Ascorbate and glutathione homeostasis in vascular smooth muscle cells: cooperation with endothelial cells

Ilia Voskoboinik, Karin Söderholm, and Ian A. Cotgreave

Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, 17177 Stockholm, Sweden

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human umbilical vein smooth muscle cells (HUVSMCs) utilize extracellular cystine, glutathione (GSH), and N-acetylcysteine (NAC) to synthesize cellular GSH. Extracellular cystine was effective from 5 µM, whereas GSH and NAC were required at 100 µM for comparable effects. The efficacy of extracellular GSH was dependent on de novo GSH synthesis, indicating a dependence on cellular gamma -glutamyltransferase (glutamyl transpeptidase). Coculture of syngenetic HUVSMCs and corresponding human umbilical vein endothelial cells (HUVECs) on porous supports restricted cystine- or GSH-stimulated synthesis of HUVSMC GSH when supplied on the "luminal" endothelial side. Thus HUVSMC GSH rapidly attained a steady-state level below that achieved in the absence of interposed HUVECs. HUVSMCs also readily utilize both reduced ascorbate (AA) and oxidized dehydroascorbate (DHAA) over the range 50-500 µM. Phloretin effectively blocked both AA- and DHAA-stimulated assimilation of intracellular AA, indicating a role for a glucose transporter in their transport. Uptake of extracellular AA was also sensitive to extracellular, but not intracellular, thiol depletion. When AA was applied to the endothelial side of the coculture model, assimilation of intracellular AA in HUVSMCs was restricted to a steady-state level below that achieved by free access.

human vascular smooth muscle cell antioxidant regulation; endothelial cell-smooth muscle cell interactions

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OXIDATIVE STRESS ARISES from an imbalance between prooxidant and antioxidant states in the cell. Oxidative stress has been implicated in a growing number of physiological and pathophysiological processes in human cells and tissues (12). For instance, it is now well established that oxidative stress is tightly associated with the injurious events within the cells of the vascular wall occurring during atherosclerosis (25), ischemia-reperfusion (8), and acute or chronic inflammations (16). It is thus of considerable clinical interest to be able to manipulate the antioxidative capacity of cells undergoing oxidative stress during such processes. To this end, many xenobiotic principles have been synthesized that function as antioxidants or antioxidant mimetics. Most notable of this latter class are the superoxide dismutase mimetics (9) and glutathione (GSH) peroxidase mimetics (4). An alternative strategy to the use of xenobiotic principles would be to utilize endogenous antioxidant principles and/or their metabolic precursors. Two antioxidants that have attracted considerable clinical interest as both markers of oxidative stress and potential therapeutic agents are the tripeptide GSH and ascorbate. However, before one can fully realize the potential of manipulating endogenous antioxidant defenses in a "natural" manner, one must understand the basic biochemical processes that control the intercellular supply and disposition of these molecules in human tissues.

The cellular composition of the human vascular wall mainly consists of the endothelial cells lining the vessel wall and the smooth muscle cells surrounding these in a variety of layers, separated by various basal membranes, or intimae. Endothelial cells provide a selective barrier to the uptake of circulatory substances, such as regulatory molecules and metabolic precursors, for example, for antioxidant defense. Furthermore, myoendothelial cooperation in such transport processes is an essential prerequisite for the further passage of the substances to the tissues. We have previously studied the amino acid precursor uptake and specificity for GSH synthesis in human umbilical vein endothelial cells (HUVECs). These endothelial cells demonstrated effective uptake of cystine from the circulation (3), through a combination of the Xc- transporter and gamma -glutamyltransferase (glutamyl transpeptidase) (7), which was optimized to the plasma concentration of the disulfide (25-50 µM). On the other hand, the uptake of cysteine was relatively inefficient, and the cells lacked the ability to utilize extracellular methionine (3). We also demonstrated the ability of the cells to utilize extracellular GSH itself for the synthesis of the tripeptide, although the concentrations required for this were nonphysiologically high, in the millimolar range. The HUVECs are also able to deacetylate the cysteine precursor thiol drug N-acetylcysteine (NAC) and utilize the resultant cysteine for GSH synthesis (6).

In another study, we monitored the characteristics of uptake of reduced ascorbate (AA) and dehydroascorbate (DHAA) into HUVECs. We demonstrated rapid, energy-dependent uptake of AA, with a dependence on a cellular glucose transporter. On the other hand, the uptake of DHAA and its reduction to ascorbate in the cells by GSH- and/or NADPH-dependent processes were shown to be relatively inefficient. The relative efficacies of these uptake processes reflect the respective plasma levels of these essential molecules, with the majority (>95%) of plasma ascorbate being in the reduced form (50-200 µM). Thus it has been suggested that these two redox forms of the antioxidant are transported into cells by distinct transporters (26).

Recently we described the isolation and culture of human umbilical vein smooth muscle cells (HUVSMCs) (1). This provides for several opportunities for further systematic study of the disposition of ascorbate and GSH precursors in the human vessel wall. First, little is known of the amino acid precursor specificity and uptake mechanism for GSH synthesis in human smooth muscle cells (21). Similarly, although the effects of extracellular ascorbate on a number of smooth muscle cell-specific biochemical processes have been studied (11), there have been no studies on the mechanism of uptake of AA and DHAA into human smooth muscle cells. Thus we report on the use of monolayer cultures of HUVSMCs for the study of the mechanism of sulfur amino acid/GSH and AA/DHAA assimilation into human smooth muscle cells. In addition, we have also previously described methods for the coculture of HUVECs and HUVSMCs, derived from the same individual, on either side of fixed, porous supports (1). Thus, using such cocultures, we investigated the potential role of endothelial cells in regulating the flow of metabolic precursors of intracellular AA and GSH in the underlying smooth muscle cells. The results are discussed in terms of the overall mechanisms of intercellular cooperation in the maintenance of ascorbate and GSH levels in the cells of the human vascular wall.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals and cell culture reagents. Reduced GSH, AA, DHAA, all amino acids, monobromobimane, and buthionine sulfoximine (BSO) were all purchased in >99% purity from Sigma Chemical (St. Louis, MO). M199 and DMEM cell culture media and fibronectin (human) were purchased from GIBCO (Inchinnan, UK). Sulfur amino acid-free M199 (M199-) was obtained from Nord Junter and Cell AB (Bromma, Sweden). Collagenase (fraction 2, trypsin free) was purchased from Boehringer-Mannheim (Mannheim, Germany). Human serum (B+ve) was obtained from the blood donor center of Sabbatsberg Hospital (Stockholm, Sweden). All other materials for the culture of cells were obtained from Falcon (Becton Dickinson Labware, Lincoln Oak, NJ). Chemicals and reagents for routine analyses were obtained from local suppliers in the highest grade available.

Cell isolation and culture. Human umbilical cords were obtained from the delivery center of Karolinska Hospital (Stockholm, Sweden) and were kept on sterile PBS at 4°C until collection. The endothelial cells were obtained by collagenase perfusion of the vein as described by Jaffe et al. (15), with minor modifications (3). Endothelial cells were maintained in M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), fungizone (1.25 µg/ml), L-glutamine (2 mM), and human serum (10%) and passaged by conventional trypsinization, and the cells were generally frozen, after reaching confluency, for further use in coculture experiments with syngenetic HUVSMCs.

The smooth muscle cells were prepared from the umbilical cord vein, after removal of the endothelial cells, essentially according to Bohlin et al. (1). Briefly, the vein was refilled with collagenase (326 U/mg, 2 mg/ml in PBS, supplemented with 0.5 mM Ca2+ and 0.4 mM Mg2+) and incubated at 37°C for 30 min. The solution was flushed out and the vein was then cut open and trimmed free from the amnion. Sections were placed into explant culture, luminal face down, in DMEM supplemented with antibiotics and human serum as for the endothelial growth medium. The explants were removed after 3 days, and the adherent HUVSMCs were grown to confluency and passaged by trypsinization. Cells at passages 3-7 were routinely used for monolayer experiments and coculture experiments with HUVECs.

Cell incubations. Confluent monolayers of HUVSMCs, on either 12- or 24-well plates, were exposed to AA or DHAA at a variety of concentrations in the DMEM culture medium for increasing times up to 24 h. After incubation, cells were washed three times in PBS, and the cell-associated ascorbate level was determined by HPLC as described below. Control experiments were performed in which it was determined that three washes were required to remove "loosely" associated ascorbate from the cells. In some experiments, phloretin (2), a potent inhibitor of the plasma membrane glucose transporter, was introduced during the incubation 5 min before the addition of AA- or DHAA-supplemented medium.

For the utilization of sulfur amino acid precursors of GSH biosynthesis, HUVSMCs were depleted of >90% of their GSH by an 18-h incubation in sulfur amino acid-free M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), and human serum (10%). The medium was then changed, and the same medium was reapplied to the cells but was supplemented with cysteine, cystine, or methionine at increasing concentrations. The cells were then incubated for increasing times up to 24 h and analyzed for intracellular GSH content as described below. Control measurements of cellular GSH levels before and after the initial depletion of GSH were also taken.

In some experiments, HUVECs and HUVSMCs were cocultured on polyethylene teraphthalate permeable supports (Costar; 3-µm pore diameter, 8 × 105 pores/cm2) precoated with human plasma fibronectin (5 µg/ml in PBS) for 30 min at 37°C. The smooth muscle cells were seeded first at a density of 5 × 104 cells/cm2 on the outside of the insert. The cells were allowed to attach for 4 h, and then the insert was inverted, placed in a culture well in a 12-well plate, filled with 1 ml of the M199 endothelial cell culture medium, and seeded with HUVECs at a density of 5 × 104 cells/ml. The cells were then cultured until both cell layers approached confluency. Parallel plates of each cell type were cultured to estimate confluency of each cell type, which was finally assessed by the measurement of transmembrane resistance as described previously (1). A cutoff resistance of 12-14 Omega /cm2, indicating maximal transcellular resistance, was used to determine the suitability of the coculture for further experimentation.

Biochemical assays. AA was assayed in cells and medium according to Honneger et al. (13) after extraction of the samples with TCA (1.25%). The molecule was separated by HPLC (Waters Associates) on a Supelcosil L-18 column (150 × 4.5 mm; 3 µm) and quantitated by electrochemical detection using an Antec Model CU-03 detector, with the electrode set in the oxidative mode at +0.5 V. Samples were analyzed as soon as possible after generation. Intracellular GSH was analyzed following in situ derivatization of the cells with the membrane-permeant thiol reagent monobromobimane, exactly as described previously (5). Cellular protein was determined by the method of Peterson (23).

Statistical methods. As appropriate, groups of data were compared for significant differences using a two-tailed, paired Student's t-test using an Instat software package. Significance was noted at the level of P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To study the sulfur amino acid precursor specificity of GSH synthesis in HUVSMCs, the cells were initially depleted of GSH either by BSO (25 µM in control medium) treatment or by incubation in M199- for 18 h, from 35.2 ± 2.1 or 32.6 ± 1.7 nmol GSH/mg protein (n = 3 for all observations) to 2.2 ± 0.2 or 3.6 ± 0.3 nmol GSH/mg protein, respectively, without morphological changes to the cells. In both cases, the half-life of turnover of GSH in HUVSMCs appears to lie between 3 and 6 h. Reincubation in control medium of the cells depleted in M199- caused an immediate and linear increase in cellular GSH to 34.3 ± 1.8 nmol/mg protein over the initial 9 h of incubation, rising to 37.7 ± 1.4 nmol/mg protein by 24 h. On the other hand, the levels of GSH in BSO-pretreated cells remained essentially unaltered after washing and reincubation with control medium for up to 9 h (5.8 ± 0.4 nmol/mg protein) and rose to 29.4 ± 2.1 nmol/mg protein by 24 h of incubation.

The incubation of GSH-depleted HUVSMCs with M199- containing cystine as the sole sulfur source resulted in rapid resynthesis of GSH. The initial levels of GSH achieved with cystine between 10 and 50 µM were similar. However, after reaching a plateau at 6 h, cellular GSH levels began to decline, but they were maintained at higher levels with increasing concentrations of the disulfide (Fig. 1). It will be noted that methionine (1 mM) induced only minimal resynthesis of GSH in HUVSMCs under these conditions (Fig. 1). Interestingly, the thiol drug NAC was also shown to be a precursor of GSH synthesis; however, the efficacy of the compound was inferior to that of cystine or cysteine, based on the number of cysteine equivalents in the medium (Fig. 2). Finally, the presence of extracellular GSH supported the elevation of intracellular GSH in depleted HUVSMCs in a concentration-dependent manner. It will be noted that 100 µM GSH was nearly as effective as 50 µM cystine in this respect (Fig. 3 vs. Fig. 1). The presence of BSO (25 µM) in the extracellular medium greatly reduced the capacity of the cells to utilize extracellular GSH to elevate intracellular levels of the tripeptide, indicating the need for the activity of glutamate-cysteine ligase (gamma -glutamyl cysteine synthetase) in this effect (Fig. 3).


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Fig. 1.   Cystine- and methionine-dependent synthesis of glutathione (GSH) in GSH-depleted human umbilical vein smooth muscle cells (HUVSMCs). Confluent HUVSMCs were depleted of GSH by an 18-h incubation in M199 lacking sulfur-containing amino acids (M199-). Cells were then washed twice with PBS and resupplied with M199- supplemented with increasing concentrations of cystine or with methionine, as sole sulfur source. Intracellular GSH was assayed at various intervals thereafter as described in MATERIALS AND METHODS. Intracellular GSH levels were 32 ± 2 nmol/mg cell protein before depletion. Data are means ± SD; number of measurements made on separate culture dishes (n) = 3 for cystine and 1 for methionine.


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Fig. 2.   N-acetylcysteine (NAC)-dependent synthesis of GSH in GSH-depleted HUVSMCs. All details are as in Fig. 1 except that NAC was supplied for GSH synthesis as sole sulfur source.


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Fig. 3.   Extracellular GSH supports intracellular GSH elevation in HUVSMCs in a manner dependent on intracellular GSH synthesis. All details are as in Fig. 1 except that GSH was used as sole sulfur amino acid source. In 1 series, cells were coincubated with buthionine sulfoximine (BSO; 25 µM) in presence of extracellular GSH.

The HUVSMCs lack measurable levels of AA when cultured under normal conditions. However, the presence of extracellular AA facilitated the assimilation of AA within the cells. The initial rates of appearance of AA in the cells over the first 1 h were fairly similar over the range 50-500 µM extracellular AA, whereas by 6 h a clear dose-dependent correlation between extracellular and intracellular ascorbate was evident (Fig. 4). It will be noted that control experiments demonstrated that the cells required two washes in PBS before assay to remove AA loosely associated with the cells (data not shown). In contrast, the presence of DHAA in the extracellular medium also stimulated the appearance of AA associated with the cells, in a concentration-dependent manner. However, the kinetics of AA assimilation were somewhat different from those achieved with AA itself, with maximum concentrations of cellular AA being achieved by 60 min of incubation and declining thereafter (Fig. 5). Additionally, on a molar basis, DHAA was less effective than AA in supporting the elevation of intracellular AA in the cells (Fig. 4 vs. Fig. 5). Both DHAA-supported and AA-supported assimilation of AA into the cells were almost totally inhibited by the presence of the glucose transporter inhibitor phloretin (1 mM) in the medium (Fig. 6).


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Fig. 4.   Ascorbate-dependent elevation of intracellular ascorbate levels in HUVSMCs. Confluent HUVSMCs were incubated with control M199 supplemented with reduced ascorbate (AA) at increasing concentrations. At various time points up to 6 h, cells were washed 3 times in PBS and intracellular AA was determined as described in MATERIALS AND METHODS. Data are means ± SD; n = 3 for all points.


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Fig. 5.   Dehydroascorbate (DHAA)-dependent elevation of intracellular ascorbate in HUVSMCs. All details are as in Fig. 4 except that cells were incubated with DHAA.


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Fig. 6.   Involvement of glucose transporter in uptake of ascorbate and DHAA into HUVSMCs. Confluent HUVSMCs were incubated with AA (500 µM) or DHAA (500 µM) in absence or presence of the specific glucose transporter inhibitor phloretin (1 mM). Intracellular AA levels were assayed as described in MATERIALS AND METHODS.

Interestingly, when cells were incubated with AA or DHAA in M199 lacking all sulfur amino acid precursors, the levels of AA that accumulated in the cells in each case were far lower than for control medium (Fig. 7, A and B, respectively). However, cells depleted of their GSH by an overnight incubation with M199 lacking sulfur precursors before incubation with AA or DHAA in the presence of BSO (25 µM) also displayed normal accumulation of AA in the cells in each case (Fig. 7, A and B, respectively).


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Fig. 7.   Depletion of extracellular sulfur-containing amino acids, but not of intracellular GSH, inhibits extracellular ascorbate- and DHAA-dependent assimilation of ascorbate in HUVSMCs. Control cells were incubated with ascorbate (AA; 500 µM; A) or DHAA (500 µM; B) in either control M199 or M199-. In other experiments, control cell GSH (31 ± 3 nmol GSH/mg protein, n = 3) was depleted >90% by an overnight incubation in M199- and exposed to AA or DHAA in control medium in presence of BSO (25 µM). Samples were taken at regular intervals for determination of intracellular ascorbate. Data are means ± SD; n = 3 for all points.

In the intact blood vessel wall, the circulation almost certainly serves as the major source of continued supply of metabolic precursors of smooth muscle cell GSH and AA. Thus it follows that the endothelial cells lining the vessel wall play an important role in regulating the levels of these antioxidants in smooth muscle cells. To begin probing the complexities of these interactions in the intact vessel wall, we have employed syngenetic cocultures of HUVECs and HUVSMCs on fixed, porous supports, in order to establish the role of an intermittent endothelial cell layer in controlling the disposition of GSH and AA in smooth muscle cells supplied with precursors from the "luminal" side of the endothelial cells. It will be noted that, under the conditions of coculture, conditions of high electrical resistance of the bilayer were maintained throughout, indicating tight-junctional integrity, particularly in the endothelial layer. To study GSH assimilation in the smooth muscle cells of the bilayer, both cell types were depleted of their GSH by an overnight incubation in medium lacking sulfur amino acids (M199-) (3). Control experiments showed that this treatment did not affect the morphology of either cell type on the filter. In Fig. 8A, it can be seen that resynthesis of HUVSMC GSH using extracellular cystine or GSH as a precursor on the luminal side of the endothelial cells was retarded in both cases, compared with the respective rates in HUVSMCs cultured alone on the filters. Similarly, Fig. 8B shows that, when AA was supplied to the luminal side of the HUVECs, the assimilation of AA in smooth muscle cells was again retarded, reaching a steady-state level by 60 min of incubation, which was maintained for up to 3 h and declined thereafter. In the absence of HUVECs, AA levels in HUVSMCs rose linearly over the 5-h test incubation period. In both cases, control experiments indicated that HUVSMCs did not release their complement of AA or GSH during their removal from the supports by trypsinization (data not shown).


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Fig. 8.   Influence of endothelial cells on assimilation of GSH (A) and ascorbate (B) in HUVSMCs supplied with either cystine or GSH or with AA, respectively, on luminal side of coculture with human umbilical vein endothelial cells (HUVECs). Both HUVSMCs and HUVECs were seeded and grown to confluency on either side of fixed, porous Transwell supports. A: cells were simultaneously depleted of GSH by an 18-h incubation in M199- lacking sulfur amino acids, without affecting cell viability or morphology. Cocultures were then incubated on luminal side of HUVECs with either cystine (25 µM) or GSH (100 µM) in M199- as sole sulfur source, and HUVSMCs were analyzed for intracellular GSH after removal by trypsinization. Control experiments showed that trypsinization did not affect intracellular GSH content of cells. Control experiments were performed in which HUVSMCs only were grown on 1 side of filter and sulfur amino acid-containing M199- was applied to other side of membrane. B: AA (250 µM) was applied to HUVEC-HUVSMC cocultures or HUVSMCs alone as for A, and intracellular AA in smooth muscle cells was determined. Again, controls indicated no loss of intracellular AA under conditions of assay employed. Data are means ± SD; n = 4 for all points. All matched data points at specific times show significant differences (P < 0.005, except for 0.25 and 1 h in B, where P < 0.01), using a 2-tailed, paired Student's t-test.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies in monolayer cultures of HUVECs have shown that the synthesis of intracellular GSH is largely dependent on the uptake of cystine and its reduction to cysteine (3). The uptake of the disulfide was shown to be matched to the plasma concentration of the disulfide and also to be dependent on the activity of both gamma -glutamyltransferase and the Xc- carrier (7). Interestingly, we show here that the smooth muscle cells, positioned immediately under the endothelial cells, also readily utilize cystine as a precursor of GSH synthesis. However, the cells appear to have an ability to utilize the disulfide far more efficiently than do the endothelial cells (3), with considerable GSH synthesis supported by levels as low as 5 µM. This indicates that HUVSMCs in the intact vessel seem to be adapted to levels of cystine in the extracellular fluid around them that are lower than the levels to which endothelial cells are normally exposed in the plasma. This perhaps indicates a reliance on carriers other than the gamma -glutamyltransferase and Xc- systems operating in HUVECs (7).

Other differences in precursor uptake behavior also exist between HUVSMCs and HUVECs. For instance, like HUVECs, HUVSMCs can utilize extracellular GSH for the synthesis of intracellular GSH. However, considerable resynthesis of GSH in GSH-depleted cells was achieved at a reduced tripeptide level of 100 µM (Fig. 4), whereas millimolar concentrations were required with HUVECs (3). Additionally, extracellular GSH was only effective in HUVSMCs if cellular glutamate-cysteine ligase was operative. This is unlike the situation with HUVECs, in which BSO had no inhibitory effect on the ability of extracellular GSH to elevate intracellular levels of the tripeptide (3). These data strongly suggest that gamma -glutamyltransferase is expressed on the surface of HUVSMCs and is active in the initial uptake of GSH from the extracellular space. Furthermore, as with HUVECs, it appears that HUVSMCs lack one or more enzymatic components of the cystathionase pathway and are unable to utilize extracellular methionine for GSH biosynthesis. Finally, the drug NAC was a less effective precursor of GSH synthesis in HUVSMCs, on a molar thiol basis, than cystine or GSH (Fig. 2 vs. Figs. 1 and 3). However, considerably lower concentrations of the thiol were required to support substantial GSH in the smooth muscle cells than in HUVECs (6). This suggests that the activity of the N-deacetylase enzyme liberating cysteine from the drug may be higher in HUVSMCs than in HUVECs. The therapeutic significance of this during systemic NAC therapy is uncertain. However, if NAC passes the endothelium intact, it may reach the smooth muscle cells and provide considerable clinical benefit by protecting smooth muscle cell GSH levels.

The above experiments detailing the mechanisms of uptake of precursors of intracellular GSH and AA into smooth muscle cells lack one important dimension. In the intact blood vessel wall, the supply of sulfur-containing amino acid precursors to the intracellular environment of the smooth muscle cells may be achieved by active uptake from the pericellular fluid on specific membrane-bound carriers, e.g., gamma -glutamyltransferase or the Xc- carrier, as discussed above. These precursors may be maintained in the intimal fluid either indirectly, by active uptake into endothelial cells from the circulation and subsequent secretion over the basal side of the plasma membrane, or directly, via alterations in tight-junctional integrity. In the former process, one particular mechanism that is of particular current interest is that of vesicular transport in and secretion from the endothelium, mediated by the vesiculo-vacuolar organelle (VVO) pathway (10, 19). Alternatively, after active uptake into the endothelial cells, metabolic cooperation may occur via myoendothelial gap junctions (14). In any event, the use of simple monolayer culture models neither gives an accurate insight into the true levels of GSH in smooth muscle cells in the intact vessel wall nor allows us to delineate any intercellular interactions with endothelial cells that may control precursor supply to smooth muscle cells.

The data detailed in Fig. 8A show the influence of an intermittent HUVEC layer on extracellular cystine- and GSH-dependent assimilation of GSH in HUVSMCs. In both cases, intracellular GSH levels were rapidly restricted to a plateau level that was considerably lower than those levels achieved during direct cellular contact with extracellular precursors. Thus the endothelial cells seem to exert a controlling influence on the actual levels of GSH achieved in the smooth muscle cells. It will be noted, however, that the efficacy of resynthesis of GSH was inferior to that achieved with cells grown on plastic. Indeed, culture-specific alterations of the GSH levels of HUVECs have been previously noted (27). Irrespective of the molecular mechanism by which this control is achieved, these data clearly show that GSH levels achieved in cultured monolayers of HUVSMCs may have little relevance to the actual levels of the tripeptide maintained in these cells in the intact vessel wall.

The mechanisms underlying the effects of the intermittent endothelial cell layer are, as yet, obscure. It may be suggested that the endothelial cells simply offer a physical barrier to the passage of precursors of HUVSMC GSH biosynthesis. On the other hand, a mechanism involving uptake by the endothelial cells, followed either by secretion into the intima and subsequent uptake of precursors into the smooth muscle cells or by direct passage of GSH and/or its precursors via myoendothelial gap junctions, may be postulated. Preliminary data (unpublished observations) demonstrate that neither lipopolysaccharide (300 ng/ml) nor tumor necrosis factor-alpha (20 ng/ml) is able to affect the kinetics of reassimilation of GSH in the HUVSMCs in the bilayer coculture model, in response to either GSH or cystine supplied on the luminal side of the HUVECs. Under these conditions, the agents are known to cause total closure of myoendothelial gap junctions in a mixed monolayer coculture (14). It may be argued, however, that such gap junctional contacts are not present in the bilayer model presently employed. However, ultra-structural studies of similar smooth muscle cell-endothelial cell models, often of discordant vessel and genetic origins, clearly demonstrate the presence of profuse numbers of gap junctions spanning the gap between the cells within the pores of membranes similar to those used in the present studies (24). However, this matter awaits further clarification in the present model, as does the possible involvement of VVO-dependent transport, although the latter process is generally regarded as being specific for macromolecules (10).

Thus it seems likely that HUVSMCs, which are clearly able to utilize lower concentrations of extracellular sulfur-containing precursors than the overlying HUVECs, assimilate their intracellular GSH in the intact vessel wall primarily as a result of active transport from a pool of sulfur amino acid precursors in the intimal fluid, which is maintained by secretion from overlying endothelial cells. In view of the potential role of a pool or pools of thiol precursors in the intimal fluid between endothelial and smooth muscle cells, it should be mentioned that very little is known of the actual levels of cystine and GSH in this extracellular compartment. This hampers prediction of which concentrations of the agents are relevant for the characterization of the respective active uptake processes. Thus experimental concentrations over a wide range were used in each case, the upper levels of which are clearly not relevant with respect to plasma concentrations of the respective agents. This quandary does not apply to endothelial cells, as they are in direct contact with the circulation, where relevant concentrations are easily amenable to assay.

Turning to ascorbate, from the data in Fig. 4 it is clear that AA is readily assimilated into HUVSMCs, in a concentration-dependent manner, when supplied extracellularly. However, unlike the case with HUVECs, levels reached saturation after 4-6 h and were generally much lower, on a nanomole AA per milligram protein per hour basis, than those achieved with corresponding concentrations of AA in the case of the HUVECs (26). Interestingly, on a molar basis, HUVSMCs were also able to utilize extracellular DHAA for the assimilation of intracellular AA nearly as effectively as extracellular AA itself (Fig. 5). The kinetics of assimilation were, however, somewhat different, with peak levels of AA achieved within the first 1 h of incubation and declining levels thereafter. This behavior is similar to that detected in HUVECs (26).

In terms of the relationships between the concentrations of AA and DHAA utilized to probe the biochemical processes detailed above, it should be mentioned that normal plasma levels of AA lie between 20 and 50 µM (22), with the corresponding levels of DHAA usually being lower than this (18). In the present experiments, both AA and DHAA have been utilized over the range 50-500 µM, which may be deemed unphysiologically high. However, these concentrations were chosen partly to maintain compatibility with our previous experiments with HUVECs and partly to accentuate those biochemical processes involved in the uptake of these materials into the smooth muscle cells. Additionally, little is known of the actual concentrations of AA and DHAA in the intimal fluid between these cells in vivo. However, AA concentrations in extracellular fluids, such as the epithelial lining fluid of the lung, clearly fall into this range (17). Furthermore, AA concentrations of up to 200 µM are often used in cell culture experiments studying the formation in and secretion of extracellular matrix materials from cells derived from connective tissues (11, 20).

In terms of the mechanism(s) of assimilation of intracellular AA in HUVSMCs, it appears that the activity of a glucose transporter is vital to the process, as the specific hexose transporter inhibitor phloretin (2) considerably hampered both AA- and DHAA-dependent assimilation of AA in the cells (Fig. 6). Previous studies have indicated a role for a glucose transporter(s) in other diploid cell types (2, 28, 29), and a hexose carrier has been shown to be responsible for the uptake of both AA and DHAA in some cell types (28). Interestingly, in the present experiments, both AA-dependent and DHAA-dependent assimilation of intracellular AA were also shown to be sensitive to the presence of sulfur-containing amino acids (reduced and/or oxidized) in the extracellular milieu. However, the depletion of intracellular GSH had no effect on this process (Fig. 7, A and B, respectively). The reasons underlying this are, however, obscure. It may be that optimal activity of the glucose transporter requires the presence of reduced and/or oxidized thiols at its extracellular surface.

Analogously to the above discussion of GSH assimilation in the HUVSMCs, precursors of AA are, in all probability, presented to the cells from the systemic circulation, via the overlying endothelial cells. From our studies using the HUVEC-HUVSMC bilayer cocultures it was again evident that an intermittent HUVEC layer strongly influenced the assimilation of AA in the HUVSMC when AA was supplied to the luminal side of the HUVECs (Fig. 8B). Thus it is again evident that intercellular cooperation between both cell types serves to maintain, over several hours, a steady-state level of AA in the smooth muscle cells that is well below levels achieved by free access to AA at all time points tested. This cooperative regulation may be of prime importance to the antioxidant function of AA in the smooth muscle cells, as previous work in HUVECs has shown that an optimal intracellular AA concentration is required in order for the molecule to elicit a protective antioxidant activity against hydrogen peroxide toxicity. When intracellular concentrations of AA were allowed to rise above this optimal level, then AA actually potentiated peroxide-dependent cytotoxicity, probably by acting as a prooxidant in supporting iron-dependent generation of reactive oxygen metabolites (26).

In terms of the molecular mechanism(s) involved in controlling the supply of AA equivalents to HUVSMCs in the intact vessel wall, preliminary experiments indicate that movement of AA via myoendothelial gap junctions may not be involved, as the assimilation of AA in HUVSMCs cocultured with HUVECs on fixed, porous supports (1) was not affected by preincubation with either lipopolysaccharide or tumor necrosis factor-alpha (14) (unpublished observations). However, the same arguments and restrictions in interpretation of these data are indicated as for the case with GSH above. Thus, in view of the apparent efficacies of the uptake of both AA and DHAA into HUVSMCs, it seems likely that intracellular AA is assimilated and maintained in the smooth muscle cells in the intact vessel wall by endothelial cell-dependent secretion of AA and/or DHAA into the intimal fluid, perhaps as a result of a VVO-mediated mechanism (10), coupled to active uptake of these compounds into the smooth muscle cells.

In summary, we show that HUVSMCs are able to maintain their intracellular antioxidative capacity through the active uptake of extracellular precursors of GSH synthesis and AA. However, a number of aspects of these processes differ strongly from the HUVECs with which they have intimate contact in the intact vessel wall. These differences may represent adaptations to the availability of these endogenous agents in the extracellular fluid at the intima, compared with the plasma, which seems to be the major source of supply of these important antioxidant precursors to this particular cell. At a more integrated level of biological organization, however, the data also suggest that contact with an overlying endothelial cell layer may exert a controlling influence on the assimilation of these important water-soluble antioxidants in the smooth muscle cells, at least in the case of those juxtaposed to the endothelium. This may perhaps occur through endothelial cell-specific regulation of intimal concentrations of the appropriate metabolic precursors by active secretion processes. Further studies with this syngenetic human endothelial-smooth muscle cell coculture system may provide insight into the mechanisms of these cooperative metabolic interactions between the cells of the intact blood vessel wall, as well as revealing the overall regulatory role of vessel wall cells in the supply of systemic precursors of AA and GSH assimilation in underlying tissues. This is vital in order to develop a rational use of endogenous water-soluble antioxidants in the therapy of human disease.

    ACKNOWLEDGEMENTS

These studies were supported by grants from the Swedish Medical and Natural Sciences Research Councils, as well as from the Swedish Committee for Alternatives to Laboratory Animals in Research.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: I. A. Cotgreave, Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, 17177 Stockholm, Sweden.

Received 24 February 1998; accepted in final form 25 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(4):C1031-C1039
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