Metabolism and Transport of Nitrate, Nitrite, and Nitric Oxide

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have now largely completed our work on platelet and blood clotting inhibition by nitrite by studying the effects of changes in ambient oxygen levels on the processes in rodent models using the TEG methodologies. We have also measured the effects of red cell reduction of plasma nitrite on platelet signaling because of some confusion in the literature and have confirmed that VASP phosphorylation occurs with the generation of NO but that this signal is very dependent on the kinetics of reaction and the exact experimental conditions, especially red cell concentrations. The VASP phosphorylation appears to be an order of magnitude more sensitive than the direct measurements of cGMP that we have used in the past. These measurements have been very valuable for our collaborators in Bangkok, Thailand who have found such changes in circulating platelets in certain patients after sodium nitrite inhalation (see DK 025104). Because of the recent interest in the results of VASP phosphorylation studies we have published further on this process and produced a "Journal of Visualized Research" tutorial so others may better use this assay, as well as other methodological information. Our studies of skeletal muscle nitrate began during our studies of NO metabolism in rodents in which we discovered that nitrate levels in skeletal muscle were much higher than in blood or any other organ, indeed muscle appears to be the main reservoir for nitrate in the body. We then showed that during exercise the nitrate could be reduced to nitrite and then NO which we believe are the main pathway controlling the massive increase in blood flow with exercise, known for 150 years but without a good explanation until now. This work has also focused on better understanding of the mechanisms of these reductive processes and how muscle obtains such high levels of nitrate. So far our results are still most compatible with xanthine oxido-reductase as the major enzyme involved in the reductive processes. Dietary and genetic manipulations of rodents has shown that NOS 1 (nNOS) and myoglobin knockouts have markedly reduced levels of skeletal muscle nitrate suggesting that, as we predicted, both proteins are involved in these high levels. However, we also find that dietary limitations of nitrate and nitrite lower these levels greatly (more than in blood or liver) and that return of these ions to the diet results in rapid accumulation and, indeed, in some cases an "overshoot" of the levels. These result raise the possibility of some active transport mechanisms in the muscle, perhaps by the protein sialin which transports nitrate into the saliva from blood. We are also currently studying these processes in muscle cells, primary and continuous lines, in culture, including in cells which can be caused to differentiate from myoblasts to myocytes and myotubes. Our results show uptake of nitrate and nitrite in all developmental stages of muscle cell but that differentiation of muscle cells is required for enhancing nNOS reduction to nitrite. We are now quantitating the roles of nitric oxide synthase enzymes, myoglobin, sialin, a chloride transporter protein, and other proteins in affecting the levels of nitrate in muscle tissues. We have worked out quantitative PCR measurements for each of these proteins and are correlating these results with Western blots of the proteins themselves. We are planning to do proteomic analyses of these cells to identify new molecules that may be involved in these processes and we are also studying how hypoxia affects the relative contributions of nNOS, reductive processes and transport processes in determining levels of nitrate, nitrite and NO in muscle under specified conditions and if smooth (diaphragm and bladder) and cardiac muscle have similar mechanisms. Our initial results do confirm the existence of these reductive pathways in smooth muscle as well as the high levels of nitrate found in skeletal muscle. In other recent studies, we have used 15-N nitrate to study the uptake of nitrate into blood and other organs (muscle and eyes in particular) of rodents and pigs and find that this process is very rapid and efficient. These studies are being expanded as these pre-clinical studies should mesh well with our clinical studies (in the accompanying Annual Report) to clarify uptake and metabolism in human subjects as well as animal models.