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Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism.

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 minutes and an apparent transport Km of 21 μM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.

Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine β-hydroxylase (DβH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 minutes, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 minutes of incubation with 100 μM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2 – 0.5 mM, which fits well with the literature Km for ascorbate of DβH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 μM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 μM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells, that it does so at physiologic intracellular concentrations in accord with the kinetics of DβH, and that it both protects cells from superoxide and by providing electrons to DβH.

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N(ω)-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.

Mildly oxidized low density lipoprotein (mLDL) acutely increases the permeability of the vascular endothelium to molecules that would not otherwise cross the barrier. This study has shown that ascorbic acid tightens the permeability barrier in the endothelial barrier in cells, so this work tested whether it might prevent the increase in endothelial permeability due to mLDL. Treatment of EA.hy926 endothelial cells with mLDL decreased intracellular GSH and activated the cells to further oxidize the mLDL. mLDL also increased endothelial permeability over 2 h to both inulin and ascorbate in cells cultured on semi-permeable filters. This effect was blocked by microtubule and microfilament inhibitors, but not by chelation of intracellular calcium. Intracellular ascorbate both prevented and reversed the mLDL-induced increase in endothelial permeability, an effect mimicked by other cell-penetrant antioxidants. These results suggest a role for endothelial cell ascorbate in ameliorating an important facet of endothelial dysfunction caused by mLDL.

Ascorbic acid improves endothelial barrier function by decreasing the permeability of endothelial cells cultured on semi-porous membrane filters. This decrease was not due to enhanced collagen synthesis and was mimicked by the collagen synthesis inhibitor ethyl-3, 4-dihydroxybenzoic acid (EDHB). Since EDHB is known to chelate intracellular free iron, the effects of two membrane-permeant iron chelators were tested on endothelial permeability. Both 2,2′-dipyridyl and desferrioxamine decreased trans-endothelial permeability in a concentration-dependent manner. Increasing intracellular iron with a chelate of 8-hydroxyquinoline and ferric iron prevented effects of both EDHB and intracellular ascorbate. That EDHB and ascorbate did in fact chelate intracellular iron was supported by finding that they both decreased the cellular fluorescence quenching of the iron-sensitive dye Phen green SK. These results show that chelation of intracellular iron decreases endothelial barrier permeability and implicate this mechanism in the ability of EDHB and possibly intracellular ascorbate to tighten the endothelial barrier.

Objectives: The reducing capacity of erythrocytes has been used clinically as to estimate resistance to oxidant stress. In this work we targeted the antioxidant capacity of pyridine nucleotide disulfide reductases of these cells by measuring their ability to reduce the disufide α-lipoic acid. Methods: Erythrocyte reduction of α-lipoic acid and related disulfides was measured as reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) outside the cells. Results: Lipoic acid-dependent DTNB reduction by human erythrocytes required d -glucose and consumed NADPH, but not NADH. This activity was inhibited by carmustine and phenylarsine oxide, as expected if α-lipoic acid is reduced by the glutathione and thioredoxin reductase systems. Reduction of hydroxyethyl disulfide, which provides an estimate of total erythrocyte disulfide reduction capacity, was similar to that of α-lipoic acid. Erythrocytes incubated with α-lipoic acid also reduced extracellular ferricyanide, although rates of dehydroascorbate reduction were several-fold greater, probably because intracellular GSH can recycle ascorbate but not α-lipoic acid in erythrocytes. Conclusion: These results show that α-lipoic acid-dependent DTNB reduction provides a simple method to selectively assess the capacity of pyridine nucleotide disulfide reductases of human erythrocytes. When coupled with other non-destructive assays, such as reduction of hydroxyethyl disulfide and ferricyanide, this assay provides a comprehensive approach to assessing erythrocyte reducing capacity in a variety of clinical conditions associated with oxidant stress.

Erythrocytes have the same intracellular concentration of ascorbate as plasma, which is much lower than that of nucleated cells. To determine why erythrocytes are unable to concentrate ascorbate, we tested for the presence of ascorbate transporters in these cells. Human erythrocytes had very low rates of uptake of radiolabeled ascorbate, which was accounted for by the lack of ascorbate transporter SVCT2 in immunoblots. Using a cell culture model of Friend virus-infected mouse erythroblasts, immunoblots showed that the SVCT2 was present in the erythroblast stages, but was lost following extrusion of the nucleus in the formation of the reticulocyte stage. Rates of specific ascorbate transport correlated with the presence of the SVCT2. These results show that mature erythrocytes fail to concentrate ascorbate due to the loss of SVCT2 during maturation in the bone marrow.

Mitochondria are the major source of potentially damaging reactive oxygen species in most cells. Since ascorbic acid, or vitamin C, can protect against cellular oxidant stress, we studied the ability of mitochondria prepared from guinea pig skeletal muscle to recycle the vitamin from its oxidized forms. Although ascorbate concentrations in freshly prepared mitochondria were only about 0.2 mM, when provided with 6 mM succinate and 1 mM dehydroascorbate (the two-electron-oxidized form of the vitamin), mitochondria were able to generate and maintain concentrations as high as 4 mM, while releasing most of the ascorbate into the incubation medium. Mitochondrial reduction of dehydroascorbate was strongly inhibited by 1,3-bis(chloroethyl)-1-nitrosourea and by phenylarsine oxide. Despite existing evidence that mitochondrial ascorbate protects the organelle from oxidant damage, ascorbate failed to preserve mitochondrial alpha-tocopherol during prolonged incubation in oxygenated buffer. Nonetheless, the capacity for mitochondria to recycle ascorbate from its oxidized forms, measured as ascorbate-dependent ferricyanide reduction, was several-fold greater than total steady-state ascorbate concentrations. This, and the finding that more than half of the ascorbate recycled from dehydroascorbate escaped the mitochondrion, suggests that mitochondrial recycling of ascorbate might be an important mechanism for regenerating intracellular ascorbate.

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 µM and complete inhibition at 50 µM. Loading cells with 100 µM ascorbate also decreased the basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 µM L-NAME (but not D-NAME) as well as by 30 µM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema.

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism.

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 microM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (Km of 113 microM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on D-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80-100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 microM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 microM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.

Ascorbic acid is one of the most sensitive cellular defenses against oxidant damage. However, it requires a sodium- and energy-dependent transporter to enter cells against a concentration gradient. To test the hypothesis that ascorbate transport is sensitive to redox stress, we studied changes in transport of the vitamin in response to sulfhydryl modification of the protein and to GSH depletion in cultured endothelial cells. Transport of ascorbic acid, measured as the uptake of radiolabeled ascorbate, was inhibited by the membrane-impermeant sulfhydryl reagents thorin, p-chloromercuribenzene sulfonic acid, and 5,5 � -dithiobis-(2- nitrobenzoic acid) in a dose-dependent manner without significant depletion of intracellular GSH. Sulfhydryl reagents capable of penetrating the plasma membrane, including phenylarsine oxide, p-chloromercuribenzoic acid, and N-ethylmaleimide, inhibited transport and lowered cellular GSH. Diamide, which induces disulfide formation, increased ascorbate transport over a narrow concentration range under conditions in which GSH was not depleted. On the other hand, specific depletion of intracellular GSH by several different mechanisms did inhibit transport. Together, these results suggest that the ascorbate transporter is sensitive to redox modulation. This relates in part to sulfhydryl groups exposed on the exofacial ascorbate transporter, and to sulfhydryl groups that are sensitive to changes in the redox state of intracellular GSH.

The thiazine dye methylene blue has long been used to stimulate cellular redox metabolism. To determine the extent to which it also generates oxidant stress in cells, its effects in cultured human-derived endothelial cells were studied. As expected, low concentrations of the dye (2-20 microM) activated the pentose phosphate pathway and oxidized both NADPH and NADH. Methylene blue enhanced extracellular ferricyanide reduction, indicating that the reduced form of the dye was present outside the cells. This reduction was greater when ferricyanide was added just before rather than 15 min after methylene blue, confirming that the dye is at least initially reduced at the cell surface. In the absence of glucose, methylene blue at concentrations above 5 microM increased intracellular oxidant stress, as manifest by oxidation of dihydrofluorescein and cellular GSH. Inclusion of glucose protected against these effects. In cells that had been loaded with ascorbate, the dye caused progressive oxidation of ascorbate, even in the presence of D-glucose. Loading cells with ascorbate also partially prevented oxidation of dihydrofluorescein by methylene blue. These results suggest that concentrations of the dye above 5 microM generated intracellular reactive oxygen species that were scavenged by ascorbate and GSH. Further, although D-glucose enhanced reduction of methylene blue, it ameliorated the oxidant stress generated by the dye.

Alpha-lipoic acid, which becomes a powerful antioxidant in its reduced form, has been suggested as a dietary supplement to treat diseases associated with excessive oxidant stress. Because the vascular endothelium is dysfunctional in many of these conditions, we studied the uptake, reduction, and antioxidant effects of alpha-lipoic acid in cultured human endothelial cells (EA.hy926). Using a new assay for dihydrolipoic acid, we found that EA.hy926 cells rapidly take up and reduce alpha-lipoic acid to dihydrolipoic acid, most of which is released into the incubation medium. Nonetheless, the cells maintain dihydrolipoic acid following overnight culture, probably by recycling it from alpha-lipoic acid. Acute reduction of alpha-lipoic acid activates the pentose phosphate cycle and consumes nicotinamide adenine dinucleotide phosphate (NADPH). Lysates of EA.hy926 cells reduce alpha-lipoic acid using both NADPH and nicotinamide adenine dinucleotide (NADH) as electron donors, although NADPH-dependent reduction is about twice that due to NADH. NADPH-dependent alpha-lipoic acid reduction is mostly due to thioredoxin reductase. Pre-incubation of cells with alpha-lipoic acid increases their capacity to reduce extracellular ferricyanide, to recycle intracellular dehydroascorbic acid to ascorbate, to decrease reactive oxygen species generated by redox cycling of menadione, and to generate nitric oxide. These results show that alpha-lipoic acid enhances both the antioxidant defenses and the function of endothelial cells.

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 hours with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.

High glucose concentrations due to diabetes increase leakage of plasma constituents across the endothelial permeability barrier. We sought to determine whether vitamin C, or ascorbic acid (ascorbate), could reverse such high glucose-induced increases in endothelial barrier permeability. Human umbilical vein endothelial cells and two brain endothelial cell lines cultured at 25 mM glucose showed increases in endothelial barrier permeability to radiolabeled inulin compared to cells cultured at 5 mM glucose. Acute loading of the cells for 30–60 min with ascorbate before the permeability assay prevented the high glucose-induced increase in permeability and decreased basal permeability at 5 mM glucose. High glucose-induced barrier leakage was mediated largely by activation of the receptor for advanced glycation end products (RAGE), since it was prevented by RAGE blockade and mimicked by RAGE ligands. Intracellular ascorbate completely prevented RAGE ligand-induced increases in barrier permeability. The high glucose-induced increase in endothelial barrier permeability was also acutely decreased by several cell-penetrant antioxidants, suggesting that at least part of the ascorbate effect could be due to its ability to act as an antioxidant.

Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.

Ascorbic acid may be involved in the defense against oxidant stress in endothelial cells. Such a role requires that the cells effectively recycle the vitamin from its oxidized forms. In this work, we studied the ability of cultured bovine aortic endothelial cells (BAECs) to take up and reduce dehydroascorbic acid (DHA) to ascorbate, as well as the dependence of ascorbate recycling on intracellular GSH. BAECs took up and reduced DHA to ascorbate much more readily than they took up ascorbate. Although BAECs in culture did not contain ascorbate, ascorbate accumulated to concentrations of 2-3 mM in BAECs following incubation with 400 microM DHA. Extracellular ferricyanide oxidized intracellular ascorbate, which was recycled by the cells. Reduction of DHA, either when added to the cells or when generated in response to ferricyanide, caused significant decreases in intracellular GSH concentrations. Depletion of intracellular GSH with 1-chloro-2,4-dinitrobenzene, diethylmaleate, and diamide almost abolished the ability of the cells to reduce DHA to ascorbate. DHA reduction by thioredoxin reductase was evident in dialyzed cell extracts, but occurred at rates far lower than direct GSH reduction of DHA. These results suggest that maximal rates of DHA reduction, and thus recycling of ascorbate from DHA, are dependent upon GSH in these cells.

Diazobenzene sulfonic acid (DABS) has been used to label thiols and amino groups on cell-surface proteins. However, we found that in addition to inhibiting an ascorbate-dependent trans-plasma membrane oxidoreductase in human erythrocytes, it also depleted alpha-tocopherol severely in the cell membrane. When erythrocytes were loaded with ascorbate, DABS-dependent loss of alpha-tocopherol was decreased, despite little change in intracellular ascorbate content. Sparing of alpha-tocopherol also was seen in erythrocyte ghosts resealed to contain ascorbate, although this was accompanied by loss of intravesicular ascorbate, probably due to the inability of ghosts to recycle ascorbate. A transmembrane transfer of electrons from ascorbate was confirmed by electron paramagnetic resonance spectroscopy, in which extracellular DABS was found to generate the ascorbate free radical within cells. When the membrane content of alpha-tocopherol was decreased to 20% of the initial value by DABS treatment, lipid peroxidation ensued, manifest by generation of F(2)-isoprostanes in the cell membranes. Intracellular ascorbate also strongly protected against F(2)-isoprostane formation. These results show that DABS causes an oxidant stress at the membrane surface that is transmitted within the cell, in part by an alpha-tocopherol-dependent mechanism, and that ascorbate recycling of alpha-tocopherol can protect against loss of alpha-tocopherol and the ensuing lipid peroxidation.

We investigated the possibility that human erythrocytes can reduce extracellular ascorbate free radical (AFR). When the AFR was generated from ascorbate by ascorbate oxidase, intact cells slowed the loss of extracellular ascorbate, an effect that could not be explained by changes in enzyme activity or by release of ascorbate from the cells. If cells preserve extracellular ascorbate by regenerating it from the AFR, then they should decrease the steady-state concentration of the AFR. This was confirmed directly by electron paramagnetic resonance spectroscopy, in which the steady-state extracellular AFR signal varied inversely with the cell concentration and was a saturable function of the absolute AFR concentration. Treatment of cells N-ethylmaleimide (2 mM) impaired their ability both to preserve extracellular ascorbate, and to decrease the extracellular AFR concentration. These results suggest that erythrocytes spare extracellular ascorbate by enhancing recycling of the AFR, which could help to maintain extracellular concentrations of the vitamin.

Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b5 reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.

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 microM aurothioglucose, doubled by 5 microM E. coli thioredoxin, and had an apparent Km 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. GSH-dependent 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.

Ascorbate has been reported to increase intracellular hydrogen peroxide (H2O2) generation in human erythrocytes. In the present work, the basis for this prooxidant effect of the vitamin was investigated in the context of erythrocyte defenses against H2O2. Ascorbate added to erythrocytes caused a dose-dependent increase in intracellular H2O2, which was measured as inactivation of endogenous catalase in the presence of 3-amino-1,2,4-triazole (aminotriazole). Ascorbate-induced catalase inactivation was not observed when only the intracellular ascorbate concentration was increased, when cells were incubated with ascorbate in plasma, or when extracellular Fe3+ was chelated. Together, these results suggest that the observed ascorbate-induced H2O2 generation is due to Fe3+-catalyzed oxidation of extracellular, as opposed to intracellular, ascorbate by molecular oxygen. Rather than generate an oxidant stress in erythrocytes, ascorbate was one of the most sensitive intracellular antioxidants to H2O2 coming from outside the cells. On the other hand, intracellular ascorbate contributed little to the detoxification of H2O2, which was found to be mediated by both catalase and by the GSH system.

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.

A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythrocyte ghosts. Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F2-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F2-isoprostane formation due to extravesicular ferricyanide. This protection was associated with a rapid transmembrane oxidation of intravesicular ascorbate by extravesicular ferricyanide. Transmembrane electron transfer, which was measured indirectly as ascorbate-dependent ferricyanide reduction, correlated with the content of α-tocopherol in the ghost membrane in several respects. First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous α-tocopherol in the ghost membrane. Second, when exogenous α-tocopherol was incorporated into the ghost membrane during the resealing step, subsequent ferricyanide reduction was enhanced. Last, incubation of intact erythrocytes with soybean phospholipid liposomes, followed by resealed ghost preparation, caused a proportional decrease in both the membrane content of α-tocopherol and in ferricyanide reduction. Incorporation of exogenous α-tocopherol during resealing of ghosts prepared from liposome-treated cells completely restored the ferricyanide-reducing capacity of the ghosts. These results suggest that the transmembrane transfer of ascorbate-derived electrons in erythrocyte ghosts is dependent in part on α-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell.

Lipophilic derivatives of ascorbic acid may protect lipid bilayers and micelles against lipid peroxidation. In this work the binding, accessibility, and reducing capacity of ascorbate 6-palmitate (A6P) were studied in human erythrocyte membranes. In contrast to less lipophilic carbon-6-modified ascorbate derivatives, A6P bound to erythrocyte membranes in a concentration-dependent manner. This binding was preserved following centrifugation washes, but was largely reversed by extraction with bovine serum albumin. Most of the ascorbyl groups of membrane-bound A6P were readily accessible to oxidation by water-soluble oxidants. Ferricyanide quantitatively oxidized membrane-bound A6P, but the latter spared endogenous tocopherols from destruction. In EPR studies, A61? was much more effective than ascorbate in reducing nitroxide spin labels positioned at either carbon-5 or carbon-16 of membrane-bound stearic acid in both intact cells and in membranes. A6P, thus, appears to intercalate into the erythrocyte membrane with the ascorbyl group located superficially, but with access to the hydrophobic membrane interior, and with the ability to recycle endogenous a-tocopherol during oxidant stress.

Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.

The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate-dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.

Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [14C]ascorbate to [14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.

Endothelial dysfunction is an early manifestation of atherosclerosis caused in part by oxidized LDL (oxLDL). Since vitamin C, or ascorbic acid, prevents several aspects of endothelial dysfunction, the effects of oxLDL on oxidative stress and regulation of the ascorbate transporter, SVCT2, were studied in cultured EA.hy926 endothelial cells. Cells cultured for 18 h with 0.2 mg/ml oxLDL showed increased lipid peroxidation that was prevented by a single addition of 0.25 mM ascorbate at the beginning of the incubation. This protection caused a decrease in intracellular ascorbate, but no change in the cell content of GSH. In the absence of ascorbate, oxLDL increased SVCT2 protein and function during 18 h in culture. Although culture of the cells with ascorbate did not affect SVCT2 protein expression, the oxLDL-induced increase in SVCT2 protein expression was prevented by ascorbate. These results suggest that up-regulation of endothelial cell SVCT2 expression and function may help to maintain intracellular ascorbate during oxLDL-induced oxidative stress, and that ascorbate in turn can prevent this effect.

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo.

Arginine vasopressin (AVP), oxytocin (OT), and angiotensin-II (AII) elicit a biphasic ACTH secretory response by perifused anterior pituitary cells consisting of an initial transient (less than 3-min) spike phase and a subsequent sustained plateau phase. In contrast, CRF produces a monophasic sustained plateau type of ACTH secretory response. We have previously demonstrated that 1) influx of extracellular Ca2+ (Cae2+) via L-type voltage-sensitive Ca2+ channels is involved in both the response to CRF and the sustained phase of the response to AVP and OT; 2) release of intracellular Ca2+ (Cai2+) is involved in the spike phase of the response to AVP, OT, and AII; and 3) activation of protein kinase-C is required for the sustained phase, but not for the spike phase, of the response to AVP. CRF action is mediated by activation of protein kinase-A. In this study we further examined the role of Cai2+ by exploiting the fact that a low concentration (1 microM) of ionomycin, a potent Ca2+ ionophore, releases Cai2+ from nonmitochondrial inositol-1,4,5-trisphosphate (IP3)-sensitive Cai2+ stores without causing Cae2+ influx. Pretreatment with ionomycin for 10 min decreased the spike phase of the response to 100 nM AVP, OT, and AII, but had no effect on the response to 10 nM CRF or the sustained phase of the responses to the other agonists. The combination of CRF plus AVP induced a biphasic and synergistic release of ACTH. Ionomycin pretreatment reduced the spike phase, especially the first 1 min, without any effect on the sustained phase. These results indicate that Cai2+ release, but not Cae2+ influx, is involved in the spike phase of the response to AVP, OT, and AII and that Cai2+ is not involved in the synergistic effect of the combination of CRF plus AVP. Having established these relationships, we examined the effect of 2-h perifusion with 100 nM dexamethasone (DEX) on stimulated ACTH release. DEX pretreatment reduced the total response to CRF, the sustained phase of the responses to AVP and OT, and the sustained phase of the synergistic response to CRF plus AVP. However, DEX had no effect on the spike phase of the responses to AVP, OT, or AII or the spike phase of the response to CRF plus AVP. These results indicate that DEX inhibits ACTH release mediated by activation of either protein kinase-A or protein kinase-C, but does not affect inositol-1,4,5-trisphosphate/Cai2(+)-mediated ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

To assess the disulfide reduction capacity of intact cells, EA.hy926 endothelial cells were incubated with alpha-lipoic acid in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Alpha-lipoic acid was reduced within cells to dihydrolipoic acid, which could be quantified upon efflux from the cells as reduction of DTNB. Uptake of both alpha-lipoic acid and alpha-lipoamide occurred at least in part via a medium chain fatty acid transporter, based on inhibition by octanoate. Alpha-lipoic acid was reduced within cells by pyridine nucleotide-disulfide oxidoreductases, since it is not reduced by GSH and since its reduction was inhibited by carmustine. Nonetheless, reduction was also dependent on the cellular redox environment, since it was inhibited by the redox cycling of menadione, by decreasing intracellular GSH, and by reduction of dehydroascorbate. Together, these results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to assess the disulfide-reducing capacity of intact cells, especially as determined by pyridine nucleotide-disulfide oxidoreductases.

Nitrite is a breakdown product of nitric oxide that in turn is oxidized to nitrate in cells. In this work, we investigated whether reactive oxidant species mightbe generated during nitrite metabolism in cultured EA.hy926 endothelial cells. Nitrite was taken up by the cells in a time- and concentration-dependent manner and oxidized to nitrate, which accumulated in cells to concentrations almost 10-fold those of nitrite. Conversion of low millimolar concentrations of nitrite to nitrate was associated with increased oxidant stress in the cells. This manifested as increased oxidation of dihydrofluorescein in tandem with depletion of both GSH and ascorbate. Further, loading cells with ascorbate or treatment with desferrioxamine prevented nitrite-induced dihydrofluorescein oxidation. Nitrite within cells also increased the fluorescence of 4-amino-5-methylamino-2',7'-difluorofluorescein and inhibited the activity of cellular glyceraldehyde 3-phosphate dehydrogenase, which are markers of intracellular nitrosation reactions. Intracellular ascorbate partially prevented both of these effects of nitrite. Although ascorbate can reduce nitrite to nitric oxide at low pH, in endothelial cells loaded with ascorbate, its predominant effect at high nitrite concentrations is to prevent potentially damaging nitrosation reactions.

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5 mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB+) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB+. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB++-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB+-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB+, which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB+, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB+reduction may correspond to MB+-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.

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 μM 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 μM, 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.

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.

Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle alpha-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes decrease steady-state concentrations of the AFR in a protein- and NADH-dependent manner. The AFR reductase has a high apparent affinity for both NADH and the AFR (2 microM). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.