Superoxide Generation from Endothelial Nitric-oxide Synthase

It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (O⨪2) under conditions ofl-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O⨪2. Moreover, the mechanism and control of this process are not fully understood. Therefore, we performed electron paramagnetic resonance spin-trapping experiments to directly measure and characterize the O⨪2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O⨪2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O⨪2formation required Ca2+/calmodulin and was blocked by the specific NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) but not its non-inhibitory enantiomerd-NAME. A parallel process of Ca2+/calmodulin-dependent NADPH oxidation was observed which was also inhibited by l-NAME but notd-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O⨪2 generation. BH4 exerted dose-dependent inhibition of the O⨪2 signals generated by eNOS. Conversely, in the absence of BH4 l-arginine did not decrease this O⨪2 generation. Thus, eNOS can also catalyze O⨪2formation, and this appears to occur primarily at the heme center of its oxygenase domain. O⨪2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than l-arginine.

nNOS and eNOS are constitutively present in cells, iNOS expression requires the stimulation of microbial endotoxins or cytokines. Activation of nNOS and eNOS requires Ca 2ϩ /calmodulin, hence NO production from these two isoforms is initiated and modulated by elevated intracellular free Ca 2ϩ . Because iNOS has a tightly bound calmodulin and is fully active under basal cytosolic Ca 2ϩ concentrations, NO formation from iNOS appears to depend primarily on the levels of enzyme transcription (4). Three NOS isoforms have considerable similarity in their structure and catalytic function. They share 50% homology in their amino acid sequences and structurally resemble NADPH cytochrome P-450 reductase. All NOSs use L-arginine, oxygen, and NADPH as substrates to synthesize NO as well as the co-product L-citrulline. Tetrahydrobiopterin (BH 4 ), calmodulin, FAD, and FMN are the requisite cofactors for this catalytic process (5).
Similar to NO synthesis, O 2 . generation from nNOS is dependent on the presence of Ca 2ϩ /calmodulin. In L-arginine-depleted cells, activated nNOS generates both O 2 . and NO leading to peroxynitrite (ONOO Ϫ )-mediated cell injury (8). Recently, iNOS was also found to produce O 2 . as well as ONOO Ϫ under L-arginine depletion, and it was shown that these oxidants can contribute to the antibacterial activity of macrophages (9). In light of the structural similarity among NOSs, it would be expected that eNOS might also produce O 2 . just as the other two isoforms. However, there has been controversy regarding whether or not eNOS can also synthesize O 2 . (10 -12). Previous functional studies suggested that eNOS might generate O 2 . in vasculature under pathological conditions (10,11). However subsequently it was reported that purified eNOS exhibits only minor uncoupling of NADPH oxidation in the absence of Larginine or BH 4 (12).
eNOS Purification-Recombinant human wide-type eNOS was prepared using a baculovirus expression system as described previously (14,15). In brief, eNOS-transfected cells were harvested and sonicated in buffer A (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 20 mM CHAPS, 10% glycerol, 1 M antipain, 1 M leupeptin, 1 M pepstatin, and 1 mM phenylmethylsulfonyl fluoride). After centrifugation (100,000 ϫ g for 60 min), the supernatant was applied to a 2Ј,5Ј-ADP-Sepharose 4B column (1.5 ϫ 2 cm) pre-equilibrated in buffer A. The column was washed with 25 ml of buffer A containing 0.5 M NaCl and followed by 10 ml of buffer A. Then the protein was eluted with 30 mM adenosine 2Ј,3Ј-monophosphate in buffer B (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 5 mM CHAPS, and 10% glycerol). The eluate was concentrated using a Centriprep 100 (Amicon) and then applied to a 10-DG column (Bio-Rad). The eNOS-containing fractions were pooled, concentrated, and stored in the buffer with 10% glycerol in liquid nitrogen. Protein content was assayed with Bradford reagent (Bio-Rad) using bovine serum albumin as standard (16). The purity of eNOS was determined by SDS-polyacrylamide gel electrophoresis (SDS/PAGE) and visualized with Coomassie Blue staining. eNOS activity was approximately 130 nmol/min/mg at 23°C assayed by monitoring the conversion of L-[ 14 C]arginine to L-[ 14 C]citrulline as described below.
EPR Spectroscopy and Spin Trapping-Spin-trapping measurements of oxygen free radicals were performed in 50 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM NADPH, 0.5 mM Ca 2ϩ , 10 g/ml calmodulin, 15 g/ml purified eNOS, and 50 mM spin trap DMPO. EPR spectra were recorded in a quartz flat cell at room temperature (23°C) with a Bruker ER 300 spectrometer operating at X-band with a TM 110 cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20 milliwatts, and microwave frequency of 9.785 GHz as described (8,19). The microwave frequency and magnetic field were precisely measured using an EIP 575 microwave frequency counter and Bruker ER 035 NMR gauss meter.
NADPH Consumption by eNOS-NADPH oxidation was followed spectrophotometrically at 340 nm (13). The reaction systems were the same as described in EPR measurements, and the experiments were run at room temperature. The rate of NADPH oxidation was calculated using a molar extinction coefficient of 6.22 mM Ϫ1 cm Ϫ1 .

RESULTS
Recombinant human eNOS was expressed in a baculovirus system and isolated using affinity chromatography. The purity and catalytic activity of the preparations were assayed. As shown in Fig. 1A, purified protein preparations exhibited one prominent major band (Ͼ90% pure) on SDS-PAGE with a molecular mass of 135 kDa, which is in accordance with the molecular mass for native eNOS as previously reported (4,5). By monitoring the conversion of L-arginine to L-citrulline, strong NOS activity was measured from this recombinant protein (Fig. 1B). The catalytic activity was dependent on addition of Ca 2ϩ and could be blocked by the NOS inhibitor, L-NAME (1 mM), confirming that it was derived from eNOS.
We then performed EPR spin-trapping experiments to determine whether eNOS generates O 2 . using the well characterized spin trap DMPO. In the control experiments without the enzyme, no signals were detected from the reaction mixtures containing DMPO and NADPH, as well as Ca 2ϩ /calmodulin ( Fig. 2A, Control). However, after adding purified eNOS (15 g/ml), strong EPR signals were seen ( Fig. 2A,  is delineated in Fig. 2B. As shown, the signals were detected immediately after the beginning of the reaction and rapidly increased over the first 5ϳ10 min with continued gradual increases over the next 20 min. In the presence of SOD, the process of O 2 . generation was totally quenched. To further demonstrate that the observed O 2 . signals were generated by eNOS, the enzyme was treated with the specific NOS blocker L-NAME. In the presence of 1 mM L-NAME, the O 2 . -derived signals were decreased by more than 90% (Fig. 3, L-NAME), whereas the non-inhibitory enantiomer D-NAME had no effect on the O 2 . signals (spectrum similar to Fig. 3, eNOS, not shown). These  generation from eNOS was decreased by more than 80% (Fig. 3,  NaCN). Another heme ligand, imidazole (1 mM) also blocked O 2 .
generation. These data suggest that eNOS-catalyzed O 2 . generation occurs primarily at the heme center of its oxygenase domain.
In NOS-catalyzed reactions, the co-substrate NADPH is oxidized and serves as an electron donor for NO or O 2 . synthesis (1)(2)(3)(4)(5). Therefore, synchronous NADPH consumption always takes place accompanying O 2 . generation. Indeed, marked NADPH oxidation was seen in the reaction mixtures containing eNOS in the absence of BH 4 and L-arginine (Fig. 4). Consistent with the O 2 . generation measured in the EPR studies, eNOS-mediated NADPH oxidation also depended on the presence of Ca 2ϩ /calmodulin. L-NAME but not D-NAME largely prevented this NADPH oxidation, reconfirming that NADPH oxidation was catalyzed by eNOS. Together, the findings that eNOS consumed NADPH in the absence of the NO-generating substrate L-arginine provided another line of evidence  The composition of the reaction mixture was the same as described in the legend to Fig. 2. eNOS, control spectrum showing O 2 . generation by eNOS; L-NAME, in the presence of 1 mM L-NAME; Ca 2ϩ /CaM free, in the absence of Ca 2ϩ /calmodulin; NaCN, in the presence of 100 M NaCN. EPR spectra were recorded in presence of 50 mM DMPO as described in the legend to Fig. 2, and representative spectra were shown from triplicate measurements. signals and catalase had no effect reconfirmed that O 2 . was the primary oxygen free radical generated. This O 2 . formation could be blocked by the NOS inhibitor L-NAME but not by its noninhibitory enantiomer D-NAME, further proving that O 2 . was synthesized from eNOS. O 2 . formation was also demonstrated by the fact that eNOS can cause marked NADPH oxidation in the absence of BH 4 and L-arginine. These findings are in disagreement with the results reported by List et al. (12). In that study, List et al. (12) reported that eNOS did not catalyze appreciable NADPH oxidation in the absence of L-arginine or BH 4 , and based on this they presumed eNOS would not produce Although derived from distinct genes and chromosomes, the three NOS isoforms share similarity in their structure and catalytic mechanisms (4,5). They are all bidomain enzymes consisting of a C-terminal reductase and N-terminal oxygenase domain. The reductase domain contains NADPH, FAD, and FMN binding sites and exhibits 58% homology to NADPH cytochrome P450 reductase (3,22). Heme, BH 4 , and L-arginine bind at the oxygenase domain. The catalytic mechanisms of NOSs involve a flavin-mediated electron transport from Cterminal-bound NADPH to the N-terminal heme center where oxygen is reduced and incorporated into the guanidino group of L-arginine giving rise to NO and L-citrulline. Calmodulin binds to a consensus sequence in the NOS enzymes and serves to position the two domains allowing the electron transfer from FMN to heme (5,23).
generation from eNOS is not similarly affected by L-arginine. In the absence of BH 4 , O 2 . production from eNOS was essentially unchanged even in the presence of high levels of L-argi-FIG. 4. NADPH consumption by eNOS. NADPH oxidation was monitored spectrophotometrically at 340 nm in the reactions containing 50 mM Tris-HCl, pH 7.4, 50 M NADPH, 0.5 mM Ca 2ϩ , 7.5 g/ml eNOS, and in the presence and absence of 10 g/ml calmodulin. As shown, eNOS caused marked NADPH oxidation in the absence of BH 4 and L-arginine (filled circles). This required Ca 2ϩ /calmodulin (unfilled circles, Ca 2ϩ /calmodulin-free) and could be largely blocked by 1 mM L-NAME (unfilled triangles) but not by the same amount of D-NAME (filled triangles). Data shown are the means of the results from three experiments. . /NO generation from eNOS is of particular interest in understanding the mechanism of vascular endothelial dysfunction. Impaired endothelial function, represented as declined NO production and elevated oxidant accumulation, plays a fundamental role in the pathogenesis of a number of cardiovascular diseases including hypercholesterolemia, atherosclerosis, hypertension, and ischemia/reperfusion injury. Despite extensive study, it remains poorly understood how this NO/oxidant imbalance takes place. Our current findings suggest that BH 4 may play an important role. BH 4 is unstable at physiological pH and prone to decompose in oxygenated solutions (25). Oxidants from other enzymatic pathways could also serve to deplete BH 4  levels may provide an important therapeutic approach to those diseases associated with endothelial dysfunction.