Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, PC70

Poster Communications

Intracellular Interactions of Superoxide and Nitric Oxide in Skeletal Muscle.

G. K. Sakellariou1, D. Pye1, A. Vasilaki1, L. Zibrik1, J. Palomero1, T. Kabayo1, F. McArdle1, H. Van Remmen2, A. Richardson2, A. McArdle1, M. J. Jackson1

1. School of Clinical Sciences, Pathophysiology Research Unit, Liverpool, United Kingdom. 2. University of Texas, Center at San Antonio and Barshop Institute for Longevity and Aging Studies, San Antonio, Texas, United States.

Superoxide (O2−.) and Nitric Oxide (NO) are the primary species generated within skeletal muscle both at rest and during contractile activity [1]. While further reaction of both species generates secondary reactive oxygen molecules such as hydrogen peroxide and hydroxyl radicals, the reaction of O2−. with NO to form peroxynitrite (ONOO-) during normal muscle metabolism is unknown. To determine the interaction of O2−. and NO, we examined their intracellular activities using fluorescence microscopy in single isolated mature skeletal muscle fibres from SOD1 knockout mice (Sod1−/−) and mice overexpressing the neuronal nitric oxide synthase (nNOSTg) at rest and following a period of contractile activity. Male adult C57Bl/6 Sod1−/− [2], C57Bl/6 nNOSTg mice [3] and control C57Bl/6 wild type (WT) mice were used. Mice were killed by cervical dislocation and the flexor digitorum brevis muscles were dissected. Muscles were incubated in collagenase to isolate single muscle fibres. Fibres were then plated on culture dishes which had been previously coated with collagen and cultured for 24h [4]. Fibres were loaded with 4-Amino-5-Methylamino-2’, 7’-Difluorofluorescein Diacetate (DAF-FM DA) and Dihydroethidium (DHE), intracellular fluorescent probes for the assessment of NO and O2−., respectively. Contractile activity was induced by electrical stimulation [4]. 3-nitrotyrosine (3-NT) in proteins was determined as an indicator of ONOO- formation through western blotting. Data were initially analysed by analysis of variance followed by Tukey post-hoc analysis, statistical significance was set at p<0.05. No increase in DHE oxidation occurred in resting fibres from Sod1-/- mice, but these fibres showed decreased DAF-FM fluorescence indicating a reduced NO content compared with fibres from the WT mice. Muscles from Sod1-/- mice showed increased 3-NT content of carbonic anhydrase III indicating increased ONOO- formation compared with WT mice. During contractions, fibres from WT but not from Sod1-/- mice showed an increase in ethidium fluorescence and DAF-FM fluorescence. Inhibition of nitric oxide synthases using NG-monomethyl-L-arginine did not affect DHE oxidation in fibres from WT or Sod1-/- mice either at rest or during contractions. In contrast, transgenic mice with nNOS overexpression in skeletal muscle had increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibres compared with fibres from the WT group. These data indicate that reaction of O2−. with NO to generate ONOO- is a major route for O2−. metabolism in muscles of mice lacking SOD1 and an increase in NO availability reduces O2−. activity in muscle fibres.

Where applicable, experiments conform with Society ethical requirements