Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH (>70%) using 1) buthionine-(S,R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (~75%) of eNOS catalytic activity measured as L-[3H]arginine-to-L-[3H]citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity.
thiols; nitric oxide; antioxidants; endothelium; nitric oxide synthase; glutathione
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INTRODUCTION |
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NITRIC OXIDE (NO) is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric oxide synthase (NOS; EC 1.14.13.39). Endothelial production of NO is crucial in the control of vascular tone (16), arterial pressure (13, 24, 26), smooth muscle cell proliferation (6, 25), and platelet adhesion to the endothelial surface (1). The bioactivity of endothelium-derived NO is impaired in many vascular diseases (4, 18, 30), and this defect is thought to contribute to the clinical manifestations of vascular disease (2, 17).
The action of NO is subject to modulation by cellular antioxidant defenses. For example, inactivation of Cu/Zn superoxide dismutase (SOD) results in impaired release of bioactive NO from endothelial cells (19, 20, 22), and arteries with reduced SOD activity demonstrate defective vasodilation to exogenous sources of NO (20, 22). The production of NO in endothelial cells is enhanced by ascorbic acid(9, 12), perhaps through an increase in the NOS cofactor tetrahydrobiopterin (12). In platelets, vitamin E status is an important determinant of platelet NO production (3). In animal models and patients with atherosclerosis and diabetes, the bioactivity of endothelium-derived NO is also enhanced by antioxidants such as vitamin E, ascorbic acid, and SOD (reviewed in Ref. 11).
Despite the link between cellular antioxidant defenses and endothelium-derived NO bioactivity, the role of intracellular thiols and glutathione (GSH) in this regard is controversial. The downstream signals elicited by NO are subject to thiol modulation as soluble guanylyl cyclase contains a critical thiol group (32). With respect to NO bioactivity, the agonist-induced release of NO from cultured bovine endothelial cells is impaired by alkylation with N-ethylmaleimide (NEM) or oxidation with 2,2'-dithiodipyridine (DTDP; Ref. 8). Similar studies with porcine endothelial cells found no correlation between thiol depletion and reduced NO production (21). Inhibition of bovine endothelial cell GSH synthesis with L-buthionine-(S,R)-sulfoximine (BSO) had no effect on the release of NO (20). In contrast, human umbilical vein endothelial cells treated with 1-chloro-2,4-dinitrobenzene (CDNB) demonstrate a dose-dependent reduction of both cellular GSH and NO production (7). Conversely, bolstering cellular GSH with glutathione monoethyl ester resulted in enhanced NO production (7). In patients with coronary artery disease, endothelium-derived NO bioactivity is impaired, and this defect is reversed by L-2-oxo-4-thiazolidine carboxylate (29), an agent that increases intracellular GSH levels (31). Thus there are conflicting data on the role of GSH in endothelium-derived NO bioactivity. The purpose of this study was to investigate the implications of thiol manipulation on endothelium-derived NO bioactivity with mechanistically distinct thiol-modulating agents and to identify the mechanisms involved.
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METHODS |
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Materials. Medium M-199, minimal essential medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY). L-[3H]arginine (10.2 Ci/mmol ) and [14C]ascorbic acid (8 mCi/mmol ) were obtained from Dupont NEN Life Science Products (Boston, MA). Tetrahydrobiopterin [6-(L-erythro-1,2- dihydroxypropyl)-5,6,7,8-tetrahydropteridine (BH4)] was from Research Biochemical International (Natick, MA). Dowex AG 50W-X8 resin was from Bio-Rad Laboratories (Hercules, CA). All other chemicals were obtained from Sigma (St. Louis, MO). The baculovirus vector for expression of bovine eNOS was kindly provided by Richard Venema (27, 28).
Cell culture. Porcine aortic endothelial cells (PAECs) were harvested from pig aorta using standard techniques and grown in M-199 supplemented with 15% FBS, 10 µg/ml heparin sulfate, and antibiotics. Cells were grown in T75 flasks coated with fibronectin and passaged using calcium- and magnesium-free Hanks' balanced salt solution and trypsin-EDTA. Cultures were used up to passage 6 and exhibited typical endothelial cell morphology, positive staining for factor VIII-related antigens.
Assay of endothelium-derived NO.
We assayed endothelium-derived NO bioactivity as the
accumulation of cGMP in response to 1 µmol/l A-23187. Confluent PAECs in six-well plates were treated for 1 h with thiol-modulating agents in HEPES-buffered physiological salt solution (PSS) containing (in mmol/l) 22 HEPES, pH 7.4, 124 NaCl, 5 KCl, 1 MgCl2, 1.5 CaCl2, 0.16 HPO70°C
until analysis. Determination of cGMP in supernatants was performed as
described (14). The cell pellet protein content was
determined by the bicinchoninic acid (BCA) protein assay (Pierce) after
solubilization with NaOH.
Intracellular thiol. Intracellular thiol was estimated from the acid-soluble supernatant of PAECs lysed with 5% metaphosphoric acid-0.1 mmol/l diethylenetriamine pentaacetic acid (DTPA) using an Ellman assay modified as described (33).
Expression and purification of eNOS.
Bovine eNOS was expressed and purified using baculovirus-infected Sf9
insect cells without dithiothreitol (DTT) to minimize its interaction
with the thiol-modulating agents as described previously
(12). Isolated enzyme was used immediately after purification and typically demonstrated a specific activity for citrulline production of 50-100
nmol · mg1 · min
1.
PAEC membrane preparations. Confluent PAECs in a 100-mm dish were trypsinized in PBS and washed twice with PBS. Harvested cells were sonicated in lysis buffer consisting of 50 mmol/l Tris · HCl, pH 7.5, 100 µmol/l DTPA, 10% glycerol, and protease inhibitors. The homogenate was centrifuged at 65,000 rpm for 60 min using a Beckman Ti70 rotor. The pellet was then resolubilized in lysis buffer with 1% Triton X-100 by sonication. The protein concentration in solubilized membrane preparations was determined by the BCA protein assay (Bio-Rad), and membrane fractions prepared in this manner had no detectable nonprotein thiol by Ellman assay.
L-[3H]arginine-to-L-[3H]citrulline conversion. For intact cell assays, PAECs in six-well plates were treated with thiol-modulating agents for 60 min, washed in PSS, and equilibrated with HEPES-buffered PSS containing 50 µmol/l L-arginine with 25 µCi L-[3H]arginine. After 15 min, cells were stimulated with 5 µmol/l A-23187 for 15 min and lysed, and the lysate was subjected to anion-exchange chromatography as described below. Parallel controls included PAECs treated with 200 µmol/l NG-nitro-L-arginine methyl ester (L-NAME). Total L-[3H]arginine uptake was determined by liquid scintillation counting of lysate aliquots before anion-exchange chromatography and did not vary as a function of thiol modulation status. Isolated enzyme assays (100 µl) contained 50 mmol/l Tris, pH 7.5, 10 µmol/l BH4, 1 mmol/l CaCl2, 10 µg/ml calmodulin, 1 µmol/l FAD, 1 µmol/l flavin mononucleotide (FMN), 50 µmol/l L-[3H]arginine (~105 cpm), 0.5 mmol/l NADPH, and 100 µmol/l DTPA with or without added thiol modulating agents. Reactions were initiated by the addition of 0.1-0.25 µg of bovine eNOS or 150-200 µg of membrane protein and were terminated by 1 ml ice-cold stop buffer containing (in mmol/l) 20 sodium acetate, pH 5.5, 1 L-citrulline, 2 EDTA, and 2 EGTA. Samples were then applied to 0.8-ml Dowex AG50W-X8 columns preequilibrated with stop buffer. L-Citrulline was eluted twice with 1 ml of H2O, and the 3-ml eluent was collected for determination of L-[3H]citrulline by scintillation counting. Reactions were inhibited >95% by 200 µmol/l L-NAME or the omission of CaCl2. The eluate typically contained <1% L-[3H]arginine based on parallel run controls without enzyme.
Spectrophotometry and glutathione reductase assays. NADPH (100 µmol/l in 0.01 N NaOH, pH 10.0) and NADP+ (50 µM in 0.1 M phosphate buffer, pH 7.6) were incubated in a 2-ml quartz cuvette with and without the addition of 0-200 µmol/l CDNB. Wavelength scans were obtained before and after CDNB addition using a Cary 3E dual-beam spectrophotometer (Varian Instruments, Sugar Land, TX). The cofactor activity of NADPH was assayed in the reduction of glutathione disulfide (GSSG) to GSH by glutathione reductase. Incubations were performed in 0.1 M PBS, pH 7.6, containing 0.01 M EDTA, glutathione reductase (5 U/ml), GSSG (5 mmol/l), and NADPH (50 µmol/l) with or without CDNB (100 µmol/l). NADPH consumption was determined by absorbance at 340 nm. In some incubations, glutathione reductase had been pretreated with 100 µmol/l CDNB in PBS followed by extensive dialysis in PBS to remove any unreacted CDNB.
Data analysis. Values are presented as means ± SE. Dose-response relationships were evaluated using one-way ANOVA and an appropriate post hoc comparison. Instances involving only two comparisons were evaluated with a Student's t-test. Statistical significance was accepted if the null hypothesis was rejected with P < 0.05.
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RESULTS |
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Endothelial cell thiol status and NO bioactivity.
We observed a dose-dependent decrease in PAEC-soluble thiol content
with BSO, diamide, and CDNB (Fig. 1,
A-C). Consistent with previous reports
(12, 20), PAEC GSH levels are not an important determinant
of endothelium-derived NO bioactivity since reducing GSH by up
to 73% did not alter A-23187-stimulated cGMP accumulation (Fig. 1,
A and D). In contrast, PAEC treatment with diamide reduced cGMP accumulation in response to both
endothelium-derived and exogenously administered NO (Fig.
1E). Moreover, the response to atrial natriuretic peptide
was also impaired by diamide (Fig. 1E), consistent with a
defect in the catalytic activity of guanylyl cyclase. PAECs treated
with 1-chloro-2,4-dinitrobenzoic acid (CDNB) demonstrated a selective
impairment in cGMP accumulation in response to A-23187, consistent with
impaired production of NO. Thus thiol-modulating agents alter
endothelial cell NO bioactivity.
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Endothelial cell thiol status and NO production.
To determine if thiol-modulating agents altered NO bioactivity as a
function of NO production, we examined their effect on PAEC conversion
of L-[3H]arginine to
L-[3H]citrulline. Vehicle-treated cells
demonstrated an increase in the production of
L-[3H]citrulline from 10 ± 25 to
270 ± 42 pmol/106 cells in response to 5 µmol/l
A-23187 (P < 0.001; n = 3). This response was inhibited in a dose-dependent manner by PAEC exposure to
diamide (Fig. 2A;
P < 0.01 by 1-way ANOVA). The effect of diamide was
also evident in homogenates and membrane preparations derived from
diamide-treated cells even in the presence of exogenously added
cofactors (Fig. 2A), consistent with a direct effect on eNOS
rather than cofactor availability. Treatment of PAECs with CDNB also
inhibited eNOS catalytic activity with a maximum inhibition of ~40%
(Fig. 2B; P < 0.01 by 1-way ANOVA). In
contrast to diamide, the inhibitory effect of CDNB on eNOS activity was
not evident in homogenates or membrane preparations derived from
CDNB-treated PAECs (Fig. 2B). Neither diamide nor CDNB had
any effect on the cellular uptake of
L-[3H]arginine during the time course of
these experiments (Fig. 2, C and D).
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Direct effects of thiol-modulating agents on eNOS enzymatic
activity.
To investigate any direct effects of thiol modulation on eNOS, we
incubated bovine recombinant eNOS with either CDNB or diamide and
examined L-[3H]citrulline production. As
shown in Fig. 3A, incubation
of eNOS with either diamide or CDNB produced a dose-dependent loss of enzymatic activity. The effect of diamide was almost completely reversible with DTT, consistent with eNOS thiol oxidation as the mechanism of diamide inhibition (Fig. 3B). In contrast, CDNB
inhibition of eNOS activity was not reversible with DTT (Fig.
3B), suggesting a distinction in the mechanism of these two
agents.
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Effect of CDNB on eNOS cofactors.
Because the inhibitory effect of CDNB on eNOS activity was lost after
the addition of exogenous cofactors and was not reversible with DTT, we
considered an effect of CDNB on eNOS cofactors. Using reconstitution
experiments, we found that increasing doses of calmodulin,
BH4, FAD, and FMN were ineffective in reversing the inhibitory effect of CDNB on eNOS activity (Fig.
4). In contrast, increasing
concentrations of NADPH completely reversed the inhibitory effect of
CDNB, suggesting an interaction between these two compounds.
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CDNB renders NADPH inactive as a cofactor.
We next sought to determine if the interaction of NADPH and CDNB
prevents NADPH from serving as a source of reducing equivalents. The
reduction of GSSG by glutathione reductase is associated with the
oxidation of NADPH to NADP+. In the presence of CDNB,
however, NADPH is no longer able to serve as a cofactor for glutathione
reductase and its oxidation is significantly impaired (Fig.
6). We observed no direct effect of CDNB
on glutathione reductase as CDNB treatment of the enzyme followed
by dialysis yielded fully active enzyme.
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DISCUSSION |
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In this study, we found that thiol-modulating agents can alter endothelial NO bioactivity through distinct mechanisms. In particular, we found that simple manipulation inhibition of GSH synthesis with BSO has no material effect on endothelial cell NO bioactivity. In contrast, both diamide and CDNB reduced endothelial cell NO production in concert with the reduction in intracellular GSH. With diamide, a thiol-oxidizing agent, we found a direct effect on eNOS catalytic activity that was readily reversed with DTT, suggesting that diamide oxidizes eNOS thiols critical for enzymatic activity. The situation with CDNB was quite distinct as it also reduced eNOS catalytic activity, but only with intact cells. The addition of exogenous eNOS cofactors to endothelial cell membrane preparations restored catalytic activity of the enzyme. It appears that CDNB reacts with NADPH, rendering the pyridine nucleotide unable to serve as a cofactor for eNOS. These data suggest that endothelial cell NO bioactivity is relatively insensitive to the manipulation of glutathione status and that prior observations to the contrary likely reflect the pleiotropic effect(s) of many thiol-modulating agents.
The role of endogenous GSH in the action and metabolism of endothelium-derived NO has been examined in some detail, but not without controversy. Bradykinin-induced release of NO from cultured bovine endothelial cells was impaired by alkylating thiols with NEM or oxidizing intracellular thiols with DTDP (8). Treatment with DTDP was not associated with a demonstrable reduction in the endothelial cell GSH, suggesting that the level of this antioxidant, per se, is not important in the release of NO (8). One proposed alternative mechanism for these effects of NEM and DTDP was the oxidation state of critical thiols involved in calcium homeostasis (8). Other studies with cultured porcine (21) or bovine (20) endothelial cells also failed to demonstrate a correlation between thiol depletion and NO production. The data presented here are in agreement with these early reports in that we found that reducing intracellular thiol levels >70% with BSO had no material effect on endothelial NO bioactivity (Fig. 1A).
One particular study in human endothelial cells contrasts sharply with the data discussed above. Treatment of human umbilical vein endothelial cells with CDNB was associated with a dose-dependent reduction of both cellular GSH and NO production (7). In the present study using porcine endothelial cells, we also observed a reduction in NO bioactivity with CDNB that closely paralleled the drop in cellular thiol content (Fig. 1C). In contrast to Ghigo and colleagues (7), we used other means of thiol manipulation and observed discordance between thiol levels and NO bioactivity, prompting an exploration of the operative mechanisms. We found the effect of CDNB was reversed after the addition of exogenous eNOS cofactors (Fig. 2B) and that excess NADPH alone could reverse the effect of CDNB (Fig. 4). Consistent with NADPH as the target for CDNB, we found that CDNB altered the spectral characteristics of NADPH (Fig. 5) and disrupted its activity as a cofactor for glutathione reductase (Fig. 6). These data indicate that the results of Ghigo and colleagues are best explained by CDNB-mediated modification of NADPH and not a reduction in intracellular GSH.
Treatment of PAECs with diamide was associated with reduced NO bioactivity measured as A-23187-stimulated cGMP accumulation (Fig. 1E). Although this reduction in NO bioactivity was closely associated with a loss of intracellular thiol (Fig. 1B), our data do not support a role for GSH in this process since we did not find any effect on NO bioactivity with BSO-mediated manipulation of GSH levels (Fig. 1D). The effect of diamide was also characterized by an impaired cGMP response to exogenous NO (Fig. 1E) that can also hardly be attributed to GSH levels. In this regard, the issue of protein thiol oxidation by diamide merits particular attention. The catalytic site of guanylyl cyclase contains critical thiol groups that are involved in its activation by NO (15) and are sensitive to oxidative inactivation (5). Thus one plausible explanation for diamide-mediated inhibition of the PAEC cGMP response to exogenous NO is oxidation of critical thiols in guanylyl cyclase. In fact, the same mechanism may also be germane to the effect of diamide on eNOS catalytic activity. We found that diamide impaired the activity of eNOS using intact cells, membrane preparations (Fig. 2A), or a baculovirus expression system (Fig. 3A). This effect was reversed by excess DTT, suggesting protein thiol oxidation as the mechanism of diamide action. These findings are in good agreement with those of Patel and Block (23) demonstrating a loss of eNOS catalytic activity with NEM and diamide in porcine endothelial cells; the latter was reversed by either DTT or thioredoxin/thioredoxin reductase. Thus protein thiol oxidation with diamide has important implications for both NO production and guanylyl cyclase activity in the endothelium.
The relative lack of a relation between endothelial cell GSH status and NO bioactivity stands in stark contrast to the situation with ascorbic acid, another important component of intracellular redox state. Endothelial cells in culture rapidly take up ascorbic acid from the media, and one consequence of this phenomenon is enhanced NO bioactivity (9, 12). The effect of ascorbic acid on NO bioactivity appears to be due to the production of NO as endothelial cells loaded with ascorbic acid demonstrate enhanced eNOS catalytic activity (9, 12). The effect of ascorbic acid appears mediated by tetrahydrobiopterin since endothelial cells loaded with ascorbate demonstrate an increase in this eNOS cofactor (12), perhaps through chemical stabilization of this reduced pterin (10). Although GSH is also a major determinant of intracellular redox state, manipulation of intracellular GSH is not associated with any change in the tetrahydrobiopterin status of endothelial cells (12).
In summary, the data presented here indicate that endothelial cell NO bioactivity is relatively insensitive to manipulations of intracellular GSH. In contrast, we found that two common thiol-manipulating agents altered endothelial NO bioactivity through mechanisms distinct from changes in intracellular GSH. In particular, protein thiol oxidation with diamide appears to have important implications for endothelial cell NO bioactivity by virtue of a direct effect on eNOS catalytic activity. With CDNB we observed an unexpected effect of this agent to react with NADPH and render it incapable of serving as a cofactor for NO synthesis. These data emphasize the pleiotropic effects often associated with thiol-manipulating agents and help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants HL-53398 and HL-52936 to J. A. Vita and HL-55854, HL-59346, and DK-55656 to J. F. Keaney, Jr. J. A. Vita and J. F. Keaney, Jr. are Established Investigators of the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. F. Keaney, Jr., Boston Univ. School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St., Rm. W507, Boston, MA 02118 (E-mail: jkeaney{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 January 2001; accepted in final form 4 April 2001.
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