© 2003 American Thoracic Society
Superoxide production during sepsis developmentTo the Editor:Javesghani and colleagues (1) characterized a constitutively active superoxide-generating reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase in the ventilatory muscles. They clearly demonstrated its contribution to the basal reactive oxygen species production. Its role in superoxide production during sepsis development is less clear. Superoxide production in the diaphragm of septic rats was significantly higher than that of control animals, and the authors speculate that NADPH oxidase contributes significantly to this rise. We believe that there are no data to support this hypothesis. The authors cannot demonstrate a difference in reduced nicotinamide adenine dinucleotide (NADH)-stimulated superoxide production in septic diaphragm in relation to control. A significant elevation of NADH-stimulated superoxide production was only observed when nitric oxide synthase activity was inhibited. The authors suggest that nitric oxide production in septic muscles might have masked the increase in superoxide, but a more complex experimental approach should be made to define this point. In contrast, published data indicate the mitochondria as the major site of production of reactive oxygen species during sepsis development. We demonstrated an increase in mitochondrial superoxide production in major organs involved in septic response (submitted data). Boczkowski and colleagues (2) demonstrated that diaphragm mitochondria from endotoxemic rats show a progressive increase in hydrogen peroxide production associated with uncoupling and decrease phosphorylating capacity. Uncoupling of oxidative phosphorylation and the increase in superoxide production rate are probably related to multiple effects of endotoxin upon mitochondrial electron transfer and ATP synthesis. One of these effects should be an inhibition of the electron transfer at the ubiquinone-cytochrome b region, which is required to elicit a significant production of superoxide (3). Nitric oxide could reversibly bind to ubiquinone–cytochrome b uncoupling electron transfer, leading to superoxide production (3). Superoxide could react with nitric oxide to form peroxynitrite, which in turn binds irreversibly to ubiquinone–cytochrome b, initiating a cascade of mitochondrial superoxide production. Mitochondrial function is significantly impaired during acute sep-sis, and this impairment is strongly associated with the extent of mitochondrial ultrastructural abnormalities present in the tissues (4). In addition, lipopolysaccharide treatment reduced the rate of state 3 respiration, especially at complex IV, and increased the rate of state 4 respiration, reflecting partial uncoupling of oxidative phosphorylation (4). We believe that multiple intracellular sites could be responsible to the superoxide production during sepsis. Mitochondrial (2), NADPH oxidase (3), and Rac1 (5) pathways could be integrated during sepsis development. More research is needed to elucidate the mechanism through which sepsis increases superoxide production to define the exact role of each proposed molecular pathway.
Universidade Federal do Rio Grande do Sul, Porto Alegre Hospital de Clínicas de Porto Alegre, Porto Alegre Universidade do Extremo Sul Catarinense, Criciúma Brazil REFERENCES
From the Authors:With respect to the letter by Ritter and colleagues about our article (1), we agree with their statement that multiple intracellular sites could be responsible for the superoxide production during sepsis. In addition to the well described mitochondrial role, other molecular sources of superoxide production in skeletal muscles include xanthine oxidase and reduced NADPH oxidase. We observed that NADPH oxidase subunits are localized inside muscle fibers and that superoxide levels were inhibited by diphenyleneiodonium and apocynin. We stressed that the exact contribution of NADPH oxidase to total muscle superoxide production remains unclear. Although many reports described that mitochondrial hydrogen peroxide production increases significantly in septic muscles (2, 3), the contribution of other molecular sources such as NADPH oxidase could not be excluded.We disagree with Ritter and colleagues' statement that there are no data to support our speculation that NADPH oxidase contributes significantly to the rise to superoxide production by the septic diaphragms. Our speculation (which remains as such) is based on the following: (1) exogenous reduced NADH, which stimulates superoxide production by intact muscle strips, does not cross the sarcolemma; (2) NADH-stimulated superoxide production is not influenced by rotenone or antimycin; (3) NADH-stimulated superoxide production by septic diaphragmatic strips was elevated when nitric oxide synthases were inhibited by NG-nitro-L-arginine methyl ester. Although several reports credited increased mitochrondrial hydrogen peroxide production to nitric oxide (3, 4), we propose that the role of nitric oxide in muscle superoxide production is dependent on the type and localization of various nitric oxide synthases inside muscle fibers. In normal muscles, the neuronal and endothelial nitric oxide synthases are localized at the sarcolemma and mitrochrondria, respectively (5, 6). The inducible nitric oxide synthase has also been localized in normal skeletal muscles. Although mitochondrial oxidant production may be influenced by the endothelial and/or inducible nitric oxide synthases, it is unlikely that mitochondrial superoxide production in skeletal muscles is regulated directly by the neuronal nitric oxide synthase simply because this isoform is anchored to the dystrophin complex at the sarcolemma. We speculate that the response of NADH-stimulated muscle superoxide production to inhibition of nitric oxide synthases in septic animals reflects an interaction between the neuronal nitric oxide synthase and NADPH oxidase complex. This speculation is based on our histochemistry results showing NADPH oxidase subunit expression in close proximity to the sarcolemma (where the neuronal nitric oxide synthase is localized), and is based on our recent finding of enhanced activity and expression of the neuronal nitric oxide synthase in septic muscles.
McGill University Montreal, Quebec, Canada REFERENCES
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