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The effect of nitric oxide on the detection of superoxide generated by mouse skeletal muscle fibres in vitro
Author(s) -
Pearson Tim,
Jackson Malcolm J
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1078.21
Subject(s) - superoxide , nitric oxide , chemistry , nadph oxidase , mitochondrion , biophysics , contraction (grammar) , skeletal muscle , reactive oxygen species , biochemistry , microbiology and biotechnology , anatomy , biology , endocrinology , enzyme , organic chemistry
Superoxide is generated from several pathways within muscle cells, including NADPH oxidase and the mitochondrial electron transport chain. Muscle derived superoxide has been claimed to exert multiple effects on muscle fibres including influencing force production, adaptive responses to contractions and in excess contributes to loss of function and cell death. Muscle superoxide activities were examined in single isolated fibres from the flexor digitorum brevis muscles of C57Bl6 mice using fluorescent confocal microscopy. Fibres were loaded with either dihydroethidium (5μM) or MitoSox Red (125nM) to determine cytoplasmic or mitochondrial superoxide respectively whilst in the presence or absence of L‐NAME (100μM), a nitric oxide synthase inhibitor. Images were acquired using Ex405 and Em605/15nm from fibres either at rest or subjected to two 9 minute contraction protocols at ten and forty minutes. 2‐hydroxyethidium fluorescence increased acutely by 20–25% (1 st , P<0.02) and 17–26% (2 nd , P<0.001) after the first and second contraction period compared with fibres at rest (n=8) in both the presence and absence of L‐NAME. In contrast, the presence of L ‐ NAME significantly increased MitoSox fluorescence as compared with untreated fibres. The pattern of fluorophore oxidation argued that the contraction‐induced increase in cytoplasmic superoxide was not due to release from the mitochondria. Supported by MRC.