Mechanism of Superoxide Generation by Neuronal Nitric-oxide Synthase*

Sovitj Pou, Lori Keaton, Wanida Surichamorn, and Gerald M. RosenDagger

From the Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201

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Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (Obardot 2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO·). Yet the competition between O2 and L-arginine for electrons, and by implication formation of Obardot 2, has until recently remained undefined. Herein, we investigated this relationship, observing Obardot 2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced Obardot 2 production in the presence of L-arginine because this antagonist attenuated NO· formation. Whereas diphenyliodonium chloride inhibited Obardot 2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of Obardot 2 at concentrations that inhibited NO· formation from L-arginine. Taken together these data demonstrate that NOS I generates Obardot 2 and the formation of this free radical occurs at the heme domain.

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At a time before the physiologic properties of endothelium-derived relaxation factor were associated with nitric oxide (NO·)1 (1-3), it was found that this free radical activated soluble guanylate cyclase from crude homogenates of brain tissue (4). The significance of this observation would, surprisingly, remain dormant for nearly a decade, even though it was known that L-arginine was the endogenous activator of this enzyme (5). With the purification and characterization of a unique monooxygenase, NOS I, capable of oxidizing L-arginine to L-citrulline, and NO· (6, 7), a new class of small molecules (8) acting as transient second messengers in the brain was discovered (9). The versatility of this free radical in controlling a myriad of brain functions will undoubtedly result in new and provocative findings. Of special interest will be research that can distinguish NO· from NOS I-secreting neurons versus NO· from NOS III-containing endothelial cells and NO· from NOS II-stimulated microglial cells. Until the recent development of specific antagonists for each of the NOS isozymes (10-12), it has been difficult to address this question, which is of particular significance when one considers, as described in this article, that NOS I, NOS II, and NOS III may produce NO· and Obardot 2 under differing cell conditions.

Nitric-oxide synthase is known to catalyze the production of NO· from L-arginine (13, 14). In the absence of substrate, we have previously demonstrated that purified NOS I can use O2 as the terminal electron acceptor, generating Obardot 2 (15). These findings have been confirmed because Obardot 2 has been spin-trapped in L-arginine-depleted NOS I-transfected human kidney cells (16). During the course of our earlier studies (15), we noted to our surprise that purified NOS I appeared to produce Obardot 2 even in the presence of L-arginine. The current study, therefore, explores this phenomenon in depth. Herein, we demonstrate that NOS I, like NOS II (17) and NOS III (18-20), can generate Obardot 2 and NO· despite saturating levels of substrate. However, unlike NOS II, the heme of NOS I is the locus for the production of both free radicals. Finally, we discuss the implications of our findings; particularly relevant is the ability of L-NMMA to enhance NOS I-derived Obardot 2 even in the presence of saturating concentrations of L-arginine.

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Materials-- NADPH, calmodulin, L-arginine, phenylmethylsulfonyl fluoride, diethylenetriaminepentaacetic acid, ferricytochrome c, xanthine, NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl L-arginine (L-NMMA), EGTA, HEPES, and penicillin G-streptomycin solution were purchased from Sigma. Imidazole, 1-phenylimazole, sodium cyanide, and diphenyliodonium chloride (DPI) were obtained from Aldrich. Tetrahydrobiopterin was purchased from Alexis Biochemicals (San Diego, CA). Cation exchange resin Dowex 50W-X8 hydrogen form resin was obtained from Bio-Rad. 2',5'-ADP-Sepharose was obtained from Pharmacia (Uppsala, Sweden). L-[14C]Arginine was purchase from ICN Radiochemicals (Costa Mesa, CA). Dulbecco's modified Eagle's medium:nutrient mixture F-12 (1:1) and phosphate-buffered saline were obtained from Life Technologies, Inc. Bovine calf serum was purchased from Hyclone (Logan, UT). Superoxide dismutase and xanthine oxidase were obtained from Boehringer Mannheim. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was synthesized according to the procedure of Bonnett et al. (21).

NOS I Purification-- NOS I-transfected kidney 293 cells were cultured in Dulbecco's modified Eagle's medium:nutrient mixture F-12 containing 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 µg/ml). NOS I was purified from these cells by the method of Bredt and Snyder (6). Briefly, cells were removed from the culture flasks and washed three times with phosphate-buffered saline via centrifugation. The pellet was resuspended in buffer containing phenylmethylsulfonyl fluoride (4 mg/ml) and homogenized with a Polytron (Brinkmann Instruments, model PCU-2 at setting 2 for 10 s). The remaining mixture was centrifuged at 15,000 rpm for 20 min to separate unbroken cells, and the supernatant was applied to a 2',5'-ADP-Sepharose affinity column. After washing the column three times with 0.45 M NaCl and standard buffer, NOS was eluted with standard buffer containing 10 mM NADPH. Excess NADPH was removed by washing and concentrating the eluate with CentriCell-30 (Polysciences, Warrington, PA) until the concentration of NADPH was approximately 1-1.5 mM as assessed spectrophotometrically at 340 nm (epsilon  = 6.2 × 103 M-1 cm-1). Protein concentration was determined by the Bradford method (22) using bovine serum albumin as a standard.

Estimation of NOS I Activity-- NOS I activity was assayed by measuring the formation of L-[14C]citrulline from L-[14C]arginine as described previously (23). Briefly, purified NOS I was added into a reaction mixture containing L-[14C]arginine (0.6 µCi/ml), L-arginine (1 mM), NADPH (1 mM), calmodulin (100 units/ml; 23.6 µg/ml), free calcium ions (CaCl2, 1 µM; calculated as described in Ref. 24), and standard phosphate buffer at pH 7.4 (final volume = 0.150 ml). After incubating at room temperature for exactly 10 min, the reaction was terminated by adding HEPES (20 mM, 2 ml) containing EDTA (2 mM, pH 5.5). L-[14C]Citrulline was separated by passage through an 0.5-ml column of Dowex-50-X8 cation exchange, and radioactivity was counted using a Beckman beta  counter.

Spin Trapping and EPR Spectroscopy-- Spin trapping experiments with purified NOS I were conducted by mixing all the components described in the figure legends to a final volume of 0.25 ml. The experiment was initiated by adding freshly purified NOS I. Reaction mixtures were then transferred to a flat quartz cell and fitted into the cavity of the EPR spectrometer (Varian Associates, model E-109), and spectra were recorded at room temperature 1 min after addition of the enzyme. Instrumentation settings are presented in the figure legends.

Superoxide Detection-- The assessment of the effect of L-NAME on the ability of xanthine/xanthine oxidase to produce Obardot 2 was evaluated as described previously (25). Briefly, xanthine oxidase was added to a solution containing xanthine (400 µM) and ferricytochrome c (80 µM) such that the rate of Obardot 2 formation, measured as the SOD-inhibitable reduction of ferricytochrome c at 550 nm (26) was 1 µM/min. The effect of various concentrations of L-NAME on the rate of Obardot 2 generation was estimated.

NADPH Oxidation-- The rate of NADPH oxidation by NOS I was determined spectrophotometrically at 340 nm (epsilon  = 6.2 × 103 M-1 cm-1).

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Before determining the ability of NOS I to generate Obardot 2, our initial series of studies were devoted to determining the Km and the Vmax for NOS I, because these constants will help dictate future experimental designs. Based on these studies, the Km and the Vmax were found to be 3.47 µM and 216 nmol min-1 mg-1, respectively, well within the ranges (Km of 2-4.3 µM and Vmax of 74-3400 nmol min-1 mg-1) reported by others (27).

In the absence of L-arginine, NOS I has previously been shown to generate Obardot 2 (15, 16), whereas at high concentrations of L-arginine (1 mM), complete inhibition of Obardot 2 production had been noted (15). We therefore examined the effect that intermediate concentrations of L-arginine would have on the ability of NOS I to generate Obardot 2. As shown in Fig. 1, L-arginine, in a dose-dependent manner, blocked Obardot 2 secretion from NOS I, exhibiting an EC50 = 5 µM. Inhibition of NOS I-generated Obardot 2 at 100 µM L-arginine was nearly complete. Under these conditions, the rate of NADPH oxidation decreased as L-arginine concentration increased from 1.96 µmol/min/mg protein in the absence of substrate to 1.53 µmol/min/mg protein at 100 µM L-arginine. Thereafter, the rate of NADPH consumption remained constant. This result suggests that for NOS I the transfer of electrons from NADPH to O2 in the absence of substrate is faster than the transfer of electrons to catalyze the formation of NO· and L-citrulline from L-arginine. In contrast to NOS I, Obardot 2 generation by purified NOS II was unaffected by 100 µM L-arginine. In fact, it was still possible to detect this free radical even in the presence of 1 mM L-arginine (17). Similar findings have been reported for NOS III (18, 19). These are surprising observations, considering the fact that the Km for NOS II varies between 2.8 to 32 µM, whereas for NOS III, the Km has been reported to be 2.9 µM (27). These data nevertheless point to a substantial difference between the three isozymes of NOS with respect to Obardot 2 secretion. For NOS II, it was found that Obardot 2 was generated by electron leakage from the flavin domain (17), based on the finding that 100 µM NaCN did not significantly inhibit Obardot 2 generation as estimated by spin trapping experiments (17). In contrast, either 100 µM NaCN (19) or 1 mM NaCN (18) blocked Obardot 2 secretion from NOS III. These data suggest that the heme domain of NOS III is the site of Obardot 2 production (18, 19).


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Fig. 1.   Effect of L-arginine on Obardot 2 production by purified NOS I as assessed by spin trapping/EPR spectroscopy. Data are presented as percentages of control levels in the absence of L-arginine. A typical reaction mixture contained DMPO (100 mM), free calcium ion (1 µM), calmodulin (100 units/ml; 23.6 µg/ml), NADPH (124 µM), and NOS (6.8 µg/ml). Each data point represents a specific experiment (n = 3) on the same purified preparation of NOS I.

The initial step in the generation of NO· by NOS is the transport of electrons from NADPH to the oxidized flavin, FAD, resulting in FADH2, after abstraction of a proton from the surrounding milieu. Disproportionation between the flavins leads to FADH·/FMNH·. The electron donation from FMNH· to Fe3+ gives the reduced heme, Fe2+ and FADH·/FMN iff  FAD/FMNH· (28). Binding of O2 in the sixth ligand position would give the hypothetical intermediate [Fe2+-O2] (29). In the absence of L-arginine, O2 accepts an electron from NOS, generating Obardot 2 (15, 17-19). When L-arginine is present, however, there is a binding of the guanidino nitrogen in an ordered position near the heme (30), which allows the oxidation of L-arginine to proceed. Based on a Km of 3.47 µM for the NOS I oxidation of L-arginine and on data presented in Fig. 1, NO· and Obardot 2 are both generated. The rate of each free radical produced, however, cannot accurately be estimated by spin trapping. Fig. 1 indicates that NOS I at L-arginine concentration around the Km is capable of producing Obardot 2 at about 60% of the rate of that generated in the absence of L-arginine. For NOS II, electron transport from FMNH· to Fe3+ appears not to be so tightly coupled, because some leakage from the flavin domain results in the formation of Obardot 2, even in the presence of 1 mM L-arginine (17). For NOS I, the site of Obardot 2 generation has not yet been defined. We suggest, however, that during the oxidation of L-arginine to L-citrulline and NO·, direct competition with O2 results in Obardot 2 formation. At a fixed concentration of O2, the ratio of NO· and Obardot 2 is dependent, therefore, on the concentration of L-arginine.

To explore the mechanism of Obardot 2 generation by NOS I, it is important to determine how electrons are transferred through the enzyme. Thus, we undertook a series of inhibition experiments exploring the effects of two well known inhibitors of NOS, L-NAME and L-NMMA, on Obardot 2 production. Before conducting these experiments, we estimated the capacity of these NOS inhibitors to block L-citrulline formation and, by implication, the generation of NO·. The dose-response curves for L-[14C]citrulline formation from L-[14C]arginine by NOS I in the presence of L-NAME and L-NMMA are shown in Fig. 2. L-NMMA was found to be slightly more potent (EC50 = 5 µM) than L-NAME (EC50 = 10 µM). For NOS III, L-NAME has been reported to be a more effective inhibitor of this isozyme than L-NMMA (31). With these data in mind, we then defined the ability of L-NAME and L-NMMA to prevent Obardot 2 production by NOS I. Fig. 3 shows the effect of increased concentrations of L-NAME on the ability of NOS I to generate Obardot 2. Similar to L-arginine, L-NAME antagonized the spin trapping of this free radical by DMPO in a dose-dependent manner, with an EC50 = 40 µM. In contrast, L-NMMA did not appreciably depress the formation of Obardot 2 by NOS I even at concentrations as high as 10 mM (Fig. 3). Consistent with these findings, the rate of NADPH oxidation by NOS I was inhibited by >70% as the concentration of L-NAME reached 100 µM, whereas the rate of NADPH oxidation remained constant at about 50% of control with increasing concentration of L-NMMA up to 10 mM (Fig. 4). These results suggested that L-NAME, but not L-NMMA, inhibited the formation of Obardot 2 by impeding the electron transport to O2. To confirm this hypothesis, the effect of L-NAME (1 mM) on the xanthine/xanthine oxidase production of Obardot 2, as measured by the SOD-inhibitable reduction of cytochrome c, was assessed. Within experimental error, this rate was unchanged. These data indicate that L-NAME did not scavenge Obardot 2 but rather that L-NAME acted specifically on NOS I, inhibiting generation of this free radical. Because L-NMMA is a potent antagonist of NOS-generated NO·, we explored whether L-NMMA could block the ability of L-arginine to inhibit NOS I production of Obardot 2. These findings are presented in Fig. 5. As expected, L-arginine (100 µM) almost completely inhibited the NOS I formation of Obardot 2 (Fig. 5B), which confirmed earlier studies (15, 32). Surprisingly, L-NMMA, in a dose-dependent manner, reversed the inhibitory properties of L-arginine (Fig. 5, C-E), almost reaching control values, in the absence of L-arginine, at 1 mM (Fig. 5, E and F). Our findings support the theory that L-NAME antagonizes the transfer of electrons to either L-arginine or O2, whereas L-NMMA prevents the oxidation of L-arginine by competing for the same binding site on the enzyme (33). Although NO· production is inhibited by the presence of L-NMMA, NOS I still has the capacity to transfer electrons from NADPH to O2 (Fig. 4 and 5).


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Fig. 2.   Effect of L-NMMA (black-diamond ) and L-NAME (box-dot ) on the generation of L-citrulline (and by implication on NO·) by purified NOS I. Data are presented as percentages of control levels in the absence of L-arginine, as described under "Experimental Procedures." Each data point represents a typical experiment (n = 3) on the same purified preparation of NOS I.


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Fig. 3.   Effect of L-NMMA (black-diamond ) and L-NAME (box-dot ) on Obardot 2 generation by purified NOS I as assessed by spin trapping/EPR spectroscopy. Data are presented as percentages of control levels in the absence of either L-NMMA or L-NAME. Reaction mixtures contained DMPO (100 mM), free calcium ion (1 µM), calmodulin (100 units/ml; 23.6 µg/ml), NADPH (124 µM), and NOS (14.4 µg/ml for L-NMMA; 15.8 µg/ml for L-NAME). Each data point represents a typical experiment (n = 3) on the same purified preparation of NOS I.


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Fig. 4.   Effect of L-NMMA (black-diamond ) and L-NAME (box-dot ) on the rate of NADPH oxidation by purified NOS I, as described under "Experimental Procedures." For L-NAME, NOS (4.53 µg/ml) was added into a quartz cuvette containing free calcium ion (1 µM), calmodulin (100 units/ml; 23.6 µg/ml), and NADPH (124 µM). The control rate of NADPH oxidation by NOS was 1.27 µmol/mg/min. For L-NMMA, NOS (10.55 µg/ml) was added into a quartz cuvette containing free calcium ion (1 µM), calmodulin (100 units/ml), and NADPH (124 µM). The control rate of NADPH oxidation by NOS was 1.64 µmol/mg/min. Each data point for L-NAME and L-NMMA represents a typical experiment (n = 3) on the same purified preparation of NOS I.


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Fig. 5.   Effect of L-NMMA on Obardot 2 generation by purified NOS I in the presence of L-arginine as assessed by spin trapping/EPR spectroscopy. Typical EPR spectra corresponding to DMPO-OOH under a variety of experimental conditions are shown. The mixture in scan A consists of DMPO (100 mM), free calcium ion (1 µM), calmodulin (100 units/ml), NADPH (124 µM), and NOS (14.4 µg/ml). Scans B-E were recorded under identical conditions to scan A except for the addition of L-arginine (100 µM), L-arginine (100 µM) + L-NMMA (10 µM), L-arginine (100 µM) + L-NMMA (100 µM), and L-arginine (100 µM) + L-NMMA (1 mM), respectively. Scan F was recorded under identical conditions to scan A except for the addition of L-NMMA (1 mM). Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G, sweep time was 12.5 G/min, response time was 1 s, and receiver gain was 10 × 104.

Next, experiments were designed to further determine the site on NOS I where Obardot 2 production takes place. Because cytochrome P-450 and NOS I are members of the same superfamily of enzymes (34), we looked to cytochrome P-450 to gain insight as to potential loci for NOS I-derived Obardot 2. There are two sites on cytochrome P-450 and NOS I where Obardot 2 may be generated: the flavins of the reductase and the iron of the heme. For cytochrome P-450, the heme domain is the origin of Obardot 2 formation (35). Would NOS I behave in a similar manner? First, we needed to demonstrate that our NOS I preparation was capable of transferring electrons from the flavin site to the heme domain. For this purpose we investigated the effect of DPI, a compound known to block the electron flow at the flavin locus (36), on NO· and Obardot 2 formation by NOS I. As shown in Fig. 6, DPI is a potent inhibitor of L-citrulline generation by NOS I with an EC50 = 10 µM. Similarly, DPI at 10 µM almost completely inhibited the spin trapping of NOS I-secreted Obardot 2 (Fig. 7B). In contrast, DPI (10 µM) did not inhibit the reaction of DMPO with Obardot 2 generated from FMN/NADPH (Fig. 8D). Even though these data indicate that the electron flow is through the flavin domain, these experiments cannot establish whether Obardot 2 is produced solely at this site or at the heme domain. To further address this query, we investigated the effect of NaCN and two imidazoles (known to inhibit cytochrome P-450 and NOS (37, 38) by blocking the heme site) on the generation of this free radical. First, however, we had to demonstrate that NaCN, imidazole, and 1-phenylimidazole inhibited the metabolism of L-arginine to L-citrulline by NOS I. Fig. 6 depicts the dose-response curves for L-[14C]citrulline formation from L-[14C]arginine by NOS I in the presence of NaCN, imidazole, and 1-phenylimidazole. NaCN was the least potent of these inhibitors with an EC50 = 10 mM, whereas imidazole and 1-phenylimidazole were considerably more potent with values for EC50 = 20 µM and 80 µM, respectively. Once completed, we were confident that if similar inhibitory properties toward Obardot 2 generation were revealed, then the heme was most likely the site of this free radical formation. At 1 mM NaCN, there was a minimal decrease in the peak height of DMPO-OOH (Fig. 7C), whereas at 10 mM NaCN inhibition of Obardot 2 was nearly complete (Fig. 7D). In an independent series of experiments, NaCN at 10 mM was found to inhibit (by 50%) the spin trapping of Obardot 2 from the model Obardot 2 generating system consisting of FMN/NADPH (Fig. 8E). Because NaCN is known to react with DMPO (21), thereby decreasing the effective concentration of DMPO available to react with Obardot 2, the findings in Fig. 8E can only suggest that the heme domain in NOS I was the site of Obardot 2 production. Alternative antagonists for NOS were sought. Recently, imidazoles have been reported to block the heme site of NOS (38). Thus, we investigated the ability of imidazole and 1-phenylimidazole to attenuate Obardot 2 formation by NOS I. As shown in Fig. 7, 1-phenylimidazole (10 mM) and imidazole (10 mM) inhibited the formation of DMPO-OOH by almost 50 (Fig. 7E) and 80% (Fig. 7F), respectively, whereas, unlike NaCN, they had no effect on the spin trapping of this free radical from the Obardot 2-generating system of FMN/NADPH (Fig. 8, B and C).


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Fig. 6.   A dose-response curve measuring the effect of 1-phenylimidazole (box-dot ), imidazole (black-diamond ), DPI (black-square), and NaCN (diamond ) on the generation of L-citrulline (and by implication on NO·) by purified NOS I. Each data point represents a typical experiment (n = 3) on the same purified preparation of NOS I.


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Fig. 7.   Effect of NOS inhibitors on Obardot 2 generation by purified NOS I. Scan A was obtained in the absence of inhibitors. Scans B-F were obtained in the presence of DPI (10 µM), NaCN (1 mM), NaCN (10 mM), 1-phenylimidazole (10 mM), and imidazole (10 mM), respectively. Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G, sweep time was 12.5 G/min, response time was 1 s, and receiver gain was 6.3 × 104.


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Fig. 8.   Effect of NOS inhibitors on Obardot 2 generation by FMN (0.1 mM)/NADPH (1 mM). Scan A was obtained in the absence of inhibitors. Scans B-E were obtained in the presence of imidazole (10 mM), 1-phe nylimidazole (10 mM), DPI (10 µM), and NaCN (10 mM), respectively. Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G, sweep time was 12.5 G/min, response time was 1 s, and receiver gain was 5 × 104.

Data gathered from inhibitory experiments with the antagonists 1-phenylimidazole, imidazole, and NaCN support the heme domain as the site of Obardot 2 production by NOS I, similar to the findings for cytochrome P-450 (35) (Fig. 9). For NOS II, it appears that the reductase domain is not as tightly coupled to the heme domain as seen in NOS I and cytochrome P-450. This weak coupling allows some electron leakage to O2, generating Obardot 2, even though sufficient electron flow to the heme permits a high flux of NO· from the oxidation of L-arginine.


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Fig. 9.   A proposed model that illustrates Obardot 2 generation from NOS I in the absence of L-arginine. Initial electron flow is from NADPH to FAD, resulting in FADH2. After disproportionation with FMN, two semiquinone free radicals, FADH·/FMNH·, allow electron transport to the heme iron, giving Fe2+. In the absence of L-arginine, O2 is the preferred substrate, resulting in Obardot 2 formation. As there still remains one electron on FAD/FMNH·, the iron heme is again reduced, with subsequent transfer to O2. Thus, in the absence of L-arginine, for every mol of NADPH oxidized, there are 2 mol of Obardot 2 generated.

Finally, we investigated the role that BH4 might play in regulating production of Obardot 2 by NOS I in the absence of L-arginine. Tetrahydrobiopterin, at 10 and 100 µM, inhibited Obardot 2 secretion by NOS I by >90%. As shown in Fig. 10, B and C, the EPR spectra are characteristic of DMPO-OH and to a lesser extent of DMPO-OOH. The source of DMPO-OH appears to be BH4. At 1 mM BH4 (Fig. 10D), we were still able to observe some DMPO-OOH, even though DMPO-OH dominated the EPR scan. At 10 mM BH4, DMPO-OH (Fig. 10E) was the only spin-trapped adduct recorded. These data suggest that at low concentrations (1-100 µM), the primary effect of BH4 is to inhibit NOS I production of Obardot 2. However, at higher concentrations, in addition to the inhibition of NOS I-secreted Obardot 2, BH4 generates Obardot 2 through autoxidation. The resultant H2O2, either through metal ion catalysis or direct oxidation, produced DMPO-OH (39, 40).


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Fig. 10.   Effect of BH4 on Obardot 2 generation from NOS I in the absence of L-arginine. Scan A was obtained in the absence of BH4. Scans B-E were obtained in the presence of BH4 with increasing concentrations from 10 µM, 100 µM, 1 mM, and 10 mM, respectively. Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G, sweep time was 12.5 G/min, response time was 1 s, and receiver gain was 8 × 104 for scans A-D and 2 × 104 for scan E.

Several important observations are readily apparent from our studies. First, we were able to spin trap Obardot 2 even in the presence of saturating levels of L-arginine. This is remarkable, considering that Obardot 2 and NO· combine at near diffusion controlled rates, producing ONOO- (41, 42), whereas Obardot 2 reacts with DMPO at only 12 M-1 sec-1 (43). The ability to spin trap Obardot 2 under these experimental conditions may result from the fact that Obardot 2 and NO· are generated sequentially at the heme iron site. After Obardot 2 is produced, NOS I must cycle twice before NO· is secreted. This is a sufficiently long time to allow Obardot 2 to diffuse from the enzyme to the surrounding milieu where this free radical can react with the high concentration of DMPO included in the reaction mixture to give the observed spin-trapped adduct, DMPO-OOH. Under physiological conditions at which NOS I generates both Obardot 2 and NO·, SOD regulates cellular flux of Obardot 2, thereby preventing, or at least drastically limiting, ONOO- generation (44).

Second, despite the above findings, there are pathologic states, such as ischemia/reperfusion injury, that might promote the formation of ONOO- (45). Therefore, it is surprising that the fate of this peroxide still remains in doubt (44, 46). At the low L-arginine concentrations at which the steady-state flux of Obardot 2 would exceed NO·, in addition to ONOO-, H2O2 would arise from the dismutation of Obardot 2. Subsequent formation of HO·, either through the metal ion catalyzed Haber-Weiss reaction (47) or from decomposition of ONOO- (48-52), at sensitive cellular sites may result in cytotoxicity. Thus, under different experimental conditions, a variety of oxidants can be produced with an impact on cell function that is significant or in some cases remains to be defined.

Third, it should be noted that the reductase domain of NOS is, like cytochrome P-450 reductase, susceptible to uncoupling, shunting electrons away from the heme toward a xenobiotic. One recent example illustrates this point (53). It was discovered that o-quinones can promote Obardot 2 production by uncoupling NOS I at its reductase domain, yielding a semiquinone free radical. In the presence of O2, Obardot 2 was generated along with the parent o-quinone.

In agreement with the result obtained with NOS III (18-20), BH4, in a dose-dependent manner, was found to inhibit the generation of Obardot 2 by NOS I. Interestingly, unlike NOS III, L-arginine, independent of added BH4, inhibited Obardot 2 production by NOS I, which was reversed by L-NMMA (Fig. 5). This suggests that in the case of NOS I, BH4 is not the sole pathway for controlling Obardot 2 formation but that the competition between L-arginine and L-NMMA is an important factor in determining the generation of Obardot 2.

How does BH4 regulate the ratio of NO· and Obardot 2? The answer, of course, is not simple and depends, to a large extent, on the isozyme of NOS. For instance, one would predict, based on the findings in Ref. 18, that for an activation of a NOS III-containing cell in which L-arginine and BH4 are at normal levels, high fluxes of Obardot 2 and NO· would result. The secretion of NO· to the surrounding milieu would be tied to the ability of SOD to scavenge Obardot 2. In contrast, activation of a NOS I-containing cell, under the conditions described above, would result primarily in the formation of NO·. Thus, under cellular conditions that would impact the availability of either L-arginine or BH4, variable amounts of NO· and Obardot 2 would be generated. Evidence in support of this thesis comes from studies with NOS I-containing transfected kidney 293 cells (16). Here, when these cells were placed in a complete medium, NO·, but not Obardot 2, was spin trapped (16). However, when the cells were cultured in L-arginine-depleted medium for 24 h, it was possible to spin trap Obardot 2 at the expense of NO· (16).

Although the implications of our findings have yet to be fully realized, two recent publications may shed some light on the importance of our observations. First, during anoxia/reoxygenation of cardiomyocytes, generation of Obardot 2 was found to be markedly enhanced when L-NMMA was included (54), which supports the data presented in Fig. 5. Second, elevated levels of MnSOD were an essential element of viable NOS I containing neurons exposed to N-methyl-D-aspartate (NMDA) (55). Even though the source of Obardot 2 in these studies (54, 55) was not identified, NOS must certainly be considered a possible contributor to the origin of this free radical.

    ACKNOWLEDGEMENT

We acknowledge the technical assistance of Guan-Liang Cao.

    FOOTNOTES

* This work was supported in part by Grants AG-14829 and CA-69538 from the National Institutes of Health.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.

Dagger To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 West Lombard St., Baltimore, MD 21201. Tel.: 410-706-0514; Fax: 410-706-8184; E-mail: grosen{at}umaryland.edu.

    ABBREVIATIONS

The abbreviations used are: NO·, nitric oxide; Obardot 2, superoxide; H2O2, hydrogen peroxide; NOS I, neuronal nitric-oxide synthase; NOS II, inducible nitric-oxide synthase; NOS III, endothelial nitric-oxide synthase; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; EPR, electron paramagnetic resonance; L-NAME, NG-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl L-arginine; DMPO-OOH, 2,2,-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl; DPI, diphenyliodonium chloride; ONOO-, peroxynitrite; SOD, superoxide dismutase; BH4, tetrahydrobiopterin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
  1. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 9265-9269[Abstract]
  2. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987) Nature 327, 524-526[CrossRef][Medline] [Order article via Infotrieve]
  3. Furchgott, R. F. (1988) in Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium (Vanhoutte, P. M., ed), pp. 401-414, Raven Press, New York
  4. Miki, N., Kawabe, Y., and Kuriyama, K. (1977) Biochem. Biophys. Res. Commun. 75, 851-856[Medline] [Order article via Infotrieve]
  5. Deguchi, T., and Yoshioka, M. (1982) J. Biol. Chem. 257, 10147-10151[Abstract/Free Full Text]
  6. Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685[Abstract]
  7. Bredt, D. S., Hwang, P. M., and Snyder, S. H. (1990) Nature 347, 768-770[CrossRef][Medline] [Order article via Infotrieve]
  8. Dawson, T. M., and Snyder, S. H. (1994) J. Neurosci. 14, 5147-5159[Abstract]
  9. Jaffrey, S. R., and Snyder, S. H. (1995) Annu. Cell. Dev. Biol. 11, 417-440[CrossRef][Medline] [Order article via Infotrieve]
  10. Moore, P. K., Wallace, P., Garren, Z., Hart, S. L., and Babbedge, R. C. (1993) Br. J. Pharmacol. 110, 219-224[Abstract]
  11. Garvey, E. P., Oplinger, J. A., Furfine, E. S., Kiff, R. J., Laszlo, F., Whittle, B. J. R., and Knowles, R. G. (1997) J. Biol. Chem. 272, 4959-4963[Abstract/Free Full Text]
  12. Marletta, M. A. (1994) J. Med. Chem. 37, 1899-1907[Medline] [Order article via Infotrieve]
  13. Marletta, M. A. (1993) J. Biol. Chem. 268, 12231-12234[Free Full Text]
  14. Masters, B. S. S., McMillan, K., Sheta, E. A., Nishimura, J. S., Roman, L. J., and Martasek, P. (1996) FASEB J. 10, 552-558[Abstract/Free Full Text]
  15. Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176[Abstract/Free Full Text]
  16. Xia, Y., Dawson, V. L., Dawson, T. M., Snyder, S. H., and Zweier, J. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6770-6774[Abstract/Free Full Text]
  17. Xia, Y., Roman, L. J., Masters, B. S. S., and Zweier, J. L. (1998) J. Biol. Chem. 273, 22635-22639[Abstract/Free Full Text]
  18. Vásquez-Vivar, J., Kalyanaraman, B., Martásek, P., Hogg, N., Masters, B. S. 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]
  19. Xia, Y., Tsai, A.-L., Berka, V., and Zweier, J. L. (1998) J. Biol. Chem. 273, 25804-25808[Abstract/Free Full Text]
  20. Wever, R. M. F., van Dam, T., van Rijn, H. J. M., de Groot, F., and Rabelink, T. J. (1959) Biochem. Biophys. Res. Commun. 237, 340-344[CrossRef]
  21. Bonnett, R., Brown, R. F. C., Clark, V. M., Sutherland, I. O., and Todd, A. (1959) J. Chem. Soc. 2094-2102
  22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  23. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder, S. H. (1991) Nature 351, 714-718[CrossRef][Medline] [Order article via Infotrieve]
  24. Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505[Medline] [Order article via Infotrieve]
  25. Pou, S., Cohen, M. S., Britigan, B. E., and Rosen, G. M. (1989) J. Biol. Chem. 264, 12299-12302[Abstract/Free Full Text]
  26. Kuthan, H., Ullrich, V., and Estabrook, R. W. (1982) Biochem. J. 203, 551-558[Medline] [Order article via Infotrieve]
  27. Nathan, C. (1992) FASEB J. 6, 3051-3064[Abstract/Free Full Text]
  28. Galli, C., MacArthur, R., Abu-Soud, H. M., Clark, P., Stuehr, D. J., and Brudvig, G. W. (1996) Biochemistry 35, 2804-2810[CrossRef][Medline] [Order article via Infotrieve]
  29. Abu-Soud, H. M., Gachhui, R., Raushel, F. M., and Stuehr, D. J. (1997) J. Biol. Chem. 272, 17349-17353[Abstract/Free Full Text]
  30. Tierney, D. L., Martasek, P., Doan, P. E., Masters, B. S. S., and Hoffman, B. M. (1998) J. Am. Chem. Soc 120, 2983-2984[CrossRef]
  31. Gross, S. S., Jaffe, E. A., Levi, R., and Kilbourn, R. G. (1991) Biochem. Biophys. Res. Commun. 178, 823-829[Medline] [Order article via Infotrieve]
  32. Heinzel, B., John, M., Klatt, P., Bohme, E., and Mayer, B. (1992) Biochem. J. 281, 627-630[Medline] [Order article via Infotrieve]
  33. Abu-Soud, H. M., Feldman, P. L., Clark, P., and Stuehr, D. J. (1994) J. Biol. Chem. 269, 32318-32326[Abstract/Free Full Text]
  34. Masters, B. S. S., McMillan, K., Sheta, E. A., Nishimura, J. S., Roman, L. J., and Martasek, P. (1996) FASEB J. 10, 552-558[Abstract/Free Full Text]
  35. White, R. E. (1994) in Cytochrome P450 Biochemistry, Biophysics and Molecular Biology (Lechner, M. C., ed), pp. 333-340, John Libbey Eurotext, Paris
  36. Stuehr, D. J., Fasehun, O. A., Kwon, N. S., Gross, S. S., Gonzalez, J. A., Levi, R., and Nathan, C. F. (1991) FASEB J. 5, 98-103[Abstract/Free Full Text]
  37. Banci, L., Bertini, I., Marconi, S., Pieratteli, R., and Sligar, S. G. (1994) J. Am. Chem. Soc. 116, 4866-4873
  38. Chabin, R. M., McCauley, E., Calaycay, J. R., Kelly, T. M., MacNaul, K. L., Wolfe, G. C., Hutchinson, N. I., Madhusudanaraju, S., Schmidt, J. A., Kozarich, J. W., and Wong, K. K. (1996) Biochemistry 35, 9567-9575[CrossRef][Medline] [Order article via Infotrieve]
  39. Makino, K., Hagi, A., Ide, H., Murakami, A., and Nishi, M. (1992) Can. J. Chem. 70, 2818-2827
  40. Hanna, P. M., Chamulitrat, W., and Mason, R. P. (1992) Arch. Biochem. Biophys. 296, 640-644[Medline] [Order article via Infotrieve]
  41. Huie, R. E., and Padmaja, S. (1993) Free Radical Res. Commun. 18, 195-199[Medline] [Order article via Infotrieve]
  42. Goldstein, S., and Czapski, G. (1995) Free Radical Biol. Med. 19, 505-510[CrossRef][Medline] [Order article via Infotrieve]
  43. Finkelstein, E., Rosen, G. M., and Rauckman, E. J. (1980) J. Am. Chem. Soc. 102, 4994-4999
  44. Fukuto, J. M., and Ignarro, L. J. (1997) Acc. Chem. Res. 30, 149-152[CrossRef]
  45. Grisham, M. B., Granger, D. N., and Lefer, D. J. (1998) Free Radical Biol. Med. 25, 404-433[CrossRef][Medline] [Order article via Infotrieve]
  46. Wink, D. A., and Mitchell, J. B. (1998) Free Radical Biol. Med. 25, 434-456[CrossRef][Medline] [Order article via Infotrieve]
  47. Haber, F., and Weiss, J. (1934) Proc. R. Soc. Lond. A 147, 332-351
  48. Yang, G., Candy, T. E. G., Boaro, M., Wilkin, H. E., Jones, P., Nazhat, N. B., Saadalla-Nazhat, R. A., and Blake, D. R. (1992) Free Radical Biol. Med. 12, 327-330[CrossRef][Medline] [Order article via Infotrieve]
  49. van der Vliet, A., O'Neill, C. H., Halliwell, B., Cross, C. E., and Kaur, H. (1994) FEBS Lett. 339, 89-92[CrossRef][Medline] [Order article via Infotrieve]
  50. Pou, S., Nguyen, S. Y., Gladwell, T., and Rosen, G. M. (1995) Biochim. Biophys. Acta 1244, 62-68[Medline] [Order article via Infotrieve]
  51. Kaur, H., Whiteman, M., and Halliwell, B. (1997) Free Radical Res. 26, 71-82[Medline] [Order article via Infotrieve]
  52. Richeson, C. E., Mulder, P., Bowry, V. W., and Ingold, K. U. (1998) J. Am. Chem. Soc. 120, 7211-7219[CrossRef]
  53. Miller, R. T., Martásek, P., Roman, L. J., Nishimura, J. S., and Masters, B. S. S. (1997) Biochemistry 36, 15277-15284[CrossRef][Medline] [Order article via Infotrieve]
  54. Vanden Hoek, T. L., Becker, L. B., Shao, Z., Li, C., and Schumacker, P. T. (1998) J. Biol. Chem. 273, 18092-18098[Abstract/Free Full Text]
  55. Gonzalez-Zulueta, M., Ensz, L. M., Mukhina, G., Lebovitz, R. M., Zwacka, R. M., Engelhardt, J. F., Oberley, L. W., Dawson, V. L., and Dawson, T. M. (1998) J. Neurosci. 18, 2040-2055[Abstract/Free Full Text]


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