Stimulation of endothelial nitric oxide production by homocyst(e)ine

Atherosclerosis 132 (1997) 177 – 185

Stimulation of endothelial nitric oxide production by homocyst(e)ine Gilbert R. Upchurch Jr a,b, George N. Welch a, Attila J. Fabian a, Alessio Pigazzi a, John F. Keaney Jr a, Joseph Loscalzo a,* a

Whitaker Cardio6ascular Institute and E6ans Department of Medicine, Boston Uni6ersity School of Medicine, 80 E. Concord Street, W-507 Boston, MA 02118, USA b Department of Surgery, Brigham and Women’s Hospital, Har6ard Medical School, Boston, Massachusetts, USA Received 8 April 1996; received in revised form 11 March 1997; accepted 14 March 1997

Abstract Hyperhomocyst(e)inemia, characterized by accelerated atherosclerosis, is believed to induce endothelial cell injury and promote atherothrombosis by supporting the generation of hydrogen peroxide. Earlier observations in our laboratory demonstrated that in vitro nitrosation of homocyst(e)ine (HCY) prevents the generation of hydrogen peroxide. We, therefore, hypothesized that stimulating the production of nitric oxide (NO) by endothelial cells would detoxify HCY by forming the corresponding S-nitrosothiol, S-nitroso-homocysteine. In an attempt to prove this hypothesis, media containing 1 mM L-arginine, 1 mM bradykinin, a known NO agonist, and one of the biologically relevant thiols (HCY, cysteine, or glutathione) at concentrations of 0, 0.05, 0.5 and 5.0 mM were incubated with bovine aortic endothelial cells (BAEC) for 0.5, 1 and 4 h. S-nitrosothiol (RSNO) concentrations were measured by photolysis-chemiluminescence. Nitric oxide synthase (eNOS or isoform 3) activity and Nos 3 steady-state mRNA levels were determined by the conversion of [3H]L-arginine to [3H]L-citrulline and Northern analysis, respectively. Results demonstrate that increasing concentrations of HCY, and not cysteine or glutathione, in the presence of bradykinin at 0.5, 1, and 4 h led to significant (PB0.05 by ANOVA) time- and dose-dependent increases in RSNO produced by BAEC. Cells exposed to 1 mM calcium ionophore A23187 in the presence of 5.0 mM HCY also produced a time-dependent increase in RSNO compared to control (PB0.05 by ANOVA). In an attempt to determine if de novo synthesis was occurring, BAEC were treated with bradykinin following a 4 h pretreatment with HCY. Pretreatment with HCY followed by stimulation also led to a time- and dose-dependent increase in RSNO production (PB0.05 by ANOVA). Using high performance liquid chromatography with electrochemical detection, S-nitroso-homocysteine was identified following treatment of BAEC with HCY and bradykinin. The increase in RSNO production in the presence of bradykinin and HCY at 4 h occurred concomitantly with a 78% increase in eNOS activity and a 58% increase in steady-state Nos 3 mRNA, with no change in Nos 3 mRNA half-life, compared to control. A partial explanation for HCY’s unique ability to support an increase in NO production was demonstrated by showing that the t1/2 of HCY in media was greater than that of cysteine or glutathione. These data show that, in the presence of an NO agonist, HCY increases RSNO production in a time- and dose-dependent fashion that is reflected by an increase in eNOS activity and Nos 3 transcription. These results suggest that stimulation of endogenous NO, or provision of an exogenous NO donor, may ameliorate endothelial cell injury and thereby decrease the atherothrombotic risk of hyperhomocyst(e)inemic states. © 1997 Elsevier Science Ireland Ltd. Keywords: Homocysteine; Homocystine; Nitric oxide; Bradykinin; Calcium ionophore

1. Introduction

* Corresponding author. Tel.: +1 617 6384888; fax: + 1 617 6384066.

In 1932, Butz and du Vigneaud [1] described the discovery of homologues of cysteine and cystine, which they denoted ‘homocysteine’ and ‘homocystine’, respec-

0021-9150/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 2 1 - 9 1 5 0 ( 9 7 ) 0 0 0 9 0 - 7

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tively. Subsequently, an elevation in plasma homocyst(e)ine (HCY), derived from methionine, was first linked with atherosclerosis over 25 years ago [2]. Since McCully’s initial observation, a number of studies have confirmed the association between hyperhomocyst(e)inemia (fasting concentrations \ 11 – 12 mM; following a methionine challenge, concentrations \ 25 – 30 mM) and premature atherosclerosis of the coronary, cerebrovascular, and peripheral vascular beds [3–8]. While hyperhomocyst(e)inemia may be caused by either an enzyme or a vitamin deficiency [9 – 14], evidence suggests that, regardless of the cause, even mild elevations in HCY promote atherosclerosis. For example, the Physician’s Health Study demonstrated that patients with plasma HCY levels above 15.8 mM had a three-fold increase in the risk of myocardial infarction, thus, suggesting that even moderate increases in HCY lead to accelerated atherosclerosis [15]. Yet, the mechanism by which HCY causes endothelial cell damage, smooth muscle cell proliferation, and subsequent atherosclerotic plaque formation has only recently come under investigation. Damage to the vascular endothelium by HCY is believed to be secondary to hydrogen peroxide formation accompanying oxidation of the amino acid’s sulfhydryl group [16]. We showed previously that a derivative of homocysteine and nitric oxide (NO), S-nitroso-homocysteine, was unable to support hydrogen peroxide formation [17]. More recently, we showed that HCY impairs the basal production of NO by generating hydrogen peroxide and by decreasing intracellular glutathione peroxidase (GPx), the enzyme responsible for the reduction of hydrogen and lipid peroxides to their corresponding alcohols [18 – 20]; these mechanisms potentiate the toxic effects of HCY on the endothelium. In concert with these adverse effects on endothelial cells, other investigators have shown that the underlying smooth muscle cells respond to pathophysiologic concentrations of HCY by proliferating at an increased rate [21]. While HCY’s toxic effects on endothelial cells are well-documented, therapeutic strategies aimed at blunting the effects of HCY on endothelial cells have been limited. Earlier in vitro studies by our laboratory demonstrated that hydrogen peroxide generation from HCY is prevented by S-nitrosation of HCY, and that, in contrast to HCY itself, S-nitroso-homocysteine has potent vasodilator and antiplatelet effects [17]. At the time, we suggested that stimulation of NO production by the endothelial cell might represent a potential detoxifying mechanism through S-nitrosation of HCY. In the present study, we tested the hypothesis that stimulating the production of NO by the endothelial cell attenuates the oxidative toxicity of HCY.

2. Materials and methods

2.1. Chemicals and solutions Trypan blue 0.4%, bradykinin triacetate, calcium ionophore A23187, L-arginine, D,L-homocysteine, Lcysteine, glutathione, 5,5%-dithio-(bis-2-nitrobenzoic acid) (DTNB or Ellman’s reagent), disodium ethylenediaminetetraacetic acid (EDTA), calmodulin, tetrahydrobiopterin (BH4), calcium chloride, reduced b-nicotinamide adenine dinucleotide phosphate (NADPH), and Dowex resin AG50WX-8 were purchased from Sigma (St. Louis, MO). Hank’s buffered saline solution (HBSS) without calcium or magnesium, newborn calf serum, antibiotics, trypsin, dithiothreitol (DTT), and media were all purchased from GIBCO BRL, NY. Phosphate-buffered saline, pH 7.4, consisted of 10 mM sodium phosphate and 150 mM NaCl. Protein concentrations were determined using a BCA (bicinchoninic acid) protein assay reagent (Pierce, Rockford, IL). 2,3,4,5-[3H]L-arginine hydrochloride (58 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).

2.2. Cell culture Bovine aortic endothelial cells (BAEC) were isolated as previously described [22] and maintained in Dulbecco’s modified Eagle media (DMEM/F12) containing 20% newborn calf serum (NCS) and antibiotics (100 U/ml penicillin G sodium and 100 mg/ml streptomycin sulfate). Cells, from passages 3–15, were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere and were subcultured after treatment with 0.05% trypsin and 0.53 mM EDTA. BAEC were identified by their maintenance of density-dependent growth after serial passage, by their typical cobblestone configuration when viewed by light microscopy, and by positive indirect immunofluorescence for von Willebrand factor [23–25]. BAEC were plated onto 20 mm×100 mm tissue culture dishes (Falcon 3003, Lincoln Park, NJ) and allowed to reach confluency over 5–7 days. Phenolfree media, containing 20% NCS and 1 mM exogenous L-arginine, was substituted prior to all experiments. Experimental groups consisted of cells treated with no additional thiols (controls), or with HCY, cysteine (CYS), or glutathione (GSH) at concentrations of 0.05, 0.5, or 5.0 mM. Bradykinin was added at a concentration of 1 mM as an agonist for NO production in selected experiments [26]. In separate experiments, confluent BAEC on 20 mm×100 mm tissue culture plates were treated with the calcium ionophore A23187, a known NO agonist [27], in the presence of either 0 or 5.0 mM HCY for 0.5, 1, and 4 h. Media were collected and assayed for RSNO as described below.

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2.3. Measurement of NO The production of free NO and S-nitrosothiols (RSNO), stable nitrosated derivatives of thiols, was measured by photolysis-chemiluminescence as previously described [28– 30]. Briefly, the system detects NO using ultraviolet light to cleave the S-NO bond homolytically; free NO is then detected in a chemiluminescence spectrometer by reaction with ozone. All measured RSNO are expressed as mean S-nitrosothiol (nmol/g protein)9 S.E.M., with each experiment performed 3–5 times in triplicate using S-nitroso-glutathione as a standard. In these experiments, the exogenous addition of up to 5.0 mM HCY to media containing a known concentration of RSNO produced by BAEC did not interfere with the RSNO signal produced by photolysis-chemiluminescence.

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pepstatin A, and 2 mM leupeptin), the crude homogenate was centrifuged at 1000 × g for 20 min at 4°C. The cytosolic fraction was then combined with the following in mM: 2.0 NADPH, 1.0 CaCl2, 0.05 Larginine (cold), 0.03 BH4, as well as 30 U/ml calmodulin and 2.25 mCi/ml 2,3,4,5- [3H]L-arginine; the total reaction volume was 1.5 ml. The 2,3,4,5-[3H]L-arginine was incubated for 60 min at 37°C with the cytosolic fraction. The reaction was terminated using 1.5 ml cold wash buffer (5 mM Hepes, 2 mM EDTA, pH 5.5). The samples were passed over a 1 ml Dowex AG50WX-8 (Na + form) column which was developed with 2 ml deionized water. The eluate was collected and radioactivity was determined by liquid scintillation spectrometry. The total cpm in the ‘blank’ represented B4% of the total cpm in the experimental samples. Total protein was determined using the bicinchoninic acid assay.

2.4. Identification of S-nitroso-homocysteine by electrochemical detection S-nitrosothiols were measured by high performance liquid chromatography (HPLC) coupled to an electrochemical detector (ECD) [30]. The HPLC device consisted of a C18 reverse-phase column coupled to an ECD with a dual Au/Hg electrode set at both oxidizing ( + 0.15 V) and reducing (−0.15 V) potentials versus a Ag/AgCl reference electrode (Bioanalytical Systems, West Lafayette, IN). Using the working electrodes in series, S-nitrosothiols are detected at the reducing electrode. The mobile phase consisted of 0.1 M monochloroacetic acid, 0.125 M EDTA, 1.25 mM sodium octyl sulfate, and 1% (v/v) acetonitrile, pH 2.8. The column was developed at a flow rate of 1 ml/min. The technique reliably separates and detects low molecular weight S-nitrosothiols in the nanomolar range [30], and unequivocally discriminates RSNO from their parent thiols.

2.5. Measurement of nitric oxide synthase acti6ity Nitric oxide synthase activity was measured by the conversion of [3H]L-arginine to [3H]L-citrulline in the presence of cofactors using a modification of a method previously described [31]. Briefly, confluent BAEC were treated with media containing 1 mM bradykinin and either 0 or 5.0 mM HCY for 4 h. Cell monolayers were washed three times with Chelex-treated PBS and were removed from the culture plate surface using a disposable cell scraper (Costar 3010, Cambridge, MA). After freezing the cell pellet at − 70°C, the cells were placed in a homogenization buffer containing the following in mM: 0.32 sucrose, 0.5 EDTA, and 20 N-2-hydroxyethylpiperazine-2-ethanesulfonic acid (Hepes). After homogenization in the presence of DTT and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM

2.6. Northern analysis of endothelial NO synthase (Nos 3) mRNA Cell monolayers were grown to subconfluency and total ribonucleic acid (RNA) was isolated from monolayers of BAEC treated for (a) 4 h with no exogenous HCY and 1 mM bradykinin; (b) 4 h with 5.0 mM HCY and 1 mM bradykinin; and (c) 5 mM HCY without bradykinin. Total RNA was extracted using the Oncogene Science RNA purification system (Uniondale, NY) which is based on a guanidinium thiocyanate–phenol– chloroform extraction step. Nos 3 steady state mRNA was detected by Northern analysis using a full-length cDNA derived from a bovine clone kindly provided by Drs Thomas Michel and Santiago Lamas (accession c M89952) [32]. Ten micrograms of total RNA were loaded in each lane and electrophoresed on a denaturing 1.2% agarose gel containing 2.2 M formaldehyde. The gel was blotted onto a Nytron-N + membrane by capillary action, hybridized with the [32P]-radiolabeled probes for Nos 3 and bovine b-actin (generous gift of Dr David R. Morris), and then exposed on Kodak X-OMAT film for 3 days at − 70°C. For quantitative evaluation of Nos 3 and b-actin transcripts, phosphoimage analysis was performed using a PhosphoImager SF (Molecular Dynamics, Foster City, CA). Nos 3 mRNA stability was analyzed using BAEC treated with 0 or 5 mM HCY in the presence of 1 mM bradykinin. At the end of the 4 h incubation period, 5 mg/ml actinomycin D was added directly to the media and total RNA was isolated and electrophoresed as described above at 0, 1, 2, and 4 h after actinomycin D treatment. Northern analysis was used to quantify changes in Nos 3 mRNA.

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2.7. Measurement of reduced thiols in buffer and media The measurement of reduced thiols was performed based on methods described previously [33]. Briefly, solutions of each of the three biologic thiols (i.e. HCY, CYS, and GSH) were made in media. Fifty microliters of each thiol solution (final concentration, 0.048 mM) were added to PBS and DTNB (final concentration, 0.143 mM) was added. The samples were vortexed and absorbance read immediately at 405 nm. Readings were performed at each concentration and at each time point (0 – 4 h) in quintuplicate [33].

2.8. Cell 6iability Cell viability of confluent monolayers was determined by one of three methods. At the end of each treatment, cells were examined by light microscopy for vacuolation and other signs of cell death. Treated cells were also compared to control cells using trypan blue exclusion. By trypan blue analysis, no significant differences were apparent at 4 h between the control group and cells treated with up to 5.0 mM HCY. Measurement of total lactate dehydrogenase (LDH) in the media of cells treated with 5.0 mM HCY has been shown previously to be similar to that of cells treated with control media [19].

2.9. Statistical analyses Statistical analysis was performed on paired samples by Student’s t-test. Groups of data were compared as a function of time and concentration by analysis of variance with a Bonferroni or Newman-Keuls post-hoc test. The data are presented as the mean 9 S.E.M. with PB 0.05 considered to be statistically significant.

Fig. 1. Effect of HCY on RSNO production by BAEC stimulated by bradykinin. BAEC were treated for 4 h with 1 mM bradykinin and increasing concentrations (NE – SW lines, 0.0 mM; hatched lines, 0.05 mM; NW – SE lines, 0.5 mM; and horizontal lines, 5.0 mM) of HCY. Media were collected at 0.5, 1, and 4 h and RSNO levels were determined. Results are expressed for RSNO in nmol/g protein as the mean 9S.E.M., n =11 – 12. (* P B0.05 for 5.0 mM HCY vs. control by ANOVA).

for the agonist bradykinin or for agonist-receptor interactions, we next treated BAEC with 1 mM calcium ionophore A23187 in the presence and absence of 5.0 mM HCY. Fig. 2 shows that cells treated with 5.0 mM HCY and A23187 produce significantly more RSNO than cells treated with A23187 alone at 0.5, 1, and 4 h (PB0.05 for 5.0 mM HCY compared to control). Further experiments demonstrated that cells stimulated with bradykinin in the presence of two other relevant biological thiols, CYS and GSH, do not produce increased RSNO. Fig. 3 shows that addition of

3. Results

3.1. Stimulation of NO production by BAEC in the presence of HCY causes a time- and dose-dependent increase in RSNO production We first examined the effects of HCY on BAEC stimulated to produce NO. BAEC were incubated with increasing concentrations of HCY in the presence of 1 mM bradykinin, and media were collected at 0.5, 1, and 4 h. The results shown in Fig. 1 demonstrate that bradykinin stimulation of BAEC leads to a time- and dose-dependent increase in RSNO production in the presence of increasing concentrations of HCY (P B 0.05 for 5.0 mM HCY compared to control and 0.05 mM HCY). In an attempt to determine whether or not the stimulation of RSNO in the presence of HCY was specific

Fig. 2. Effect of HCY on RSNO production by BAEC stimulated with A23187. BAEC were treated for 0.5, 1, and 4 h with 1 mM A23187 and 0 ( ) or 5.0 (“) mM HCY. Media were collected at 0.5, 1, and 4 h, and RSNO levels were determined. Results are expressed for RSNO in nmol/g protein as the mean9S.E.M., n = 6. (* PB 0.05 for 5.0 mM HCY vs. control by ANOVA).

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Fig. 3. Effect of 3 biological thiols on bradykinin-stimulated RSNO production by BAEC. BAEC were treated with 1 mM bradykinin or media containing 5.0 mM HCY, 5.0 mM CYS, or 5.0 mM GSH and 1 mM bradykinin. Media were collected at 0.5 h and RSNO concentrations were determined. Results are expressed for RSNO in nmol/g protein as the mean9S.E.M., n= 9–12. (control, open bars; thiol, filled bars; * P B0.05 for 5.0 mM HCY vs. control by ANOVA).

CYS or GSH at concentrations of 5.0 mM in the presence of 1 mM bradykinin does not support an increase in RSNO formation by BAEC compared with control at 0.5 h (P B 0.05 for 5.0 mM HCY compared to control); similar results were obtained at 1 and 4 h (data not shown).

3.2. Pretreatment of BAEC with HCY followed by bradykinin stimulation causes a time- and dose-dependent increase in RSNO production

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analyzed using HPLC with ECD to detect S-nitrosothiols. S-nitrosothiols were identified and quantified by comparison with authentic standards. Fig. 5 is a representative chromatograph demonstrating that S-nitrosohomocysteine is produced by BAEC in the presence of 5.0 mM HCY, with a mean concentration of 9739375 nmol/g protein (n= 3). In cells treated with control media, S-nitroso-homocysteine was undetectable. S-nitroso-cysteine and S-nitroso-glutathione, with typical retention times of 3.4 and 9.7, respectively, were not detected secondary to the presence of an excessive amount of O2 in each sample of media, despite degassing, producing a sizeable peak in the chromatogram that overlapped with those of S-nitroso-cysteine and S-nitroso-glutathione.

3.4. Treatment of BAEC with HCY and bradykinin stimulates eNOS acti6ity Having demonstrated an increase in NO production in the presence of HCY and NO agonists, we next determined eNOS activity in BAEC stimulated with bradykinin in the presence of HCY. The results show that BAEC treated with 5.0 mM HCY for 4 h have a 78% increase in eNOS activity compared to cells treated with control media (2359 38 fmol [3H]L-citrulline/g protein for 5.0 mM HCY versus 1329 40 fmol [3H]Lcitrulline/g protein for control, n= 12 for each group; PB 0.005). In separate experiments, the rate of [3H]L-arginine uptake by BAEC was quantified at 0.25, 0.5, 1, and 4 h following treatment with either 0 or 5.0 mM HCY. The

We next determined whether or not the increase in NO production is secondary to an increase in the synthesis of NO or a consequence of excess thiol in the media. Following a 4 h exposure to HCY at various concentrations (0, 0.05, 0.5, and 5.0 mM), BAEC monolayers were washed with HBSS, incubated with fresh media containing 1 mM bradykinin without additional HCY, and aliquots removed at 0.5, 1, and 4 h. Fig. 4 shows that BAEC pretreated with HCY respond to bradykinin stimulation by elaborating increasing RSNO as a function of HCY concentration during the preincubation period, and these increases reach statistical significance at 0.5 and 5.0 mM HCY compared to control and 0.05 mM HCY (P B0.05). RSNO also increase at each HCY concentration over time (PB 0.05 by ANOVA from 0.5 to 4.0 h).

3.3. S-nitroso-homocysteine is produced by BAEC in the presence of HCY After confluent BAEC were exposed to PBS containing 5.0 mM HCY, 1 mM L-arginine, and 1 mM bradykinin for 30 min, the PBS was collected and

Fig. 4. Effect of HCY pretreatment on bradykinin-stimulated RSNO production by BAEC. BAEC were treated for 4 h with increasing concentrations of HCY (NE – SW lines, 0.0 mM; hatched lines, 0.05 mM; NW – SE lines, 0.5 mM; and horizontal lines, 5.0 mM) followed by stimulation with 1 mM bradykinin. Media were collected at 0.5, 1, and 4 h following bradykinin stimulation and RSNO concentrations were determined. Results are expressed for RSNO in nmol/g protein as the mean 9 S.E.M., n =9. (* PB 0.05 for 0.5 and 5.0 mM HCY vs. control and 0.05 mM HCY by ANOVA).

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Fig. 5. Chromatogram of S-nitroso-homocysteine as determined by HPLC with ECD. Confluent BAEC were treated for 30 min with 5.0 mM HCY, 1 mM L-arginine, and 1 mM bradykinin. Samples were analyzed by HPLC with ECD and compared with authentic standards. This figure shows a representative chromatogram from 3 experiments for S-nitroso-homocysteine, detected at 50 nA at a concentration of 973 9375 nmol/g protein. Typical retention times for S-nitroso-cysteine and S-nitroso-glutathione in PBS are 3.4 and 9.7 min, respectively, with the limits of detection for RSNO being 200 nM using the HPLC-ECD methodology.

results demonstrate that 5.0 mM HCY does not interfere with [3H]L-arginine uptake by BAEC (P =N.S. by ANOVA) (data not shown) compared to cells treated with control media. There also appeared to be a similar linear uptake of [3H]L-arginine in both treatment groups lending validity to the examination of eNOS activity at any single time point for treatment group comparisons. This finding suggests that the increase in eNOS activity after treatment with HCY and bradykinin is secondary to an alteration in Nos 3 steady-state mRNA levels.

demonstrates no difference in changes in levels of Nos 3 mRNA between control and HCY-treated cells over time. Coupled with the known prolonged half-life of Nos 3 mRNA [34,35], this observation implies that changes induced by HCY and bradykinin are the result of changes in Nos 3 transcription rather than mRNA stabilization.

3.5. Treatment with HCY and bradykinin alters Nos 3 steady-state mRNA in BAEC In an attempt to examine HCY’s effect on Nos 3 mRNA, Northern analyses were performed. Fig. 6 is a representative blot of BAEC total RNA probed with a bovine full-length Nos 3 cDNA and bovine b-actin cDNA. RNA was isolated from BAEC after being treated with control media containing 1 mM bradykinin for 4 h (lane C), media containing 5.0 mM HCY and 1 mM bradykinin for 4 h (lane H), or media containing 5.0 mM HCY without bradykinin (data not shown). The results of Northern blot analyses are presented in Fig. 6. Quantitative results from densitometric analysis of Northern phosphoimages demonstrate that in the presence of 5.0 mM HCY and 1 mM bradykinin, Nos 3 steady-state mRNA is increased modestly by 58% compared with BAEC treated with control media or in the presence of 1 mM bradykinin; no change in the 2 kb b-actin mRNA was observed in these experiments. BAEC treated with 5.0 mM HCY also showed an increase in Nos 3 steady-state mRNA compared to cells treated with control media or in the presence of 1 mM bradykinin (data not shown). Fig. 7 is a Northern blot of BAEC Nos 3 mRNA after treatment with either 0 or 5 mM HCY in the presence of bradykinin for 4 h, followed by treatment with 5 mg/ml actinomycin D. Phosphoimage analysis

Fig. 6. Nos 3 mRNA expression after homocysteine treatment as determined by Northern analysis. Subconfluent BAEC were treated with either control media (C) or media containing 5.0 mM homocysteine (H) for 4 h stimulated with 1 mM bradykinin. (A) An exemplary blot probed with a Nos 3 cDNA (4.5 kb) and a b-actin cDNA (2.0 kb). (B) Data are presented as the mean 9S.D. for 3 experiments each performed in duplicate (* P B0.05).

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Fig. 7. Nos 3 mRNA levels in BAEC incubated with (H) or without (C) HCY and exposed to 5 mg/ml actinomycin D. BAEC were treated with 1 mM bradykinin and control media (C) or 5 mM HCY (H) for 4 h, followed by the addition of 5 mg/ml actinomycin D. Total cellular RNA was isolated immediately after treatment (0 h), and at 1, 2, and 4 h following treatment with actinomycin D. Northern blots were probed with a full length (4.5 kb) Nos 3 cDNA.

3.6. Half-li6es of biological thiols in media The half-lives of the various thiols in media were determined in order to examine whether or not HCY might serve as a better reducing equivalent and consequently a better thiol pool for S-nitrosation, compared to two other biologically-relevant thiols. These data indicate that HCY’s half-life (t1/2 =172 min) in media used in these experiments is greater than that of GSH (t1/2 =81 min) or CYS (t1/2 =9.7 min).

4. Discussion We have previously shown that unstimulated endothelial cells exposed to increasing concentrations of HCY produce less basal NO secondary to the elaboration of hydrogen peroxide from HCY’s oxidation and to a decrease in intracellular glutathione peroxidase [18,19]. In the experiments presented here, we extend this observation by demonstrating that, in contrast to cells in the basal state, stimulated endothelial cells exposed to increasing concentrations of HCY produce more NO than control cells. Experiments with two other biological thiols, CYS and GSH, failed to produce a concentration-dependent increase in RSNO, possibly secondary to their reduced half-lives in media compared to HCY. Yet, while the ability to generate increased NO by BAEC is specific for HCY, it is independent of the NO agonist used, as evidenced by the similar effects of bradykinin and A23187, suggesting a generalized agonist effect and not a specific receptor-dependent event. In other experiments, pretreatment of BAEC with HCY followed by an NO agonist also ‘induced’ cells to produce more RSNO. The present findings suggest that, in addition to forming an RSNO in the extracellular milieu with HCY, increased RSNO are synthesized intracellularly de novo. We further demonstrated the existence of a specific S-nitroso-thiol, S-nitroso-homocysteine, in the extracellular milieu after cells were treated with HCY and an NO agonist. In contrast to

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HCY, S-nitroso-homocyst(e)ine possesses EDRF-like properties, including potent vasodilatory and antiplatelet effects, but does not support H2O2 production and is not converted to homocysteine thiolactone [17]. eNOS activity and Nos 3 steady state mRNA are also increased in cells exposed to HCY and an NO agonist, suggesting that the initial response of endothelial cells to HCY exposure is to synthesize more NO. These data provide indirect evidence that HCY, in the form of homocystine or a mixed disulfide, enters the cell, is reduced, and, in the presence of oxygen, forms an RSNO which is subsequently secreted. These data also suggest that when excess thiol, in the form of HCY, is present in media (or serum), it may actively scavenge free NO. Wink et al. [36] have suggested that thiols are important physiologic scavengers of NO in vivo and serve to detoxify reactive NOx intermediates. In a recent study, these investigators demonstrated that S-nitrosothiols are kinetically capable of forming under aerobic physiological conditions and their formation involves the nitrosating intermediate N2O3. They compared rates of formation of S-nitroso-glutathione and S-nitroso-cysteine with the competing hydrolysis of N2O3 to nitrite, and determined a selectivity ratio of 10 000/M:5000/M:1 for glutathione:cysteine:water. These data suggest that thiols, such as intracellular glutathione, found in millimolar concentrations [37] are capable of reacting with NO (as N2O3) to form the corresponding S-nitroso-derivative and do so with favorable kinetics. Kharitonov et al. [38] confirmed and extended these observations by showing that physiologically attainable concentrations of GSH (up to 5 mM) competed ‘successfully’ with water for reaction with N2O3, and that at these high concentrations of thiol almost all of the N2O3 formed is consumed by the formation of an RSNO. While the selectivity of N2O3 for HCY has not been examined, one might speculate that in view of the present observation that the t1/2 of HCY in the reduced form is greater in media than either GSH or CYS, HCY might be an even better thiol source for reacting with NOx species, although these kinetics have not yet been studied. The stimulation of BAEC to produce RSNO in the presence of up to millimolar concentrations of HCY might serve a dual role: (1) limiting the damage caused by reactive NOx species, and, conversely, (2) limiting the production of reactive O2 species such as H2O2 from oxidation of HCY. The present study uses HCY in up to millimolar concentrations to demonstrate that BAEC have the capacity, at least short-term (4 h), to synthesize RSNO in a time- and dose-responsive fashion. While clearly (patho)physiologic levels of HCY never approach 5 mM, the concept that the endothelial cell can produce and secrete endogenous NO in the form of an RSNO in order to limit the damage caused by a potentially toxic

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thiol (HCY) represents a novel mechanism of antioxidant protection that may have biologic relevance with longer term exposure to lower levels of HCY or to shorter term exposure to higher levels (post-prandially). The chemical structure and reactivity of RSNO have been reviewed by Oae et al. [39]. In 1980, Ignarro and Gruetter [40] demonstrated that RSNO, similar to original endothelium-dependent relaxing factor (EDRF) described by Furchgott and Zawadzki [41], exert their effect by activating vascular smooth muscle guanylyl cyclase. The activation of guanylyl cyclase by EDRF/ NO subsequently leads to a rise in cyclic GMP with vasodilation but only in the presence of a thiol [42]. Through the development of various new analytical methodologies [43–45] our laboratory has shown that RSNO are relatively stable compared with free NO. Ignarro et al. [42] suggested that RSNO may be stored in acidic vesicles, and upon exposure to physiologic pH (above 7), release free NO. While the release of free NO occurs, it is not required for the effects of RSNO, as they are more bioactive than the complement of NO they bear [46]. We further demonstrated that RSNO are capable of forming from either free low-molecularweight thiols (such as HCY) or from protein thiols, such as albumin [43,44], in the presence of NO and molecular O2 and that RSNO, with their EDRF-like properties, are the predominant redox forms of NO in vivo, with S-nitroso-albumin the most abundant form in plasma [29]. Based on these general observations about RSNO coupled to our recent observation that RSNO can undergo thiol–S-nitrosothiol exchange [47], we hypothesized that stimulation of BAEC to produce/secrete NO might prevent the generation of hydrogen peroxide from HCY, and, thereby, serve to detoxify this atherothrombotic thiol. Earlier studies in our laboratory demonstrated that elevated concentrations of HCY attenuate the production of NO/EDRF by the endothelium. In the present study, we demonstrate a potential mechanism by which the cell’s ability to produce and secrete NO can be used to minimize toxicity by HCY and its oxidative by-products. Future therapeutic strategies designed to stimulate endogenous NO production or to provide exogenous NO donors may help to ameliorate endothelial injury, and thereby limit the atherothrombotic risk of hyperhomocyst(e)inemia.

Acknowledgements This work was supported in part by National Institutes of Health (NIH) grants HL47416, HL48743 and P50 HL55993; a Merit Review Award from the US Veterans Administration; and by a grant from NitroMed, G.R. Upchurch is the recipient of a National Research Service Award from the NIH (HL 09124) and

is an American College of Surgeons Resident Scholar. G. Welch is supported by an NIH Cardiovascular Training Grant Fellowship (P32 HL07224). J.F. Keaney Jr. is the recipient of a Clinical Investigator Development Award (HL03195) from the NIH. We are to grateful to Stephanie Tribuna for her assistance in preparation of this manuscript. This work was presented in part at the American Society of Hematology, December 1–5, 1995, Seattle, WA and appears in abstract form in Blood 1995;86:378a.

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