Interactions of Peroxynitrite, Tetrahydrobiopterin, Ascorbic Acid, and Thiols

IMPLICATIONS FOR UNCOUPLING ENDOTHELIAL NITRIC-OXIDE SYNTHASE*

Nermin Kuzkaya {ddagger} §, Norbert Weissmann {ddagger}, David G. Harrison and Sergey Dikalov 

From the Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322 and the {ddagger}Division of Pulmonology, Justus-Liebig University School of Medicine, Giessen 57080, Germany

Received for publication, March 4, 2003 , and in revised form, April 5, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO reacted with BH4 6–10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO and BH4 was the trihydrobiopterin radical (), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO-spin trap iron-diethyldithiocarbamate. Bolus ONOO, the ONOO donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO production. Exogenous BH4 supplementation restored endothelial NO production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO prevented uncoupling of eNOS by ONOO. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles radical back to BH4.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endothelial nitric-oxide synthase (eNOS)1 is a dimeric enzyme composed of two catalytic domains: a C-terminal reductase domain, which binds NADPH, FMN, and FAD, and an N-terminal oxygenase domain, which binds a prosthetic heme group, 5,6,7,8-terahydrobiopterin (BH4) (Scheme 1), oxygen, and L-arginine (15). The catalytic production of nitric oxide involves flavin-mediated electron transfer from C-terminal bound NADPH to the N-terminal heme center. At the heme site, oxygen is reduced and incorporated into the guanidino group of L-arginine, producing NO and L-citrulline (1, 2, 6). eNOS is only catalytically active in the dimeric form, and the ability to bind BH4 is dependent on dimer formation. There is evidence that BH4 promotes dimer formation, although this is controversial (7).



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SCHEME 1.
Chemical structure of (6R)-5,6,7,8-tetrahydrobiopterin (BH4).

 

BH4 plays a critical role in allowing electron transfer from the prosthetic heme to L-arginine. In the absence of BH4, electron flow from the reductase domain to the oxygenase domain is diverted to molecular oxygen rather than to L-arginine, leading to a condition known as eNOS uncoupling (8, 9), which causes production of superoxide rather than nitric oxide.

Superoxide reacts rapidly with NO to form the peroxynitrite anion (ONOO), which is a strong biological oxidant (10) known to oxidize lipids, protein, sulfhydryls, and DNA and to cause nitration of tyrosines (1113). Recently, it has been suggested that BH4 is an important target for oxidation by ONOO (Scheme 2) (14). Treatment of purified eNOS with ONOO significantly decreases the ability of the enzyme to produce NO (15). Laursen et al. (14) and others (16, 17) demonstrated that ONOO is more potent than either superoxide or H2O2 in causing oxidation of BH4. These investigators found that ONOO dramatically increased vascular superoxide production in vessels from control mice but not in vessels from eNOS-deficient mice, suggesting that eNOS was the source of superoxide (17).



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SCHEME 2.
Mechanism of the reaction between ONOO and BH4 and the role of ascorbate (AH). ONOO oxidizes BH4 to the intermediate radical, which can decay to BH2 or can be converted back to BH4 by ascorbate.

 

Cellular BH4 levels also seem to be dependent on ascorbate. Pretreatment of endothelial cells with ascorbate increases NO production without affecting NOS expression or L-arginine uptake (18, 19). This effect of ascorbate is BH4-dependent as in the absence of BH4 it is not observed (18, 19). Whereas it is logical to assume that ascorbate may prevent oxidation of BH4, the precise mechanism whereby ascorbate can enhance cellular levels of BH4 has not been defined.

In the present study, we examined the reaction of ONOO with BH4, ascorbate, and thiols using electron spin resonance (ESR) and the spin probe 1-hydroxy-3-carboxy-2, 2,5-tetramethyl-pyrrolidine (CPH). Uncoupling of eNOS by peroxynitrite in cultured bovine aortic endothelial cells (BAECs) was investigated by measuring with new cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (20) and by measuring nitric oxide using colloidal Fe(DETC)2, which allows detection and quantification of NO with high sensitivity and specificity (21, 22). We also studied the role of ascorbate on BH4 oxidation and uncoupling of eNOS and determined whether ascorbate prevents uncoupling of eNOS by scavenging peroxynitrite or if it improves eNOS function by recycling BH4.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—(6R)-5,6,7,8-tetrahydrobiopterin and 7,8-dihydro-L-biopterin were purchased from Schircks Laboratories (Switzerland). L-Ascorbic acid, GSH, cysteine, and Me2SO were obtained from Sigma. Peroxynitrite was obtained from Cayman. The ONOO donor SIN-1 and the cyclic hydroxylamines CPH, CMH, and 2,4-diamino-6-hydroxypyrimidine (DAHP) were purchased from Alexis Corp. (San Diego, CA).

Peroxynitrite concentration was determined spectrophotometrically from its absorbance at 302 nm in 0.1 M NaOH using a molar extinction coefficient of 1670. The ESR buffer consisted of sodium phosphate buffer with 2.35 g/liter NaH2PO4 + 7.61 g/liter Na2HPO4, 0.15 g/liter NaCl, 1 g/liter glucose, 0.37 g/liter KCl, 0.2 g/liter CaCl2 and was treated for 4 h with 50 g/liter Chelex 100, a cationic resin, to minimize contamination with transition metals. Krebs-Hepes buffer contained 5.786 g/liter NaCl, 0.35 g/liter KCl, 0.368 g/liter CaCl2, 0.296 g/liter MgSO4, 2.1 g/liter NaHCO3, 0.142 g/liter K2HPO4, 5.206 g/liter Na-Hepes, and 2 g/liter D-glucose.

Preparation of Spin Probe and BH4 Stock Solutions—Stock solutions of CPH and CMH (10 mM) dissolved in 0.9% NaCl containing 1 mM diethylenetriamine-pentaacetic acid and purged with argon were prepared daily and kept under argon on ice. diethylenetriamine-pentaacetic acid was used to decrease autoxidation of hydroxylamines catalyzed by trace amount of transition metals. CPH and CMH were used in a final concentration of 1 mM. BH4 was dissolved in argon-purged PBS with diethylenetriamine-pentaacetic acid (0.1 mM) and kept under argon on ice.

Cell Culture and Treatments—BAECs (Cell Systems, Kirkland, WA) were cultured in Medium 199 (Invitrogen), containing 10% fetal calf serum (Hyclone Laboratories, Logan, UT) as previously described (23). Confluent BAECs from passages 4–7 cultured on 100-mm plates were used for ESR experiments. Cell suspensions were used for treatment with ONOO. For this purpose, BAECs were scraped and centrifuged at 1800 rpm for 10 min and resuspended in 0.2 ml of ESR buffer. Peroxynitrite (0.27 mM) was added to the cell suspension as a bolus and vortexed. ESR measurements of were made 3 min later. To test whether supplementation with BH4 could restore eNOS function after ONOO, the suspended cells were divided into two Eppendorf tubes, and one portion was incubated with 20 µM BH4 for 4 min at room temperature. Some of these cell suspensions were incubated with L-NAME (1 mM) or polyethylene glycol-superoxide dismutase (50 units/ml) for 5 min, and then the spin probe CMH was added, and the mixture vortexed. Superoxide production was determined by inhibition with 50 units/ml polyethylene glycol-superoxide dismutase, whereas superoxide generated by uncoupled eNOS was measured as L-NAME (1 mM)-inhibited 3-methoxycarbonyl-proxyl (CM) nitroxide formation. In preliminary experiments, we confirmed that BH4 (5–10 µM) did not interfere with CMH detection of by xanthine and xanthine oxidase.

Measurements of Nitric Oxide with Fe(DETC)2NO production in BAECs has been measured with colloid solution of Fe(DETC)2 as previously described (21, 22). Due to its high lipophilicity, the formed NO-Fe(DETC)2 complex is exclusively associated with cell membrane and specifically detects NO but not nitrite (21, 22). After incubation with Fe(DETC)2, medium was aspirated, and cells were harvested with a rubber policeman in Krebs-Hepes buffer, resuspended, and aspirated into 1-ml syringes, which were frozen immediately in liquid nitrogen.

ESR Measurements—Oxidation of the spin probes CPH and CMH by reactive oxygen species (ROS) forms stable nitroxide radicals 3-carboxyproxyl (CP) and CM, which can be assayed by ESR spectroscopy (20, 24, 25). The amount of nitroxide formed equals the concentration of the reacted oxidant species. The concentration of nitroxides was determined from the ESR amplitude according to a calibration curve using standard solutions of the 3-carboxyproxyl radical. ROS formation was measured from the kinetics of nitroxide accumulation by following the ESR amplitude of the low field component of ESR spectra. The rate of superoxide radical formation was determined by measuring the superoxide dismutase-inhibited nitroxide generation.

The reactivity of peroxynitrite scavengers with ONOO was studied by competition with CPH using both bolus ONOO and ONOO generated by SIN-1. BH4 and other ONOO scavengers compete with CPH to react with ONOO. The reactivity of each scavenger with bolus ONOO was determined using the formula,

(Eq. 1)
where A0 represents the ESR amplitude in the absence of ONOO scavengers, A is the ESR amplitude in the presence of ONOO scavengers, k is the reaction rate constant, and c is concentration. With SIN-1 as the ONOO donor, the reactivity of ONOO scavengers was calculated using the formula,

(Eq. 2)
where V0 is the rate of nitroxide accumulation in absence of ONOO scavengers, and V is the rate in presence of ONOO scavengers.

ESR samples were placed in a 100-µl capillary and measured at room temperature using a field scan with the following ESR settings: microwave frequency, 9.78 GHz; modulation amplitude, 2 G; microwave power, 10 dB; conversion time 164 ms; time constant, 164 ms. Peroxynitrite and ROS production by BAECs were detected by following the low field peak of the nitroxide ESR spectra using time scans with the following ESR settings: microwave frequency, 9.78 GHz; modulation amplitude, 2 G; microwave power, 10 dB; conversion time, 1.3 s; time constant, 5.2 s.

The intermediate radical was measured by direct ESR spectroscopy without a spin trap. The high resolution spectrum of the radical was detected and quantified using a microwave frequency of 9.78 GHz, modulation amplitude of 0.7 G, microwave power of 10 dB, conversion time of 82 ms, and time constant of 82 ms.

Frozen probes with NO-Fe(DETC)2 have a three-line ESR spectra whose amplitude is proportional to amount of bioactive NO produced in cells (21, 22, 26). Frozen cell samples were measured in a finger Dewar flask filled with liquid nitrogen at 77 K in field scan with the following ESR settings: field sweep, 160 G; microwave frequency, 9.39 GHz; microwave power, 20 milliwatts; modulation amplitude, 3 G; conversion time, 655 ms; time constant, 5242 ms; receiver gain, 1 x 104; number of scans, 4.

Computer Simulation of ESR Spectra—Computer simulation of the high resolution ESR spectra was used for calculation of hyperfine coupling constants. Programs for simulation of ESR spectra and spin trap data base are readily available through the Internet (on the World Wide Web at epr.niehs.nih.gov/). Details of this computer simulation program have been described elsewhere (27). Hyperfinecoupling constants are expressed as an average of ESR parameters obtained from computer simulation. The ESR spectrum of radical was simulated as a combination of five nitrogens with four protons with following hyperfine coupling constants (aN = 8.05 G, aN = 2.31 G, aN = 1.79 G, aN = 1.16 G, aN = 0.93 G, aN = 8.41 G, aN = 9.50 G, aN = 2.50 G, aN = 1.06 G).

Statistical Analysis—Data are presented as means ± S.E. Analysis with linear regression was done with the software Sigma Plot. For comparison of two groups, a one-tailed t test was employed using Excel software. Statistical significance was assumed when p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reaction of ONOO with BH4 and Other Antioxidants— Previous studies indicated that ONOO readily oxidizes BH4 in cultured cells and vessels. We therefore hypothesized that the reactivity of ONOO with BH4 would exceed that of ONOO with other common intracellular antioxidant small molecules. The reactivity of ONOO with BH4, BH2, GSH, cysteine, ascorbate, and Me2SO was studied by examining the competitive reaction between these agents and the hydroxylamine CPH. Boluses of ONOO (0.27 mM) were added to reaction mixtures containing these potential ONOO scavengers and CPH. In the absence of any scavenger, the reaction of ONOO with CPH resulted in formation of CP that could be detected as a strong ESR signal (Fig. 1A). BH4 reduced CP nitroxide generation in a concentration-dependent fashion, confirming that BH4 could prevent the reaction of ONOO with CPH (Fig. 1A). In contrast, BH2, cysteine, GSH, ascorbate, and Me2SO exhibited substantially less reactivity with peroxynitrite. For comparison of these data, CP formation by ONOO was set as 100%, and the effectiveness of the various scavengers was expressed as a percentage of this value. BH4 strongly inhibited the ESR amplitude by 94%, whereas ascorbate and thiols inhibited ESR signals to a lesser degree (69 and 63%, respectively). Me2SO minimally inhibited the reaction of ONOO with CPH (24%) (Fig. 1B).



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FIG. 1.
Comparison of peroxynitrite scavenging by BH4 and other endogenous antioxidants. A, ESR spectra of 0.27 mM bolus ONOO and CPH in the presence of 0.125, 0.5, and 1 mM BH4.BH4 competes with CPH in the reaction with ONOO and inhibits CP formation by ONOO detected by ESR. B, inhibition of CP nitroxide formation by ONOO in the presence of 0.25 mM peroxynitrite scavengers. Residual ESR signals (percentages) were compared with ESR amplitude of ONOO set as 100%. Compared with the antioxidants, BH4 competing with CPH for the reaction with bolus ONOO had the highest inhibition on CP generation by ONOO, implying high reactivity with ONOO.

 

By using the ESR amplitudes for the respective reactions as described under "Experimental Procedures" and in Equation 1, the relative reactivity of the antioxidant scavengers with ONOO was calculated (Fig. 2A). As a separate approach to quantify the reactivity of these antioxidants with peroxynitrite, the slopes of the lines presented in Fig. 2A were compared (Fig. 2B). According to these data, BH4 reacted with peroxynitrite 10 times faster than ascorbate and 6 times more rapidly than either cysteine or GSH. These data indicate that, in the concentrations employed, neither dihydrobiopterin, ascorbate, nor thiols are able to fully protect BH4 from oxidation by ONOO.



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FIG. 2.
Reactivity of potential ONOO scavengers with bolus ONOO A, inhibition of CP nitroxide formation by bolus ONOO in the presence of different peroxynitrite scavengers at varying concentrations; reactivity was calculated using the ESR amplitude (A0/A) – 1 = kSCAV/kCPH x [scav]/[CPH] as described under "Experimental Procedures." Linear regressions of the data are presented in curves. The BH4 curve has the highest slope, implying highest reactivity with ONOO. Slope of the BH4 curve was significantly different from the slopes of the other peroxynitrite scavengers, with p < 0.01. B, ratio kSCAV/kCPH calculated from the slope of (A0/A) – 1 is shown in A. S.E. were less than 5%.

 

Reactivity of Peroxynitrite Scavengers Studied with ONOO Donor SIN-1—SIN-1 generates superoxide and nitric oxide, resulting in constant production of ONOO, and therefore serves as a model for physiological ONOO production. We therefore studied reactions of various scavengers with ONOO generated by SIN-1. The rate of ONOO formation by SIN-1 (5 mM) was measured from the kinetics of CP nitroxide accumulation by following the ESR amplitude of the low field component of ESR spectra (Fig. 3A, insert). The control sample showed little nitroxide accumulation, whereas SIN-1 resulted in sharp increase in nitroxide generation (Fig. 3A). The accumulation of CP nitroxide was strongly inhibited by 0.25 mM BH4 (Fig. 3A). BH4 had the highest reactivity with SIN-1-generated ONOO followed by cysteine, GSH, ascorbate, and Me2SO as calculated using formula 2 (Fig. 3B). In keeping with our results with bolus addition of ONOO, BH4 reacted with SIN-1-generated ONOO 10 times faster than ascorbate and 6 times faster than GSH (Fig. 4).



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FIG. 3.
Comparison of reactions between various potential ONOO scavengers and ONOO generated by SIN-1. A, inset, ESR spectrum of CPH incubated with 5 mM SIN-1. Accumulation of CP nitroxide was followed by low field component of the ESR spectra shown by the arrow. Rapid accumulation of CP nitroxide in the probe with 5 mM SIN-1 was strongly inhibited by 0.25 mM BH4. B, inhibition of CP nitroxide generation by SIN-1 in the presence of peroxynitrite scavengers at varying concentrations; calculation was done using (V0/V) – 1 = kSCAV/kCPH x [scav]/[CPH] as described under "Experimental Procedures." Linear regressions of the data are presented in curves. The slope of the BH4 curve was significantly different from the other peroxynitrite scavengers with p < 0.01.

 


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FIG. 4.
Reactivity of BH4, thiols, and ascorbate with ONOO generated by SIN-1. Reactivity (kSCAV/kCPH) of various scavengers with ONOO generated by SIN-1 was calculated from the slopes of (V0/V) – 1 shown in Fig. 3B. The BH4-ONOO reaction yielded the highest slope implying that BH4 has the highest reactivity with ONOO of the reductants studied, followed by cysteine, GSH, ascorbate and Me2SO. The calculated S.E. were less than 7%, and ONOO reactivity with BH4 was significantly different from the reactivity with other peroxynitrite scavengers (p < 0.01).

 

Table I provides summary data for experiments with both bolus peroxynitrite and SIN-1. For this analysis, the reactivity of BH4 with ONOO (either as a bolus or generated by SIN-1) was set as 100% and compared with reactivities of other antioxidants with ONOO. The reactivities for cysteine, ascorbate, and GSH were similar for both systems.


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TABLE I
Reactivity of peroxynitrite scavengers with bolus ONOO- and SIN-1-generated ONOO-

Reactivity of BH4 with bolus ONOO- or ONOO- generated by SIN-1 was set at 100% ± S.E. and compared with the reactivity of the other antioxidants with ONOO-. The reactivity of ascorbate and GSH were in the same range in both systems with some variation for cysteine and Me2SO.

 

Formation of Radical and Its Reaction with Ascorbate and Thiols—The above experiments suggest that ascorbate is only marginally effective in scavenging peroxynitrite. In prior studies, however, it has been reported that ascorbate preserves BH4 content of purified eNOS permitting full catalytic function of the enzyme. It is also known that ascorbate is incapable of reducing BH2 back to BH4 (28). These data suggest that ascorbate may act as a "free radical sink" reducing the intermediate radical (29), which may be formed upon the reaction of BH4 with peroxynitrite. We therefore performed additional experiments to examine interactions between ascorbate, BH4, and ONOO. Whereas buffer containing BH4 yielded no ESR signal (Fig. 5A), the bolus addition of ONOO (0.27 mM) to BH4 resulted in formation of a five-line ESR signal (Fig. 5A). Neither decomposed ONOO nor NaOH (the solvent for ONOO) produced an ESR signal when exposed to BH4. High resolution ESR spectra revealed additional hyperfine components (Fig. 5A). Computer simulation of this high resolution spectrum confirmed assignment of the radial intermediate of BH4 oxidation by ONOO as the radical (Fig. 5A). This computer simulation of the high resolution spectra of BH4 and ONOO was further supported by analysis of the ESR spectrum of radical in D2O (data not shown), which was previously reported by Vasquez-Vivar et al. (28).



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FIG. 5.
Formation of the intermediate BH3 radical in the reaction of BH4 with ONOO. A, BH4 solution alone (18 mM) has no ESR spectra; ESR signal of 18 mM BH4 plus 0.27 mM ONOO with a five-line spectrum; the control samples with 18 mM BH4 plus decomposed ONOO and 18 mM BH4 plus NaOH as solvent of ONOO had no ESR spectra; high resolution ESR spectrum of 18 mM BH4 plus 0.27 mM ONOO recorded as described under "Experimental Procedures." The computer simulation of the high resolution verified that as the spectrum. ESR spectrum of radical (E) was simulated as a combination of five nitrogens and four protons with following hyperfine coupling constants (aN = 8.05 G, aN = 2.31 G, aN = 1.79 G, aN = 1.16 G, aN = 0.93 G, aN = 8.41 G, aN = 9.50 G, aN = 2.50 G, aN = 1.06 G). B, reactivity of radical with the antioxidants ascorbate and thiols; the effect of ascorbate or thiols at different concentrations on the ESR signal of radical is presented as a percentage of ESR amplitude. The antioxidants were added a few seconds after the reaction of BH4 with bolus ONOO. Data confirm high reactivity of radical with ascorbate but not with thiols.

 

We next sought to determine whether ascorbate or thiols could reduce the radical by adding these antioxidants 2–3 s after mixing of ONOO with BH4. Ascorbate (100 µM) inhibited the radical ESR signal by 32%, whereas 1 mM ascorbate decreased this signal by 79% (Fig. 5B). In contrast, the addition of either cysteine or GSH in concentrations of 1–10 mM only minimally reduced the ESR signal (Fig. 5B). Thus, ascorbate seems to be much more potent than thiol-containing compounds in reducing the radical.

Recovery of Enzymatic Activity of Uncoupled eNOS in ONOO-treated Endothelial Cells by BH4 Supplementation— Next, we performed experiments to determine whether BH4 was a target of ONOO oxidation in vivo. To assess function of eNOS in cultured BAECs, the spin probe CMH was used to detect . ROS production by BAECs was measured from the kinetics of CM nitroxide accumulation by following the ESR amplitude of the low field component of ESR spectra (Fig. 6A, inset). Untreated cells demonstrated minimal accumulation of nitroxide radical (Figs. 6A and 7A). In contrast, cells exposed to peroxynitrite robustly oxidized CMH to CM, and this signal was inhibited by the addition of superoxide dismutase or by preincubation of cells with the NOS inhibitor L-NAME (Figs. 6B and 7B). SIN-1 also increased BAEC ROS production, and either L-NAME or superoxide dismutase inhibited this effect (Figs. 6C and 7C). Both bolus ONOO and ONOO generated by SIN-1 uncoupled eNOS in BAECs in a similar fashion. We have previously shown that and H2O2 react minimally with BH4 (14). In keeping with these previous findings, exposure of BAECs to xanthine (50 µM) and xanthine oxidase (0.5 milliunits/ml), which generates superoxide and hydrogen peroxide, had no effect on subsequent production of by endothelial cells (Fig. 6D). Nitroxide accumulation was similar to the control cells and did not show inhibition by L-NAME, indicating that superoxide did not uncouple eNOS (Fig. 6D).



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FIG. 6.
Superoxide production by endothelial cells following exposure to ONOO or SIN-1. ROS formation was measured in BAECs after treatment with bolus ONOO, ONOO donor SIN-1, or superoxide generated by xanthine and xanthine oxidase as accumulation of CM nitroxide, which was followed by low field component of the ESR spectra shown by the arrow in the inset (A). Superoxide production was determined by inhibition with 50 units/ml polyethylene glycol-superoxide dismutase, whereas superoxide generated by uncoupled eNOS was measured as L-NAME (1 mM)-inhibited CM nitroxide formation. A, CM accumulation in nontreated control cells. The inhibition of coupled eNOS with L-NAME increased the amount of detected superoxide. B and C, ROS production in BAECs treated by bolus 0.27 mM ONOO or 0.5 mM SIN-1. The inhibition of uncoupled eNOS with L-NAME decreased the amount of detected superoxide. D, ROS production in BAECs treated by 50 µM xanthine and 0.5 milliunits/ml xanthine oxidase.

 


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FIG. 7.
Effect of BH4 supplementation on superoxide production by uncoupled eNOS in BAECs treated with ONOO, SIN-1, and DAHP. A,CM nitroxide accumulation of control cells before and after BH4 (20 µM) supplementation and the inhibition by L-NAME (1 mM). B, ROS production by endothelial cells treated with 0.27 mM ONOO and inhibition of CM accumulation by L-NAME. To test the concentration-dependent effect of BH4, we compared supplementations with 10 and 20 µM BH4 after BAECs were treated with bolus ONOO (B). C, CM accumulation in BAECs treated with 0.5 mM SIN-1. L-NAME was strongly inhibited by superoxide production in SIN-1-treated cells, whereas supplementation with BH4 abolished the effect of L-NAME. D, CM accumulation in BAECs treated with an inhibitor of BH4 synthesis, DAHP (5 mM). Supplementation with BH4 (20 µM) restored eNOS function.

 

Whereas the effects of ONOO, SIN-1, and the BH4 synthesis inhibitor DAHP on eNOS function are consistent with depletion of BH4, these agents may have nonspecific effects on endothelial cell NO and production. We therefore examined whether BH4 supplementation was capable of restoring eNOS activity. For this purpose, we measured by BAECs incubated with exogenous BH4 after the treatment with ONOO, SIN-1, or DAHP (Fig. 7). In preliminary experiments, we confirmed that BH4 (5–10 µM) did not interfere with CMH detection of generated by xanthine and xanthine oxidase.

Treatment of control cells with BH4 increased production, and this was unaffected by L-NAME (Fig. 7A). In contrast to control cells, supplementation with BH4 concentration-dependently inhibited production in cells exposed to bolus ONOO, as did L-NAME (Fig. 7B). A similar effect of BH4 and L-NAME on endothelial production was observed in cells that were exposed to either SIN-1 or the inhibitor of BH4 synthesis DAHP (Fig. 7, C and D). The addition of L-NAME to BH4 supplemented cells (Fig. 7, C and D) increased the ESR signal similar to the control cells, providing evidence that eNOS function was completely restored.

The activity of eNOS was also determined by measuring NO production in BAECs using the NO-specific spin probe colloid Fe(DETC)2. Because Fe(DETC)2 cannot be used in cell suspensions and bolus ONOO can only be used in cell suspension, we only examined the effect of SIN-1-generated ONOO on cellular NO production. Colloid Fe(DETC)2 in a cell-free sample did not yield an ESR signal. Nontreated control cells demonstrated a strong ESR signal of NO-Fe(DETC)2 consistent with coupled eNOS function (Fig. 8). Treatment of control cells with BH4 slightly reduced NO production (Fig. 8). We then treated cells with 0.5 mM SIN-1 to generate ONOO at a rate of 1–1.5 µM/min, levels similar to those observed in pathophysiological conditions (30). This significantly decreased BAEC NO production (Fig. 8). Following treatment with SIN-1, supplementation with BH4 completely restored NO production to values similar to that observed in control cells. Similar to SIN-1, treatment of cells with the BH4 synthesis inhibitor DAHP for 24 h also decreased NO production (Fig. 8), and BH4 reversed this effect (Fig. 8). Thus, by measuring and NO from cultured endothelial cells, we have shown that ONOO derived from SIN-1 and DAHP uncouples eNOS and that BH4 supplementation corrects this. These data confirm that ONOO uncoupled eNOS in BAECs by oxidation of BH4, because supplementation with BH4 after ONOO treatment fully restored eNOS function.



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FIG. 8.
Effect of ONOO donor SIN-1, DAHP, and BH4 supplementation on NO production in endothelial cells. NO production in cultured BAECs was detected as NO-Fe(DETC)2 as described under "Experimental Procedures." Production of NO in BAECs was measured in untreated control cells, control cells supplemented with 20 µM BH4, 0.5 mM SIN-1-treated cells, BH4-supplemented cells after SIN-1 treatment, 5 mM DAHP-treated cells, and BH4-supplemented cells after DAHP treatment. Amplitude of ESR signal corresponds to bioavailable NO in cells and is shown as a percentage compared with signal of control cells set as 100%.

 

Ascorbate Prevention of eNOS Uncoupling—Our previous data have shown that BH4 administration could recover eNOS function after uncoupling by ONOO and also that ascorbate recycles BH4 after its reaction with ONOO via reducing the intermediate radical. It was of interest to determine whether exogenous BH4 or ascorbate could protect eNOS against ONOO in intact endothelial cells. Treatment of control cells with ascorbate did not affect NO production (Fig. 9). As in Fig. 8, SIN-1 treatment markedly decreased endothelial cell NO production (Fig. 9). This effect of SIN-1 was not altered by pretreatment of cells with either ascorbate or BH4 (Fig. 9), indicating that saturating cells with ascorbate or BH4 does not prevent eNOS uncoupling when the cells are subsequently challenged with the ONOO donor SIN-1. In contrast, co-incubation of cells with BH4 and ascorbate during SIN-1 treatment completely prevented the effect of SIN-1 (Fig. 9) on NO production. The effect of co-incubation with either ascorbate or BH4 alone during SIN-1 treatment was approximately half that of when these agents were used together (Fig. 9). These data as well as data presented in Fig. 5B strongly support the concept that ascorbate protects eNOS from uncoupling by recycling intracellular BH4 (Scheme 2).



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FIG. 9.
Effect of ascorbate on NO production in ONOO (SIN-1)-treated BAECs. Production of NO in BAECs was measured as NO-Fe(DETC)2 in untreated control cells, 100 µM ascorbate-treated cells, 0.5 mM SIN-1-treated cells, cells incubated with ascorbate before treatment with 0.5 mM SIN-1, cells incubated with both ascorbate plus SIN-1, cells incubated with BH4 before treatment with 0.5 mM SIN-1, cells simultaneously incubated with BH4 plus SIN-1, and cells coincubated with both BH4 and ascorbate during SIN-1 treatment. Amplitude of ESR signal (percentage) was compared with ESR signal of control cells set as 100%.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we demonstrated that ONOO reacts with BH4 ~10 times faster than with ascorbate and 6 times faster than with the thiol-containing compounds glutathione and cysteine. We also showed that this reaction led to formation of the radical. The radical was found to have high reactivity with ascorbate but not with thiols. Finally, we demonstrated that peroxynitrite leads to eNOS uncoupling in cultured endothelial cells and that following exposure of endothelial cells to peroxynitrite, eNOS activity could be fully restored by treatment of the cells with tetrahydrobiopterin. Taken together, these data demonstrate that BH4 is probably a crucial target for ONOO and that even in the presence of common cellular antioxidants such as ascorbate and thiols, ONOO can lead to BH4 oxidation.

By examining the competition between oxidation of the spin probe CPH and various potential antioxidants, we were able to compare rate constants of ONOO reactions with tetrahydrobiopterin, ascorbate, and thiols. The rate constants of ONOO reactions with ascorbate, cysteine, and glutathione have been previously determined to be 236 M1s1, 103 M–1s1, and 5.8102 M–1s1 (3133). Table I compares ratios of the ONOO rate constant with BH4 to the rate constants of other antioxidants. According to these data, the rate constant of BH4 is 10 times higher than the rate constant of ascorbate and 6 times higher than the rate constants of thiols. Given our current data, it is possible to estimate the rate constant for the reaction between ONOO and BH4 as being 6103 M–1s1.

It has recently been shown that ascorbate treatment increases endothelial cell NO synthesis and tetrahydrobiopterin levels (18, 19, 34), although the mechanism whereby this occurs is not well understood. BH2 is not reduced to BH4 by ascorbate (28), and these prior studies showed that ascorbate did not affect expression of eNOS or GTP-cyclohydrolase, the rate-limiting enzyme for BH4 synthesis (18, 34). The authors of this prior study (18) indicated that ascorbate stabilized BH4 within the endothelial cell. Our present data provide further insight into this effect of ascorbate. It is unlikely that ascorbate prevents oxidation of BH4 by scavenging ONOO, since it was found to be 10-fold less reactive with ONOO than BH4. Our data indicate that the product of the reaction between ONOO and BH4 is the radical and that this radical is highly reactive with ascorbate. These data are consistent with the recent observation that the radical is reduced by ascorbate to BH4 with a rate constant of ~1.7105 M–1 s1 (29). Thus, an important mechanism whereby ascorbate stabilizes levels of BH4 seems to involve reduction of the radical back to BH4, rather than prevention of oxidation of BH4 by oxidants such as ONOO. According to our data, did not exhibit the same reactivity with thiols, a finding in agreement with previously published data on reactivity of the radical (29). It also seems that ascorbate does not preserve eNOS activity by scavenging ONOO or by preventing BH4 from reacting with ONOO but that ascorbate improves eNOS function by recycling BH4 (Scheme 2, Fig. 5B). This is supported by our experiments with SIN-1-treated BAECs (Fig. 9), where co-incubation with BH4 and ascorbate fully prevented loss of eNOS function.

Several clinical studies have shown that administration of intraarterial administration of vitamin C can improve endothelium-dependent vasodilatation in the forearms of humans with hypercholesterolemia, diabetes, and cigarette smoking (3537). The effect of vitamin C in these studies has largely been attributed to scavenging of (38). Whereas scavenging may be a mechanism for improvement in endothelium-dependent vasodilatation in these studies, our current data would indicate that another effect of vitamin C might involve recycling of the radical to BH4. Our results are consistent with the previously reported data showing that low concentrations of ascorbate stimulate nitric-oxide synthase in activated macrophages (39). Thus, recycling of the radical to BH4 by ascorbate may play an important role in preserving the activity of not only endothelial but also the inducible and neuronal isoforms of nitric-oxide synthase.

In keeping with the above findings, we observed that ONOO generated from SIN-1 led to a condition of eNOS uncoupling in cultured cells. This was reflected by a decrease in NO and a concomitant increase in production, which could be inhibited by the NOS inhibitor L-NAME.

Recently, it has been suggested that ONOO uncouples eNOS by oxidation of the zinc-thiolate complex that comprises the BH4 binding site. This in turn leads to dissociation of eNOS dimers to monomers (40). In our experiments, however, BH4 supplementation fully restored NO production in ONOO-treated cells, a finding that seems at odds with the concept that the BH4 binding site is disrupted by ONOO. If the zinc-binding site was a primary target for ONOO, eNOS would be irreversibly uncoupled, and BH4 supplementation would seem unlikely to restore endothelial cell NO production after exposure to ONOO. Our data also demonstrate that the reactivity of BH4 with ONOO substantially exceeds that of thiols with ONOO. Given these considerations, it seems unlikely that ONOO would react with the zinc-thiolate center of eNOS in preference to BH4. It is possible that both the zinc-thiolate center and BH4 are targets of oxidation by ONOO, particularly when high levels of this oxidant are present, but our data would indicate that BH4 is preferentially oxidized.

Related to the above discussion, the previous studies at low temperature SDS-PAGE show that BH4 markedly stabilized the dimer of eNOS (41, 42) by preventing dissociation of the heme (42). Low temperature SDS-PAGE itself, however, affects dimer formation. Therefore, it is unclear whether cellular eNOS in situ exists in the same monomer/dimer forms as it does in gel in vitro. Data obtained with transformed yeast suggest that eNOS does not require BH4 for dimer formation (7, 43). Nevertheless, it has been previously reported that BH4 increases the critical temperature for dissociation of eNOS dimer from 30–40 to 40–50 °C (7). Thus, BH4 may have some effect on stabilization of the eNOS dimer, and oxidation of BH4 by ONOO is a likely cause of partial dissociation of the eNOS dimer in intact endothelial cells.

BH4 has been postulated to be deficient in various conditions associated with altered endothelial function (17). Depletion of endothelial BH4 has been shown to stimulate superoxide production from the isolated eNOS enzyme (44) and from eNOS in intact endothelial cells (45). Supplementation with BH4 enhances NO production, improves endothelium-dependent vasodilatation (46), and efficiently couples NADPH oxidation to NO synthesis and inhibits superoxide and hydrogen peroxide formation (8, 44). We have shown that oxidation of BH4 by ONOO uncouples eNOS and that BH4 supplementation fully restores eNOS function after uncoupling, providing evidence that BH4 is a crucial target for peroxynitrite under physiological conditions.

It is now well established that numerous common diseases such as hypercholesterolemia, hypertension, diabetes, and heart failure are associated with a loss of NO production by the endothelium, a condition commonly referred to as endothelial dysfunction (47). In many of these conditions, eNOS uncoupling seems to be present, leading to an increase in endothelial cell production and a decrease in NO production. Our current data, together with other recent publications (14, 16, 17), strongly suggest that one mechanism leading to eNOS uncoupling is oxidation of BH4 by ONOO and similar oxidants. Of note, BH4 can be administered orally and is effective in treatment of mild phenylketonuria (48). Based on our current findings, it is possible that BH4 or more likely a combination of BH4 and vitamin C may prove useful in correcting endothelial dysfunction in these common disorders.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO-1 HL39006 and PO-1 HL058000-06. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Sonderforschungsbereich SFB 547 funded by the Deutsche Forschungsgemeinschaft. Back

To whom correspondence should be addressed: Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr., WMRB 319, Atlanta, GA 30322. Tel.: 404-712-9550; Fax: 404-727-4080; E-mail: dikalov{at}emory.edu.

1 The abbreviations used are: eNOS, endothelial nitric-oxide synthase; BH4, tetrahydrobiopterin; ONOO, peroxynitrite; , trihydrobiopterin radical; ESR, electron spin resonance; CPH, 1-hydroxy-3-carboxy-2, 2,5-tetramethyl-pyrrolidine; BAEC, bovine aortic endothelial cell; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; DAHP, 2,4-diamino-6-hydroxypyrimidine; L-NAME, L-nitro arginine methyl ester; ROS, reactive oxygen species; CP, 3-carboxy-proxyl; CM, 3-methoxycarbonyl-proxyl; , superoxide radical; SIN-1, 3-morpholinosydnonimine. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gorren, A. C., and Mayer, B. (1998) Biochemistry (Mosc.) 63, 734–743[CrossRef][Medline] [Order article via Infotrieve]
  2. Alderton, W. K., Cooper, C. E., and Knowles, R. G. (2001) Biochem. J. 357, 593–615[CrossRef][Medline] [Order article via Infotrieve]
  3. Raman, C. S., Li, H., Martasek, P., Kral, V., Masters, B. S., and Poulos, T. L. (1998) Cell 95, 939–950[Medline] [Order article via Infotrieve]
  4. Stuehr, D. J. (1999) Biochim. Biophys. Acta 1411, 217–230[Medline] [Order article via Infotrieve]
  5. Marletta, M. A. (1993) J. Biol. Chem. 268, 12231–12234[Free Full Text]
  6. Nathan, C., and Xie, Q. W. (1994) J. Biol. Chem. 269, 13725–13728[Free Full Text]
  7. Venema, R. C., Ju, H., Zou, R., Ryan, J. W., and Venema, V. J. (1997) J. Biol. Chem. 272, 1276–1282[Abstract/Free Full Text]
  8. Vasquez-Vivar, J., Kalyanaraman, B., Martasek, P., Hogg, N., Masters, B. S., Karoui, H., Tordo, P., and Pritchard, K. A., Jr. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9220–9225[Abstract/Free Full Text]
  9. Xia, Y., Tsai, A. L., Berka, V., and Zweier, J. L. (1998) J. Biol. Chem. 273, 25804–25808[Abstract/Free Full Text]
  10. Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) J. Biol. Chem. 266, 4244–4250[Abstract/Free Full Text]
  11. Sampson, J. B., Rosen, H., and Beckman, J. S. (1996) Methods Enzymol. 269, 210–218[Medline] [Order article via Infotrieve]
  12. Zhao, K., Whiteman, M., Spencer, J. P., and Halliwell, B. (2001) Methods Enzymol. 335, 296–307[Medline] [Order article via Infotrieve]
  13. Daiber, A., and Ullrich, V. (2002) Methods Enzymol. 359, 379–389[Medline] [Order article via Infotrieve]
  14. Laursen, J. B., Somers, M., Kurz, S., McCann, L., Warnholtz, A., Freeman, B. A., Tarpey, M., Fukai, T., and Harrison, D. G. (2001) Circulation 103, 1282–1288[Abstract/Free Full Text]
  15. Pasquet, J. P., Zou, M. H., and Ullrich, V. (1996) Biochimie (Paris) 78, 785–791[CrossRef][Medline] [Order article via Infotrieve]
  16. Milstien, S., and Katusic, Z. (1999) Biochem. Biophys. Res. Commun. 263, 681–684[CrossRef][Medline] [Order article via Infotrieve]
  17. Landmesser, U., Dikalov, S., Price, S. P., McCann, L., Fukai, T., Holland, S. M., Mitch, W. E., and Harrison, D. G. (2003) J. Clin. Invest. 111, 1201–1209[Abstract/Free Full Text]
  18. Heller, R., Unbehaun, A., Schellenberg, B., Mayer, B., Werner-Felmayer, G., and Werner, E. R. (2001) J. Biol. Chem. 276, 40–47[Abstract/Free Full Text]
  19. Huang, A., Vita, J. A., Venema, R. C., and Keaney, J. F., Jr. (2000) J. Biol. Chem. 275, 17399–17406[Abstract/Free Full Text]
  20. Dikalov, S., Skatchkov, M., and Bassenge, E. (1997) Biochem. Biophys. Res. Commun. 231, 701–704[CrossRef][Medline] [Order article via Infotrieve]
  21. Dikalov, S., Fink, B., Skatchkov, M., Stalleicken, D., and Bassenge, E. (1998) J. Pharmacol. Exp. Ther. 286, 938–944[Abstract/Free Full Text]
  22. Fink, B., and Dikalov, S. (2002) Free Radic. Biol. Med. 33, 366
  23. Kleschyov, A. L., Mollnau, H., Oelze, M., Meinertz, T., Huang, Y., Harrison, D. G., and Munzel, T. (2000) Biochem. Biophys. Res. Commun. 275, 672–677[CrossRef][Medline] [Order article via Infotrieve]
  24. Kleschyov, A. L., and Munzel, T. (2002) Methods Enzymol. 359, 42–51[Medline] [Order article via Infotrieve]
  25. Drummond, G. R., Cai, H., Davis, M. E., Ramasamy, S., and Harrison, D. G. (2000) Circ. Res. 86, 347–354[Abstract/Free Full Text]
  26. Dikalov, S., Landmesser, U., and Harrison, D. G. (2002) J. Biol. Chem. 277, 25480–25485[Abstract/Free Full Text]
  27. Duling, D. R. (1994) J. Magn. Reson. B 104, 105–110[CrossRef][Medline] [Order article via Infotrieve]
  28. Vasquez-Vivar, J., Whitsett, J., Martasek, P., Hogg, N., and Kalyanaraman, B. (2001) Free Radic. Biol. Med. 31, 975–985[CrossRef][Medline] [Order article via Infotrieve]
  29. Patel, K. B., Stratford, M. R., Wardman, P., and Everett, S. A. (2002) Free Radic. Biol. Med. 32, 203–211[CrossRef][Medline] [Order article via Infotrieve]
  30. Lee, C., Miura, K., Liu, X., Zweier, J. L. (2000) J. Biol. Chem. 275, 38965–38972[Abstract/Free Full Text]
  31. Squadrito, G. L., Jin, X., and Pryor, W. A. (1995) Arch Biochem. Biophys 322, 53–59[CrossRef][Medline] [Order article via Infotrieve]
  32. Sies, H., and Arteel, G. E. (2000) Free Radic. Biol. Med. 28, 1451–1455[CrossRef][Medline] [Order article via Infotrieve]
  33. Masumoto, H., Kissner, R., Koppenol, W. H., and Sies, H. (1996) FEBS Lett. 398, 179–182[CrossRef][Medline] [Order article via Infotrieve]
  34. Baker, T. A., Milstien, S., and Katusic, Z. S. (2001) J. Cardiovasc. Pharmacol. 37, 333–338[CrossRef][Medline] [Order article via Infotrieve]
  35. Ting, H. H., Timimi, F. K., Haley, E. A., Roddy, M. A., Ganz, P., and Creager M. A. (1997) Circulation 95, 2617–2622[Abstract/Free Full Text]
  36. Ting, H. H., Timimi, F. K., Boles, K. S., Creager, S. J., Ganz, P., and Creager, M. A. (1996) J. Clin. Invest. 97, 22–28[Abstract/Free Full Text]
  37. Heitzer, T., Just, H., Munzel T. (1996) Circulation 94, 6–9[Abstract/Free Full Text]
  38. Gotoh, N., Niki, E. (1992) Biochim. Biophys. Acta 1115, 201–207[Medline] [Order article via Infotrieve]
  39. Kosaka, H., Wishnok, J., Miwa, M., Leaf, C. D., Tannenbaum, S. R. (1989) Carcinogenesis 10, 563–566[Abstract]
  40. Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817–826[Abstract/Free Full Text]
  41. Leber, A., Hemmens, B., Klosch, B., Goessler, W., Raber, G., Mayer, B., and Schmidt, K. (1999) J. Biol. Chem. 274, 37658–37664[Abstract/Free Full Text]
  42. List, B. M., Klosch, B., Volker, C., Gorren, A. C., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159–165[Medline] [Order article via Infotrieve]
  43. Rodriguez-Crespo, I., Counts Gerber, Nancy, and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462–11467[Abstract/Free Full Text]
  44. Vasquez-Vivar, J., Martasek, P., Whitsett, J., Joseph, J., and Kalyanaraman, B. (2002) J. Biochem. 362, 733–739
  45. Ishii, M., Shimizu, S., Yamamoto, T., Momose, K., and Kuroiwa, Y. (1997) Life Sci. 61, 739–747[CrossRef][Medline] [Order article via Infotrieve]
  46. Heitzer, T., Krohn, K., Albers, S., and Meinertz, T. (2000) Diabetologia 43, 1435–1438[CrossRef][Medline] [Order article via Infotrieve]
  47. Zeiher, A. M., Drexler, H., Saurbier, B., and Just, H. (1993) J. Clin. Invest. 92, 652–662[Medline] [Order article via Infotrieve]
  48. Muntau, A. C., Roschinger, W., Habich, M., Demmelmair, H., Hoffmann, B., Sommerhoff, C. P., and Roscher, A. A. (2002) N. Engl. J. Med. 347, 2122–2132[Abstract/Free Full Text]