Xanthine (10 pM)
2.15 ± 0.10
1.87 ± 0.09
0.34 ± 0.03
NADH (1 mM)
0.7 ± 0.05
0.3 ± 0.03
0.11 ± 0.01
DBA (0.1 mM)
1.96 ± 0.10
0.76 ± 0.05
0.45 ± 0.04
occurring during ischemia, NOS does not function to synthesize NO, this NOS-independent NO generation could be of particular importance. Indeed, it has been shown that the acidosis occurring during ischemia results in reversible denaturation of NOS, which progresses to irreversible denaturation and enzyme degradation .
Overall, it is clear that XO-mediated NO generation can potentially be an important source of NO under ischemic conditions in biological tissues that contain substantial levels of the enzyme along with nitrite and reducing substrates. In tissues such as the liver and gastrointestinal tract, which contain high levels of the enzyme, this could be even more pronounced than for the example of the heart considered above . Beyond the obligatory need for the enzyme, the levels of tissue nitrite and enzyme-reducing substrates have a critical role in controlling this process. Nitrite is required, and overall it is the most limiting substrate, because its Km is ~ 2.5 mM, whereas typical tissue levels of nitrite are at least 2 orders of magnitude below this value. A number of factors that increase tissue nitrite levels, such as prior activation of constitutive or inducible NOS in inflammatory conditions, dietary sources, pharmacological sources, or bacterial sources, could all modulate this pathway of NO generation [19,46-52]. This pathway also requires a reducing substrate, such as NADH or xanthine. Xanthine was the most effective substrate, triggering NO generation under anaerobic conditions with a Vmax 4-fold higher than that of NADH. Although only low xanthine concentrations are required, because its Km value is about 1.5 mM, high levels of xanthine, above 20 mM, resulted in prominent substrate-mediated inhibition. If particularly high levels of xanthine accumulate, this pathway would be inhibited, and perhaps this may serve as a regulatory role to prevent overproduction of NO. Thus, XO can be an important source of NOS-independent NO generation. Under anaerobic conditions, XO reduces nitrite to NO at the molybdenum site of the enzyme with xanthine, NADH, or aldehyde substrates serving to provide the requisite reducing equivalents. The substrate-dependent rate relationship for anaerobic nitrite reduction by XO was determined, and it was demonstrated that under conditions of tissue ischemia, the rate of NO generation is greatly increased above the rate of nitrite disproportionation. This NO production from the enzyme could serve as an alternative source of NO under ischemic conditions in which NO production from NOS is impaired. NO derived from nitrite would accumulate during ischemia. Initially, it could serve to provide protection via compensatory vasodilation, whereas upon reperfusion it would react with superoxide, forming peroxynitrite, which can result in protein nitration and cellular injury [53,54].
EFFECT OF OXYGEN ON XO-MEDIATED NITRIC OXIDE GENERATION FROM NITRITE 
It is clear that XO-mediated nitrite and nitrate reduction occurs and can be an important source of NO, particularly under conditions of limited tissue perfusion and resulting acidosis. However, questions remained regarding whether XO-mediated NO generation also occurs in the presence of oxygen. In mammalian organs under normoxic conditions, O2 concentration ranges from 10 to 0.5%, with values of ~14% in arterial blood and ~5% in the myocardium. During mild hypoxia, myocardial O2 levels drop to ~1-3% or lower . Therefore, combined studies using EPR, chemiluminescence NO analyzer, and NO electrode technique were performed to measure the magnitude and kinetics of XO-mediated NO formation under different oxygen tensions. NO chemiluminescence detection from the reaction mixtures was done in a glass-purging vessel equipped with pressure-monitoring device, which allowed the maintenance of atmospheric pressure inside the purging vessel. Because purging of the released NO was performed using gas mixtures containing variable concentrations of oxygen, studies were performed to estimate the applicability of chemiluminescence measurements in the presence of oxygen. Compared with measurements performed with argon (100%), the efficiency of NO measurement with air, 10%, 5%, or 2% oxygen/nitrogen, was 91.9%, 92.5%, 96.3%, or 98.6%, respectively. Chemiluminescence linearly increased with NO generation when purging with any of these gas mixtures. Alternatively, the reaction solution (or heart tissue) for nitrite reduction was placed in purging vessel 1, and the NO released was purged out by flow of inert gas (argon) or oxygen mixtures of known concentrations to purging vessel 2 (Fig. 4). Purging vessel 2 (trap vessel) was filled with aqueous solutions containing the ferrous iron complex of MGD (N-methyl-D-glucamine dithiocarbamate), Fe-MGD, which forms the stable, water-soluble mononitrosyl adduct (MGD)2-Fe-NO, that exhibits a characteristic triplet EPR spectrum at g = 2.04 and aN = 12.8. This setup was designed to isolate the reaction solution in the purging vessel from the spin trap and thus avoid any possible perturbation caused by the reaction of (MGD)2-Fe with nitrite or with the enzyme [56,57]. Samples from purging vessel 2 were aliquoted at desired times for EPR quantitative measurements.
All three typical reducing substrates of XO triggered NO generation from XO-mediated nitrite reduction; however, their kinetics are quite different in the presence of molybdenum-site-binding substrates xanthine or DBA, compared with that in the presence of the FAD-site-binding substrate NADH. With xanthine or DBA as reducing substrates that donate electrons to XO at the molybdenum site of enzyme, the rate of NO production followed typical Michaelis-Menten kinetics, and kinetic studies show that oxygen acts as a strong competitive inhibitor of nitrite reduction.
Under aerobic conditions, with xanthine or DBA as reducing substrates, XO-mediated NO production is less than 10% of NO production under anaerobic conditions . With the FAD site binding reducing substrate, NADH, as electron donor, XO-mediated NO production is maintained at more than 70% of the anaerobic levels, and the XO-catalyzed NO generation rate only changes from ~ 0.30 nmol-mg-1s-1 under anaerobic conditions to ~ 0.22 nmol-mg-1s-1 under aerobic conditions in the presence of the same enzyme and substrate concentrations. With NADH, under aerobic conditions, XO-mediated nitrite reduction did not follow Michaelis-Menten kinetics. NADH serves as electron donor to XO at the FAD site, the same site as that for oxygen binding, whereas nitrite reduction takes place at the molybdenum site of the enzyme . With NADH as reducing substrate, the possible XO-mediated NO generation may occur through two possible processes as shown on Scheme 1.
In Process I, XO is in the reduced state. With FAD site free, both oxygen and nitrite can accept electrons from reduced XO. Thus, under aerobic conditions, oxygen is a strong competitive inhibitor to XO-mediated nitrite reduction in Process I. NO generation through Process I would decrease greatly in the presence of oxygen. However, in Process II, the FAD site is occupied by the binding of NADH, thus oxygen reduction is totally blocked; meanwhile, at the molybdenum site, XO-mediated nitrite reduction is unaffected. Thus, the rate of XO-mediated nitrite reduction would be similar in the presence or absence of oxygen.
In Process II, under aerobic conditions, less than 30% of the nitrite reductase activity of XO is inhibited, which suggests that most nitrite reduction happens while the FAD site is occupied with NADH. NADH is necessary for many biochemical reactions within the body and is found in every living cell. Brain cells contain about 50 ^g of NADH per gram of tissue, and heart cells contain 90 ^g of NADH per gram of tissue. With molybdenum-site binding electron donors xanthine or DBA, nitrite reduction is greatly inhibited by the presence of oxygen, whereas with NADH, XO-mediated NO generation remains at more than 70% of anaerobic levels. The relatively high concentration of NADH in biological systems and its inhibitive effects on the binding of oxygen strongly suggest that NADH would be the major electron donor for XO-catalyzed NO production under aerobic conditions.
Interestingly, DPI, the inhibitor of FAD site-related function, greatly increased NO generation under aerobic conditions with xanthine or DBA used as reducing substrate. It is known that oxypurinol blocks the binding of xanthine, DBA, and nitrite, whereas DPI inhibits the reduction of XO by NADH. With xanthine or DBA as reducing substrates, the presence of DPI inhibits XO-mediated oxygen reduction at the FAD site and thus increases the capability of the enzyme for nitrite reduction at the molybdenum site. Both the reduction of nitrite and the oxidation of xanthine and DBA take place on the molybdenum site of XO. The potential effects of DPI in stimulating NO generation from XO should be taken into account when DPI is used in biological systems, especially when high concentrations of nitrite are present.
In contrast to the superoxide generation, where maximum superoxide production occurs at alkali conditions (pH 8-9), the aerobic XO-mediated NO generation rates increase more than 10 times when pH values fall from 8.0 to 6.0, and further increase about 3-fold as pH values decrease from 6.0 to 5.0. With lower pH, a more rapid increase of XO-mediated NO generation rate was observed under aerobic conditions than under anaerobic conditions. This would be expected, because under aerobic conditions, the acidosis would significantly increase XO-mediated nitrite reduction and simultaneously inhibit the competitive reaction of oxygen reduction, thus facilitating NO generation under aerobic conditions. The simultaneous production of NO and superoxide can form the potent oxidant peroxynitrite. In the setting of inflammatory disease or pharmacological treatment with organic nitrates or NO-donating compounds, nitrite concentrations can rise by more than an order of magnitude [19,46-52]. Without the protection of antioxidants or antioxidant enzymes, accumulated nitrite can become an important source of peroxynitrite production that can damage cells or tissues. Superoxide dismutase in biological systems is an extremely potent antioxidant enzyme that is responsible for the elimination of cytotoxic active oxygen by catalyzing the dismutation of the superoxide radical to oxygen and hydrogen peroxide. Because NO is readily inactivated by superoxide, the bioactivity of NO is dependent upon the local activity of SOD . Also, there are numerous peroxynitrite scavengers, such as uric acid and NADH, in biological systems [42,59]. These results suggest that under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in mammalian cells and tissues. During ischemia, the myocardial NADH/NAD+ concentration ratio can increase more than 10-fold , xanthine levels can rise to the level of 10-100 ^M, with nitrite levels of about 10 ^M [19,20]; the low oxygen pressure and acidosis greatly facilitate XO-mediated NO generation and limit superoxide production. The magnitude of XO-mediated NO generation can approach that of the maximal NO production from NOS . Even with mild to moderate levels of hypoxia, as can occur with subtotal coronary lesions or regional ischemia in the presence of collateral flow, this process would be stimulated. This could allow NO to accumulate and exert a vasodilator role during ischemia. Upon reperfusion, the accumulated NO would react with XO-derived superoxide, giving a burst of peroxynitrite production that can mediate protein nitration and cellular injury . Thus, provided that the environmental conditions are appropriate, it may be possible that XOR acts as a salvage pathway to maintain levels of NO in situations where conventional constitutive NOS activity may be compromised. Such situations would include inflammatory cardiovascular conditions (atherosclerosis) with associated endothelial dysfunction and particularly myocardial infarction [1-7]. Indeed, XOR activity is upregulated during hypoxia [37,61-63], with increasing acidosis , and with atherosclerosis. In patients with coronary artery disease, endothelium-bound XOR activity is increased twofold .
Thus, XO-mediated NO generation occurs under aerobic conditions as well as under anaerobic conditions. With substrates such as xanthine or DBA that bind at the molybdenum site of the enzyme, oxygen serves as a competitive inhibitor of nitrite reduction, whereas with NADH, which binds at the FAD site, oxygen exerts only a modest inhibition of nitrite reduction. This process of aerobic XO-mediated NO generation is modulated by oxygen tension, pH, nitrite levels, and reducing substrate concentrations.
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