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, 567 (Pt 1), 113-20

Decreasing Xanthine Oxidase-Mediated Oxidative Stress Prevents Useful Cellular Adaptations to Exercise in Rats

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Decreasing Xanthine Oxidase-Mediated Oxidative Stress Prevents Useful Cellular Adaptations to Exercise in Rats

Mari-Carmen Gomez-Cabrera et al. J Physiol.

Abstract

Reactive oxygen or nitrogen species (RONS) are produced during exercise due, at least in part, to the activation of xanthine oxidase. When exercise is exhaustive they cause tissue damage; however, they may also act as signals inducing specific cellular adaptations to exercise. We have tested this hypothesis by studying the effects of allopurinol-induced inhibition of RONS production on cell signalling pathways in rats submitted to exhaustive exercise. Exercise caused an activation of mitogen-activated protein kinases (MAPKs: p38, ERK 1 and ERK 2), which in turn activated nuclear factor kappaB (NF-kappaB) in rat gastrocnemius muscle. This up-regulated the expression of important enzymes associated with cell defence (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when RONS production was prevented by allopurinol. Thus we report, for the first time, evidence that decreasing RONS formation prevents activation of important signalling pathways, predominantly the MAPK-NF-kappaB pathway; consequently the practice of taking antioxidants before exercise may have to be re-evaluated.

Figures

Figure 1
Figure 1. Xanthine oxidase (XO) activity in rat plasma increases after exhaustive exercise; allopurinol administration completely prevents this effect
Rest, n = 5; Exercised, n = 5; Exercised treated with allopurinol, n = 5. *P < 0.05 versus Rest. ‡P < 0.01 versus Exercised.
Figure 2
Figure 2. Muscle oxidative stress after exhaustive physical exercise in rats
A, allopurinol prevents oxidation of glutathione (GSSG) in exhaustive exercise. Values are means ±s.d.; Rest, n = 5; Exercised, n = 4; Exercised treated with allopurinol, n = 4. *P < 0.05 versus Rest. B, reduced glutathione (GSH) muscle levels in exhaustive exercise. Values are means ±s.d.; Rest, n = 5; Exercised, n = 4; Exercised treated with allopurinol, n = 4.
Figure 3
Figure 3. Muscle protein oxidation after exhaustive physical exercise in rats
A, Western blot showing carbonylated proteins in cytosolic extracts from gastrocnemius muscle. Representative experiments are shown. B, densitometry analysis of protein carbonylation. Values are means ±s.d.*P < 0.05 versus Rest.
Figure 4
Figure 4. Muscle protein glutathionylation after exhaustive physical exercise in rats
A, Western blot showing glutathionylated proteins in cytosolic extracts from gastrocnemius muscle. Representative experiments are shown. B, densitometry analysis of protein glutathionylation. Values are means ±s.d.*P < 0.05 versus Rest.
Figure 5
Figure 5. Exercise activates p38 phosphorylation: prevention by allopurinol
A, Western blot analysis of phospho-p38 in the cytosolic fraction of rat gastrocnemius. B, densitometry analysis of p38 phosphorylation (n = 3). Values are means ±s.d.**P < 0.01 versus Rest.
Figure 6
Figure 6. Exercise activates ERK1/ERK2 phosphorylation: prevention by allopurinol
A, Western blot analysis of phospho-ERK1/ERK2 in the cytosolic fraction of rat gastrocnemius. B, densitometry analysis of ERK1/ERK2 phosphorylation (n = 3). Values are means ±s.d.**P < 0.01 versus Rest.
Figure 7
Figure 7. Exercise activates NF-κB: prevention by allopurinol
A, EMSA analysis of NF-κB in the nuclear extracts of rat gastrocnemius. (Ø, competition assay: unlabelled competitor probe was added in 100-fold molar excess). B, densitometry analysis of NF-κB (n = 4). Values are means ±s.d.**P < 0.01 versus Rest.
Figure 8
Figure 8. Exercise-induced up-regulation of Mn-SOD and NO synthases is prevented by allopurinol
Expression of Mn-SOD (A), iNOS (B) and eNOS (C) measured by real-time RT-PCR from gastrocnemius muscle of rats at rest, after exercise, and after exercise but pretreated with allopurinol (n = 9).
Figure 9
Figure 9. Muscle protein Mn-SOD levels after exhaustive physical exercise in rats
Western blot analysis of Mn-SOD in the cytosolic fraction of rat gastrocnemius muscle.
Figure 10
Figure 10
Proposed mechanism of the role of reactive oxygen species in signalling of cell adaptations after exercise.

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