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The effect of nNOS overexpression on intracellular ROS activities in skeletal muscle fibres at rest and following a period of contractile activity
Author(s) -
Sakellariou Giorgos K,
Palomero Jesus,
Van Remmen Holly,
Jackson Malcolm J
Publication year - 2010
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.24.1_supplement.801.31
Subject(s) - intracellular , skeletal muscle , chemistry , stimulation , microbiology and biotechnology , reactive oxygen species , superoxide , nitric oxide , fluorescence microscope , biophysics , biochemistry , fluorescence , endocrinology , biology , enzyme , physics , organic chemistry , quantum mechanics
ROS serve as important signaling molecules and interact with multiple cell signaling and regulatory pathways to modulate changes in gene expression. The aim was to examine the effect of nNOS overexpression on ROS activities in skeletal muscle fibres at rest and following a period of contractile activity. To elucidate the roles of ROS, we examined their intracellular activities by using fluorescence microscopy. Mice overexpressing nNOS and age‐matched wild type (WT) mice were used in this study. Flexor digitorum brevis muscles were dissected and incubated in collagenase to isolate single muscle fibres. Fibres were plated on culture dishes and were loaded with DAF‐FM DA, DHE or DCFH DA, fluorescent probes for the assessment of nitric oxide (NO · ) superoxide (O 2 · ) and a general probe for ROS respectively. Contractile activity was induced by electrical stimulation. Control non‐stimulated fibres from the nNOS mice showed a higher rate of increase in DAF‐FM fluorescence compared with the WT group implying a higher NO · formation. Fibres from nNOS overexpressors showed a reduced ethidium fluorescence compared with fibres from WT mice at rest indicating a lower O 2 · generation. No differences were observed between groups following contractions. These results suggest that overexpression of nNOS can increase NO · ‘bioavailability’ in skeletal muscle cells and reduce O 2 · toxicity. Supported by the A Onassis Foundation (GR).