtained similar results with dialyzed hemolysates, but were able to show DHA reduction in excess of that attributable to the nonenzymatic reaction with added GSH in freshly prepared hemolysates (Fig. 4). This suggests that erythrocytes do have GSH-dependent DHA reductase activity that could account for as much as a third of the total activity observed. The enzymatic component is lost upon dialysis in oxygenated buffer. Oxidation of sensitive sulfhydryl groups on glutaredoxin6 would account for this observation. It was also possible to demonstrate NADPH-dependent DHA reductase activity in freshly prepared erythrocyte hemolysates. This activity was present in overnight-dialyzed hemolysates, albeit at markedly reduced levels, again suggesting oxidative loss of NADPH-dependent activity. Several considerations suggest that the NADPH-dependent reductase activity in hemolysates is due to TR. First, we have recently reported that purified rat liver TR has DHA reductase activity.9 Second, he-
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molysate DHA reductase activity was inhibited almost 70% by 10 mM aurothioglucose (Fig. 5), a known inhibitor of selenoenzymes.14,29 –31 The selenoenzyme glutathione peroxidase is inhibited only slightly by this concentration of aurothioglucose,14 whereas TR is inhibited by more than 80%.14 Third, 5 mM E. coli thioredoxin doubled aurothioglucose-sensitive DHA reductase activity in erythrocyte hemolysates (Fig. 5). The ability of purified rat liver TR to reduce DHA to ascorbate is tripled by addition of the same concentration of E. coli thioredoxin.9 Fourth, the aurothioglucose-sensitive component of hemolysate DHA reductase activity showed a saturable dependence on the concentration of DHA in the assay, with an apparent K m of 1.5 mM (Fig. 6). The latter is similar to that measured for purified TR.9 The results of treatment of erythrocytes with PAO also suggest that TR contributes significantly to DHA reduction in intact cells. PAO is an agent with specificity for vicinal or nearby sulfhydryl groups,24,32 which are required for catalysis in TR.33 PAO was a potent inhibitor of purified rat liver TR (Fig. 8). When incubated with intact erythrocytes, PAO inhibited the ability of cells to carry out DHA-dependent ferricyanide reduction to a much greater extent than it decreased the GSH content of cells (Figs. 2 and 3). These results suggest involvement of one or more sulfhydryl-containing enzymes, such as glutaredoxin or TR. Because treatment of intact cells with PAO inhibited aurothioglucose-sensitive DHA reductase activity by 80% in hemolysates in the presence of NADPH (Fig. 7), at least part of the effect of PAO in intact cells appears due to inhibition of TR. The extent to which enzymatic NADPH-dependent versus either enzymatic or nonenzymatic GSH-dependent DHA reduction predominates in intact erythrocytes is difficult to ascertain. In hemolysates, comparison of the normalized data in Figs. 4 and 6 at 1 mM DHA reveals that rates of GSH-dependent DHA reduction are more than 10-fold those of NADPH-dependent DHA reduction. This may not reflect the situation in the intact cell, however, as indicated by the effects of PAO discussed above. Attempts to selectively deplete cellular GSH using glutathione-S-transferase substrates, although suggestive of GSH-dependent reduction, did not provide a conclusive answer. Phorone at concentrations effective for depletion of GSH from liver and other tissues22,34 had only a small effect on erythrocyte GSH content, although it did decrease DHA-dependent ferricyanide reduction (Fig. 1A). CDNB was much more potent than phorone in decreasing both erythrocyte DHA recycling and GSH content (Fig. 1B), but CDNB is also an inhibitor of mammalian TR.35 Still, there is more evidence for GSH dependence of DHA reduction in erythrocytes than in HL-60 cells, in which the depletion of GSH has little effect on rates of DHA reduction.36 In dialysed rat liver
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hemolysates, we found both GSH- and NADPH-dependent DHA reductase activities.9 Mechanisms of DHA reduction may vary in different cells, and at least in erythrocytes and liver, also appear to be redundant.
19.
20. Acknowledgements—This work was supported by NIH Grant DK 50435, and by a Research Grant from the American Diabetes Association. The authors appreciate the gift of purified rat liver TR from Drs. Raymond Burk and Kristina Hill, and reading of the manuscript by Dr. Burk.
21. 22.
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CDNB—1-chloro-2,4-dinitrobenzene DHA— dehydroascorbic acid HPLC— high-performance liquid chromatography PBS—phosphate-buffered saline PAO—phenylarsine oxide TR—thioredoxin reductase