Skeletal muscle as an endogenous nitrate reservoir

Abstract

The nitric oxide synthase (NOS) family of enzymes form nitric oxide (NO) from arginine in the presence of oxygen. At reduced oxygen availability NO is also generated from nitrate in a two step process by bacterial and mammalian molybdopterin proteins, and also directly from nitrite by a variety of five-coordinated ferrous hemoproteins. The mammalian NO cycle also involves direct oxidation of NO to nitrite, and both NO and nitrite to nitrate by oxy-ferrous hemoproteins. The liver and blood are considered the sites of active mammalian NO metabolism and nitrite and nitrate concentrations in the liver and blood of several mammalian species, including human, have been determined. However, the large tissue mass of skeletal muscle had not been generally considered in the analysis of the NO cycle, in spite of its long-known presence of significant levels of active neuronal NOS (nNOS or NOS1). We hypothesized that skeletal muscle participates in the NO cycle and, due to its NO oxidizing heme protein, oxymyoglobin, has high concentrations of nitrate ions. We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver. The nitrate reservoir in muscle is easily accessible via the bloodstream and therefore nitrate is available for transport to internal organs where it can be reduced to nitrite and NO. Nitrate levels in skeletal muscle and blood in nNOS−/− mice were dramatically lower when compared with controls, which support further our hypothesis. Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate. We suggest that skeletal muscle participates in overall NO metabolism, serving as a nitrate reservoir, for direct formation of nitrite and NO, and for determining levels of nitrate in other organs.

Nitrate as a source of nitrite and nitric oxide during exercise hyperemia in rat skeletal muscle

“…Figures 1A and B show levels of nitrate and nitrite ions in liver, blood and skeletal muscle of sedentary and exercised Wistar rats and are very similar to our recent publication in which we reported that skeletal muscle in resting rodents have high levels of nitrate (19). To examine possible changes in these species with exercise, nitrate and nitrite levels were measured after the animals completed one exercise session which consisted of two 45 min runs on a treadmill with a 60 min rest period between runs, as described above.…”

“…In our previous study we have shown that skeletal muscle is an endogenous nitrate reservoir, perhaps the largest one in the mammalian body (19). Presumably, nitrate stored in this tissue originates from NO formed from arginine by NOS1 and its oxidation by muscle oxymyoglobin.…”

“…We have shown previously that skeletal muscle homogenate is able to reduce exogenously added nitrate into nitrite and that xanthine oxidoreductase is responsible for at least part of this reduction (19). Indeed, in 1928 Bernhein and Dixon (25) reported nitrate reduction into nitrite by muscle extracts.…”

“…In our previous study (19) we hypothesized that colocalization of functional NOS, present in skeletal muscle in large quantities, and oxymyoglobin in muscle cell leads to the oxidation of NO to nitrate and to the accumulation of nitrate in the muscle tissue. In Figure 5 we show the results of measurements of nitrate (A) and nitrite (B) levels in skeletal muscle of sedentary rats after seven days at inhibition of NOS-mediated NO formation and with no intervention.…”

“…We previously observed that skeletal muscle is a very large nitrate reservoir in the mammalian body, with a strong nitrate gradient from skeletal muscle-to-blood-to-liver in both rats and mice (19). We also demonstrated low but significant levels of nitrate reduction in muscle extracts.…”