Abstract
Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.
Highlights
Recent studies have shown that nitrite is an important storage form and source of NO in biological systems
NO generation from nitrite mainly occurs in tissues not in the blood, with xanthine oxidase (XO) and aldehyde oxidase (AO) playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations
NO production from [15N]nitrite in anoxic tissue or blood was measured by electron paramagnetic resonance (EPR) spectroscopy. 15NO generated was purged from the reaction vessel using argon to a secondary vessel containing the spin trap Fe2ϩ-Methyl-D-glucamine dithiocarbamate (MGD). 15NO is paramagnetic and binds with high affinity to the water-soluble spin trap Fe2ϩ-MGD forming the mononitrosyl iron complex that exhibits a characteristic doublet 15NO-Fe2ϩ-MGD spectrum, rather than the triplet observed with natural abundance 14NO, enabling direct and selective detection of nitrite-derived NO formation
Summary
Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations. 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 (16 –20). This NOS-independent NO production could serve as a source of NO under ischemic conditions where NO production from NOS is significantly impaired [17,18,19]. D-glucamine dithiocarbamate; EPR, electron paramagnetic resonance; PBS, phosphate-buffered saline
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