Recycling of Vitamin C from Its Oxidized Forms by Human Endothelial Cells

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Biochimica et Biophysica Acta 1640 (2003) 153–161 www.bba-direct.com

Recycling of vitamin C from its oxidized forms by human endothelial cells

James M. May*, Zhi-chao Qu, Dustin R. Neel, Xia Li

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, 715 Preston Research Building, Vanderbilt University School of Medicine, 2220 Pierce Avenue, Nashville, TN 37232-6303, USA Received 30 December 2002; received in revised form 26 March 2003; accepted 28 March 2003

Abstract

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 AM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3–1.6 mM generated intracellular DHA concentrations of 20–30 AM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms. D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Ascorbic acid; Ascorbate free radical; Dehydroascorbic acid; Oxidant stress; Endothelial cell

1. Introduction cofactor for GSH-dependent enzymes, such as glutaredoxin or protein disulfide isomerase [5]. However, whether endo- Endothelial cells, which form the barrier between the thelial cells recycle ascorbate from the AFR, and how this bloodstream and underlying tissues, are exposed to poten- compares to DHA reduction is unknown. tially damaging oxidant stress from both compartments. Of DHA concentrations in plasma are low, in the range of several lines of defense against oxidant stress in the vascular 0–2 AM, or about 1–2% of ascorbate concentrations [6– bed, ascorbic acid is one of the most sensitive, both in 9]. Plasma DHA concentrations are increased severalfold plasma [1] and in cells [2]. The ability of endothelial cells to in conditions manifesting oxidant stress, such as uncon- regenerate ascorbate from its oxidized forms, dehydroascor- trolled diabetes mellitus [6] and smoking [10]. Intracellular bic acid (DHA) and the ascorbate free radical (AFR), may DHA concentrations have been measured in human lym- be important not only for the ascorbate economy of the cell, phocytes, and found to be as much as 20% of ascorbate but also may determine the amount of the vitamin available concentrations [11]. Because ascorbate concentrations in for passage across the endothelium. Bovine aortic endothe- human blood monocytes are in the low-millimolar range lial cells reduce DHA, the two-electron oxidized form of [12], DHA concentrations might be substantial. If so, then ascorbate, by GSH-dependent mechanisms [3].GSH intracellular DHA could serve as a significant source for reduces DHA directly to ascorbate [4],orservesasa recycling to ascorbate, especially in cells undergoing oxidant stress. On the other hand, DHA is readily degraded due to Abbreviations: AFR, ascorbate free radical; DHA, dehydroascorbic irreversible ring-opening. Intracellular DHA can also be acid; Hepes, N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid; KRH, lost to the cells by efflux on the ubiquitous ‘‘GLUT’’ type Krebs–Ringer Hepes; PBS, phosphate-buffered saline * Corresponding author. Tel.: +1-615-936-1653; fax: +1-615-936- glucose transporters that mediate its uptake into cells [13]. 1667. Thus, the extent to which cells recycle the AFR relative to E-mail address: [email protected] (J.M. May). DHA is important for ascorbate economy of the cell. In

0167-4889/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-4889(03)00043-0 154 J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161 homogenates of several pig tissues, rates of AFR reduction prepared by the Cell Culture Core of the Vanderbilt Diabetes exceeded those of DHA reduction [14]. Reduction of the Research and Training Center. Cells were cultured to con- AFR is catalyzed by microsomal [15–17] and mitochon- fluence for 18–24 h in 10-cm or six-well plates and rinsed drial [18] NADH-dependent cytochrome b5 reductases, and three times in Krebs–Ringer Hepes (KRH) buffer at 37 jC by the NADPH-dependent enzyme thioredoxin reductase before use in an experiment. The latter consisted of 20 mM [19]. These enzymes have high affinity for the AFR, in the Hepes (N-2-hydroxyethylpiperazine N-2-ethanesulfonic range of 10 AM or less, which is in the range of AFR acid), 128 mM NaCl, 5.2 mM KCl, 1 mM NaH2PO4, 1.4 concentrations expected for cells undergoing oxidant stress mM MgSO4, and 1.4 mM CaCl2, pH 7.4. [20–22]. To address the question of relative rates of AFR and DHA recycling, it is necessary to compare rates at 2.3. Measurement of intracellular ascorbic acid and DHA AFR and DHA concentrations that might be expected in the cells. Following incubation of cells in six-well plates, the me- In the present work, we evaluated the relative contri- dium was aspirated, and the cells were quickly rinsed twice butions of recycling of ascorbate from the AFR and from with 2 ml of ice-cold KRH that contained 5 AM cytochalasin DHA using EA.hy926 cells. The latter is a permanent cell B. These conditions were used to prevent the efflux of DHA line derived from human umbilical vein endothelial cells on glucose transporters during the rinsing steps. For assay of that expresses factor VIII antigen [23], forms capillary-like ascorbate alone, the medium was removed and the mono- tubes in culture [24], oxidatively modifies human LDL layer was treated with 0.1 ml of 25% metaphosphoric acid [25], and shows calcium-dependent stimulation of eNOS (w/v), followed by 0.35 ml of a buffer containing 0.1 M [25]. We recently found that the redox cycling agent Na2HPO4 and 0.05 mM EDTA, pH 8.0. For assay of total menadione causes an oxidant stress that depletes both ascorbate, both solutions contained 2 mM 2,3-mercapto-1- ascorbate and DHA in these cells [26], suggesting that, propanol. The cells were scraped from the plate and the as in bovine aortic endothelial cells [3], DHA reduction is lysate was microfuged 1 min at 14,000Âg at 3 jC. Aliquots at least partially dependent on GSH. In the present work, of the supernatant were taken for assay of ascorbic acid by we compared the mechanisms of ascorbate recycling high-performance liquid chromatography with electrochem- directly using cell lysates. We found that both NADH ical detection [27], except that tetrapentylammonium bro- and NADPH supported AFR reduction, and that NADPH- mide was used as the ion pair reagent. It was determined that dependent AFR reduction was due largely to thioredoxin 2 mM 2,3-dimercapto-1-propanol did not interfere with the reductase. Maximal rates of enzyme-mediated reduction of assay of ascorbate. Further, this concentration of 2,3-dimer- DHA were comparable to those of AFR reduction. Based capto-1-propanol was found to completely reduce 100 AM on these findings and on the stability and reactivity of the DHA to ascorbate in 1 min under conditions of DHA AFR and DHA, we suggest that reduction of the AFR is reduction. Intracellular concentrations of ascorbate were adequate to recycle ascorbate, except in cells under oxidant calculated based on an intracellular water space in EA.hy926 stress, in which case DHA reduction serves as a second cells of 3.6F1.2 Al/mg protein, as previously measured in line of defense. these cells [28].

2.4. Measurement of DHA and AFR reduction in cell lysates 2. Materials and methods Cells cultured to confluence on a 10-cm plate were rinsed 2.1. Materials three times in 5 ml of KRH and scraped from the plate with a rubber spatula in 2 ml of ice-cold KRH. This suspension Ascorbate oxidase (from Curcubita species), DHA, cyto- was subjected to two freeze–thaw cycles in dry ice/ethanol chalasin B, and 2,3-dimercapto-1-propanol were from and either used directly, or microfuged for 1 min at 4800Âg Sigma/Aldrich Chemical (St. Louis, MO). The last two at 3 jC. The slightly turbid supernatant was removed and reagents were prepared and stored as 5-mM solutions in used for assay of AFR and DHA reduction at a final protein ethanol. concentration in the assay of about 0.1 mg/ml. For assay of AFR reduction, lysate was incubated in a 2.2. Cell culture and preparation for assays semi-micro cuvette that contained phosphate-buffered saline (PBS, 12.5 mM Na2HPO4, 140 mM NaCl, pH 7.0) at EA.hy926 cells were provided by Dr. Cora Edgell (Uni- 37 jC, 100 AM of either NADH or NADPH, 1 mM versity of North Carolina, Chapel Hill, NC). Cells were ascorbate, the indicated concentration of ascorbate oxi- cultured in Dulbecco’s minimal essential medium that con- dase, and other agents as noted. The cuvette was placed tained 20 mM D-glucose, 10% (v/v) fetal bovine serum, and in the sample chamber of a Beckman DU-640 recording HAT media supplement (Sigma/Aldrich Chemical). The spectrophotometer at 37 jC, and the change in absorb- latter contains 5 mM hypoxanthine, 20 AM aminopterin, ance at 340 nm was measured every 10 s for 3 min. The and 0.8 mM thymidine in culture. Cell culture media were linear rate of decrease in absorbance was calculated and J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161 155

the rate of oxidation of the nucleotide was determined using a coefficient of extinction of 6.22 mMÀ1 cmÀ1. This rate was corrected in each experiment for the rate observed with lysate and reduced nucleotide alone. This correction was less than 10% of the total rate. Assay of GSH-dependent DHA reduction was measured by incubating supernatant from microfuged lysate (0.1 mg/ ml) in PBS at pH 7.0 with 0.4 U/ml glutathione reductase, 0.1 mM NADPH, 2 mM GSH (except where indicated), and the noted concentration of freshly prepared DHA. Record- ing and calculations were carried out as described for assay of AFR reduction, and rates were corrected for rates of direct reduction of DHA measured under the same conditions in the absence of lysate.

2.5. Other methods

AFR generation from ascorbate and ascorbate oxidase was measured spectrophotometrically by its absorbance at 360 nm, using an extinction coefficient of 3.3 mMÀ1 cmÀ1 [29]. Protein was measured by the method of Bradford [30]. Results are shown as meanFS.E. Statistical comparisons were made using SigmaStat 2.0 software (Jandel Scientific, San Rafael, CA). Differences between treatment groups Fig. 1. Generation of the AFR by ascorbate and ascorbate oxidase. The were assessed by one- or two-way analysis of variance with indicated concentration of ascorbate oxidase (AO, in units per ml, U/ml) j post-hoc testing using Dunnett’s test. was incubated at 37 C with 1 mM ascorbate in PBS at pH 7.0 and the absorption at 360 nm was followed for times noted in panel A. At each time point, the concentration of the AFR was calculated as described in Materials and methods. Results are shown as meanFS.E. from nine experiments. In 3. Results panel B, the average AFR concentration over the total 3-min incubation from these data is plotted as a function of the ascorbate oxidase 3.1. Generation and measurement of the AFR concentration.

To study the ability of EA.hy926 cells to regenerate its oxidase. These changes reflect enzyme-dependent AFR ascorbate from the AFR, it was necessary to use cell lysates, reduction. However, NADH was oxidized at only about in which the AFR can be generated by the combination of half the rate observed for NADPH. The possibility that ascorbate and ascorbate oxidase. The latter oxidizes ascor- NADH-dependent activity present in particulate fractions bate to the AFR, which then can be reduced back to was lost during a 1-min microfuge step to clear large cell ascorbate, oxidized further to DHA, or can dismutate to fragments was tested by repeating the studies without the form a molecule of ascorbate and one of DHA from two microfuge step. As shown in Fig. 2B, relative rates of molecules of the AFR. First, to confirm the stability of AFR NADH disappearance increased about 5-fold under these concentrations when generated by the ascorbate/ascorbate conditions, and exceeded the rate of NADPH disappear- oxidase system, we measured AFR generation in buffer ance, which increased only about 50%. This suggests that alone by its absorbance of the AFR at 360 nm. As shown in most of the NADH-dependent AFR reductase activity was Fig. 1A, in the presence of 1 mM ascorbate, the steady-state in the particulate fractions, whereas most of the NADPH- AFR concentration over 3 min was relatively constant, dependent activity was in the soluble fraction. In an although it did decrease about 10% at the highest oxidase attempt to increase NADH-dependent AFR reduction even concentrations. The steady-state AFR concentration over 3 further, the assays were carried out in the presence of min showed a linear dependence on the concentration of non-ionic detergents. However, both 0.2% Triton X-100 ascorbate oxidase (Fig. 1B). and40mMoctyl-h-D-glucopyranoside decreased the To compare rates of AFR reduction by NADH and relative rates of NADH disappearance (Fig. 2B).The NADPH, lysates of EA.hy926 cells microfuged briefly to Triton X-100 concentration was chosen based on the clear cell debris, then incubated with 5 U/ml of ascorbate results of a previous study that showed enhanced oxidase to generate an AFR concentration of about 6 AM. NADH-dependent AFR reduction in mitochondria [9]. As shown in Fig. 2A, there was increased disappearance Lower concentrations of Triton X-100 were tried, but of both NADH and NADPH above background rates of either had no effect or were inhibitory (results not NAD(P)H disappearance in the absence of ascorbate and shown). 156 J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161

studies, aurothioglucose is a specific inhibitor of thioredoxin reductase [31]. Together, these results suggest that most of the NADPH loss is due to thioredoxin reductase, and that thioredoxin-stimulated rates of NADPH-dependent AFR reduction are comparable to those observed when NADH is the electron donor. Additional features of NAD(P)H-dependent AFR reduction are shown in Figs. 3 and 4. First, there was very little NADPH oxidized in the absence of lysate protein, and the rate of oxidation increased linearly with lysate protein (Fig. 3). Second, the apparent affinity of AFR reduction using either NADH or NADPH as the electron donor was in the low-micromolar range. This was assessed by increasing the amount of ascorbate oxidase (or the AFR) present in the incubation. Rates of both NADH- and NADPH-dependent AFR reduction were half-maximal at estimated AFR concentrations of 1–2 AM, based on non-linear fitting of the data to a rectangular hyperbola (Fig. 4). The x-axis values in this figure are the measured values and errors in the absence of cells that are shown in Fig. 1. These results show that the affinity of the NADPH-dependent thioredoxin reductase system for the AFR is similar to that of NADH-dependent reduction by enzymes in the particulate fraction.

3.2. Lysate reduction of DHA

Fig. 2. Pyridine nucleotide-dependent reduction of the AFR. Lysates (0.08– To determine the extent to which DHA reduction con- 0.15 mg protein/ml) were either microfuged briefly as described under tributes to ascorbate recycling in EA.hy926 cells, we incu- Materials and methods (panel A) or used directly (panel B). These were mixed in PBS that contained 1 mM ascorbate, 5 U/ml ascorbate oxidase, bated lysates of EA.hy926 cells with 0.1 mM DHA and a 5- and one or more of the following as noted under the graph: NADH (100 fold molar excess of GSH in the presence of glutathione AM), NADPH (100 AM), dithiothreitol (5 mM), thioredoxin (5 AM), aurothioglucose (ATG, 10 AM), Triton X-100 (0.2% v/v), or octyl-h-D- glucopyranoside (Octyl Glucoside, 40 mM). The rate of NAD(P)H disappearance was followed spectrophotometrically at 340 nm for 3 min at 37 jC and the rate of change in the concentration of NAD(P)H was calculated as described under Materials and methods. Results are shown as meanFS.E. from at least six experiments in panel A and from five experiments in panel B. An asterisk (*) indicates P<0.05 compared to other treatments in the same panel.

The mechanism of NADPH-dependent AFR reduction was further investigated in the experiments shown in Fig. 2. First, to determine the extent to which reduction of DHA (generated by dismutation of the AFR) contributed to the rate of NADPH loss, 0.5 mM dithiothreitol was included in the incubation. Dithiothreitol directly reduces DHA to ascorbate, and thus should decrease or prevent NADPH loss due to DHA reduction. As shown in the third bar from the left in Fig. 2A, dithiothreitol did not significantly affect rates of NADPH oxidation. This suggests that there is little DHA either generated or reduced under these conditions. In the presence of thio- Fig. 3. Dependence of thioredoxin-stimulated NADPH reduction on the redoxin, a protein cofactor for thioredoxin reductase, rates concentration of lysate protein. NADPH disappearance was followed in the spectrophotometer at 37 jC in PBS containing increasing amounts of of NADPH disappearance tripled. When aurothioglucose EA.hy926 cell lysates, 5 AM thioredoxin, 1 mM ascorbate, and 5 U/ml was added, rates of NADPH loss decreased by more than ascorbate oxidase. Results are shown from three experiments as meanFS.E. 75%. At the concentration of 10 AMusedinthese for both the concentration of protein and the rate of NADPH disappearance. J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161 157

Fig. 5. Dependence of DHA reduction by GSH on the lysate protein content. NADPH disappearance was followed as a function of the lysate Fig. 4. Concentration dependence of AFR reduction. NADPH disappear- protein concentration in a coupled assay that measures NADPH ance (circles) was followed in the spectrophotometer at 37 jC in PBS disappearance as oxidized GSH is recycled by glutathione reductase. containing cell lysate supernatant (0.08–0.12 mg protein/ml), 0.1 mM Reduction was followed in the spectrophotometer at 37 jC in PBS NADPH, 5 AM thioredoxin, 1 mM ascorbate, and increasing amounts of containing 0.1 mM NADPH, 0.4 U/ml glutathione reductase, 0.5 mM GSH, ascorbate oxidase, as indicated. NADH disappearance (squares) was 0.1 mM DHA, and the indicated concentration of cell lysate actually followed in the same manner except that the crude lysate was used without measured in the experiments. Results are shown from seven experiments as centrifugation (0.1–0.15 mg protein/ml) and 0.1 mM NADH was used meanFS.E. for the rate of NADPH disappearance. instead of NADPH. Results from at least six experiments are shown as F mean S.E. The x-axis shows the estimated AFR concentration generated added to the lysates. These results are corrected for non- from data of Fig. 1. enzyme-dependent DHA reduction by GSH. The increase in rates of NADPH disappearance was half-maximal at a DHA reductase and NADPH. In this system, as DHA is reduced by GSH, either directly or with the aid of one or more enzymes, the resulting GSSG is recycled by glutathione reductase with consumption of NADPH. As shown in Fig. 5, there was substantial direct reduction of DHA by GSH in the absence of lysate. However, addition of increasing amounts of lysate caused a linear increase in the extent of NADPH oxidation, indicating that one or more enzymes facilitate GSH-dependent DHA reduction. In subsequent experiments, the direct reduction of DHA by GSH was measured in the absence of lysate under the same conditions, and the rate was subtracted from the corresponding value measured in the presence of lysate. Under these conditions, 10 AM aurothioglucose failed to inhibit GSH- dependent NADPH oxidation, indicating little contribution by the thioredoxin reductase system (results not shown). Similarly, NADH did not serve as a substrate for GSH- dependent AFR reduction (results not shown). The reduction of a 0.1-mM concentration of DHA by the lysates increased with increasing GSH concentrations up to Fig. 6. Enzyme-dependent reduction of DHA by GSH in cell lysates. about 4 mM GSH (results not shown). Because the meas- Supernatants from cell lysates (0.08–0.12 mg protein/ml) were incubated at ured GSH concentration in these cells is about 2.0 mM [28], 37 jC in PBS that contained 0.1 mM NADPH, 0.4 U/ml glutathione this concentration was used to test the effects of increasing reductase, 2 mM GSH, and the indicated initial concentration of DHA. The reaction was followed in the spectrophotometer as the disappearance of amounts of added DHA on NADPH oxidation. As shown in NADPH. All values are corrected for the results in paired samples in which Fig. 6, the rate of NADPH disappearance increased in a reduction of DHA was followed in the absence of lysate. Results are shown saturable manner when increasing amounts of DHA were as meanFS.E. from five experiments. 158 J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161 concentration of 0.1F0.02 mM (based on fitting the data to ascorbate. With ascorbate loading, intracellular ascorbate a rectangular hyperbola). concentrations increased to levels similar to that observed with acute DHA loading (Fig. 7A, squares). Intracellular 3.3. Intracellular ascorbate and DHA concentrations in DHA concentrations following overnight ascorbate treat- EA.hy926 cells ment were comparable to those seen with acute DHA loading (Fig. 7B, squares). Thus, although intracellular The question remains as to what concentrations of DHA DHA concentrations were a small fraction of ascorbate might be expected in endothelial cells. Therefore, intra- concentrations, DHA was present after either acute loading cellular ascorbate and DHA concentrations were measured of the cells with DHA or overnight loading with ascorbate. in intact EA.hy926 cells following acute loading with DHA Further, intracellular DHA did not depend on the loading and overnight loading with ascorbate. In the absence of concentration of either DHA or ascorbate. ascorbate loading, neither ascorbate nor DHA were detected in EA.hy926 cells (results not shown). However, when cells were incubated with increasing amounts of DHA for 15 4. Discussion min, intracellular ascorbate increased into the low-millimolar range (Fig. 7A, circles). Despite the increase in intra- The potential cellular mechanisms of ascorbate recycling cellular ascorbate as a function of the DHA loading from both the AFR and DHA are diagramed in Fig. 8. concentration, the intracellular DHA concentration re- Human-derived endothelial cells reduced the AFR by both mained about 20–30 AM (Fig. 7B, circles). Because acute NADH- and NADPH-dependent mechanisms. As expected DHA reduction might be limited by depletion of cellular from previous studies showing NADH-dependent reduction reducing equivalents, ascorbate and DHA concentrations of the AFR by cytochrome b5 reductases in liver mitochon- were also measured in cells incubated overnight with dria [18] and microsomes [15–17], partial removal of the particulate fraction of lysates by a brief centrifugation also decreased the amount of NADH-dependent AFR reductase activity (Fig. 2A and B). When normalized to protein, rates of NADH-dependent AFR reduction in EA.hy926 cell lysates were similar to those observed in crude homogenates of several pig tissues by Coassin et al. [14]. In contrast to results of that study, use of non-ionic detergents decreased, rather than increased, NADH-dependent AFR reductase activity in particulate-containing fractions (Fig. 2B). This may reflect better access of substrates to the particulate enzymes in cultured endothelial cells than in crude tissue homogenates. In lysates of EA.hy926 cells that had been briefly microfuged to remove particulate matter, NADPH was a better electron donor for AFR reduction than was NADH (Fig. 2A). NADPH-dependent AFR reduction was tripled by thioredoxin, and inhibited by aurothioglucose. These results support a role for thioredoxin reductase in cellular AFR reduction. We previously found that purified rat liver thioredoxin reductase reduces the AFR with an affinity of about 2 AM [19]. The apparent affinity of thioredoxin-stimulated NADPH-dependent AFR reductase activity is 1–2 AMin EA.hy926 cell lysates (Fig. 4). Similarly, NADH-dependent AFR reduction has a comparable affinity (Fig. 4), and is in Fig. 7. Intracellular ascorbate and DHA concentrations in ascorbate-loaded the range observed for NADH-dependent cytochrome b5 F cells. For DHA treatment (circles, mean S.E. from six experiments), reduction of the AFR [15,18]. Further, maximal rates of EA.hy926 cells in six-well plates were rinsed twice in warm KRH to remove culture medium, and incubated for 15 min at 37 jC in KRH that NADPH- and thioredoxin reductase-dependent AFR reduc- contained 5 mM D-glucose and the indicated initial DHA concentration. For tion observed in EA.hy926 cell lysates are similar to those ascorbate treatment (squares, meanFS.E. from nine experiments), cells in previously observed by us in overnight-dialyzed rat liver six-well plates were incubated for 14–16 h in complete culture medium homogenates under similar conditions [19]. They were also with the indicated ascorbate concentration. After completion of the comparable to rates of NADH-dependent AFR reduction in incubations, the cells were prepared for assay of DHA and ascorbate as described under Materials and methods. Results of intracellular ascorbate EA.hy926 cell lysates containing cell particulate matter (Fig. measurements are shown in panel A, and those from intracellular DHA 4), indicating that the thioredoxin system contributes sig- measurements are shown in panel B. nificantly to AFR reduction in these endothelial cells. J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161 159

ity, or overestimates the DHA concentration at which activity is half-maximal. To determine whether the apparent affinity

of DHA reductase activity in EA.hy926 cells corresponds to intracellular concentrations of DHA might be present in cells containing physiologic amounts of ascorbate, we measured both DHA and ascorbate in cells that had been loaded with each form of ascorbate. As expected, intracellular ascorbate increased with the loading concentration of both DHA and ascorbate (Fig. 7A). DHA was measured at 20–30 AMin cells either loaded acutely with DHA, or loaded overnight with ascorbate (Fig. 7B). The failure of intracellular DHA concentrations to increase with increasing intracellular ascorbate concentrations could relate to a constant amount of oxidant stress present in the cells in culture or during an acute incubation. It seems clear from these results that increasing intracellular ascorbate does not induce its own oxidation. The measured intracellular DHA concentrations are appro- priate for the maximal 100 AM apparent Km of the DHA reductase activity. Further, in oxidatively stressed cells, Fig. 8. Schematic of intracellular ascorbate recycling. Ascorbate is attacked by a radical species (XÁ) that removes an electron and converts it to the DHA concentrations would likely increase severalfold, and AFR. The AFR can undergo recycling back to the AFR via NADPH- would then fit the kinetics of glutaredoxin, which has an dependent reduction by thioredoxin reductase, or via NADH-dependent apparent Km for DHA reduction of about 250 AM [34]. reduction by either the microsomal or mitochondrial AFR reductases. DHA Our results address the question of the relative activ- is formed from the AFR either by dismutation of two molecules of the AFR ities of DHA and AFR reductases in recycling ascorbate or further radical attack on the AFR (less likely). The resulting DHA can then undergo two-electron reduction back to ascorbate by several in endothelial cells. Coassin et al. [14], found that rates of mechanisms. GSH-dependent reduction of DHA can be direct, or mediated AFR reduction in homogenates of several pig tissues were by GSH-dependent enzymes glutaredoxin or protein disulfide isomerase severalfold greater than rates of enzyme-dependent DHA (PDI). NADPH-dependent DHA reduction can be mediated by thioredoxin reduction. At 2 mM GSH and 100 AM DHA, rates of reductase or by 3a-hydroxysteroid dehydrogenase [39]. enzyme-dependent DHA reduction in EA.hy926 cells (f10 nmol/mg/min) are similar to those observed for GSH-dependent reduction of DHA by EA.hy926 cell the sum of maximal NADPH- and NADH-dependent AFR lysates was assessed in a coupled reaction with glutathione reduction (f12 nmol/mg/min). Any contribution from reductase that measured the disappearance of NADPH. direct DHA reduction by GSH will add further to the However, to estimate enzyme-dependent DHA reduction, maximal capacity for GSH-dependent DHA reduction. it was necessary to correct for direct reduction of DHA by Nonetheless, enzyme-dependent AFR reduction in endo- GSH in each assay. We used a pH of 7.0 in the reaction to thelial and other cells is likely to be important because of attempt to decrease the rate of the non-enzymatic compo- the unique chemistry of the AFR. Ascorbate participates nent of DHA reduction, but it remained substantial (Fig. in one-electron reactions that necessarily generate the 5). Even after this correction, when normalized to protein, AFR and not DHA [35]. Although the one-electron corrected rates of GSH-dependent DHA reductase activity reduction potential of DHA to form the AFR is lower in endothelial cells are severalfold greater than those than for the AFR to form ascorbate [36], the AFR is measured in crude homogenates of pig organs [14]. relatively unreactive [37] and thus will not compete well Although the thioredoxin reductase system can reduce with ascorbate for scavenging of such radicals. DHA DHA to ascorbate at appreciable rates in rat liver homo- appears to be generated mostly from dismutation of two genates [32], in this and previous studies in bovine aortic molecules of the AFR to ascorbate and DHA [37,38]. endothelial cells [3] and human erythrocytes [33],we High-affinity AFR reductases would thus be expected to found little contribution of thioredoxin reductase to DHA recycle the AFR before it could dismutate to DHA. reduction relative to that mediated by GSH. Although we can measure DHA in ascorbate-loaded Whether GSH-dependent DHA reductase activity has cultured endothelial cells, in the presence of lower oxygen physiologic relevance (e.g., relative to AFR reductase activ- tensions in vivo, DHA concentrations inside cells might ity) depends on the kinetics of the reaction and the expected also be much lower. Perhaps it is best to consider intracellular DHA concentrations. DHA reductase activity ascorbate recycling as a two-step mechanism, such that was saturable, with a half-maximal effect at an initial DHA if the AFR escapes reduction, there are high-capacity, concentration of 100 AM (Fig. 6). Because the concentration low-affinity processes to reduce DHA before it escapes of DHA will decrease during the reaction due to its lack of the cells on glucose transporters or is irreversibly lost due chemical stability, this value underestimates the actual affin- to ring-opening. 160 J.M. May et al. / Biochimica et Biophysica Acta 1640 (2003) 153–161

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