Key points Nitric oxide (NO), a potent vasodilator and a regulator of many physiological processes, is produced in mammals both enzymatically and by reduction of nitrite and nitrate ions. We have previously reported that, in rodents, skeletal muscle serves as a nitrate reservoir, with nitrate levels greatly exceeding those in blood or other internal organs, and with nitrate being reduced to NO during exercise. In the current study, we show that nitrate concentration is substantially greater in skeletal muscle than in blood and is elevated further by dietary nitrate ingestion in human volunteers. We also show that high‐intensity exercise results in a reduction in the skeletal muscle nitrate store following supplementation, likely as a consequence of its reduction to nitrite and NO. We also report the presence of sialin, a nitrate transporter, and xanthine oxidoreductase in human skeletal muscle, indicating that muscle has the necessary apparatus for nitrate transport, storage and metabolism. Abstract Rodent skeletal muscle contains a large store of nitrate that can be augmented by the consumption of dietary nitrate. This muscle nitrate reservoir has been found to be an important source of nitrite and nitric oxide (NO) via its reduction by tissue xanthine oxidoreductase. To explore if this pathway is also active in human skeletal muscle during exercise, and if it is sensitive to local nitrate availability, we assessed exercise‐induced changes in muscle nitrate and nitrite concentrations in young healthy humans, under baseline conditions and following dietary nitrate consumption. We found that baseline nitrate and nitrite concentrations were far higher in muscle than in plasma (∼4‐fold and ∼29‐fold, respectively), and that the consumption of a single bolus of dietary nitrate (12.8 mmol) significantly elevated nitrate concentration in both plasma (∼19‐fold) and muscle (∼5‐fold). Consistent with these observations, and with previous suggestions of active muscle nitrate transport, we present western blot data to show significant expression of the active nitrate/nitrite transporter sialin in human skeletal muscle. Furthermore, we report an exercise‐induced reduction in human muscle nitrate concentration (by ∼39%), but only in the presence of an increased muscle nitrate store. Our results indicate that human skeletal muscle nitrate stores are sensitive to dietary nitrate intake and may contribute to NO generation during exercise. Together, these findings suggest that skeletal muscle plays an important role in the transport, storage and metabolism of nitrate in humans.

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The presence of nitric oxide (NO) synthase enzymes, mainly the NOS1 isoform, in skeletal muscle had been well established; however in the last decade it has been realized that NO may also be produced by reduction of nitrate and tissue nitrite. We have recently shown that rodent skeletal muscle contains unusually high concentrations of nitrate, compared to blood and other tissues, likely produced by oxidation of NOS1-produced NO. In the present study we measured nitrate and nitrite levels in Wistar rat leg tissue before and after acute and chronic exercise of the animals on a treadmill. We found a very large decrease of muscle nitrate levels immediately after exercise accompanied by a transient increase of nitrite levels. A significant decrease in blood nitrate levels accompanied the changes in muscle levels. Using skeletal muscle tissue homogenates we established that xanthine oxidoreductase (XOR) is at least partially responsible for the generation of nitrite and/or NO from nitrate and that this effect is increased by slight lowering of pH and by other processes related to the exercise itself. We hypothesize that the skeletal muscle nitrate reservoir contributes significantly to the generation of nitrite and then, probably via formation of NO, exercise-induced functional hyperemia. A model for these metabolic interconversions in mammals is presented. These reactions could explain the muscle-generated vasodilator causing increased blood flow, with induced contraction, exercise, or hypoxia, postulated more than 100 years ago.