Reactive oxygen species (ROS) participate in tissue injury after
ischemia-reperfusion. Their implication in leukocyte adherence and increase in permeability at the venular side of the microcirculation have been reported, but very little is known about ROS production in arterioles. The objective of this work was to evaluate, in the arteriole wall in vivo, the temporal changes in
superoxide anion production during
ischemia and reperfusion and to identify the source of this production. Mouse cremaster muscle was exposed to 1 h of
ischemia followed by 30 min of reperfusion, and
superoxide anion production was assessed by a
fluorescent probe, i.e., intracellular
dihydroethidium oxidation. During
ischemia, we found a significant increase in
dihydroethidium oxidation; however, we observed no additional increase in fluorescence during the subsequent reperfusion. This phenomenon was significantly inhibited by pretreatment with
superoxide dismutase.
Allopurinol (
xanthine oxidase inhibitor) or
stigmatellin [Q(o)-site (oriented toward the intermembrane space) inhibitor of mitochondrial
complex III] or simultaneous administration of these two inhibitors significantly reduced
superoxide production during
ischemia to 80%, 88%, and 72%, respectively, of that measured in the untreated
ischemia-reperfusion group. By contrast, no significant inhibition was found when
NADPH oxidase was inhibited by
apocynin or when mitochondrial complex I or complex II was inhibited by
rotenone or
thenoyltrifluoroacetone. A significant increase in ROS was found with
antimycin A [Q(i)-site (located in the inner membrane and facing the mitochondrial matrix) inhibitor of mitochondrial
complex III]. We conclude that a significant increase in ROS production occurs during
ischemia in the arteriolar wall. This increased production involves both a cytoplasmic source (i.e.,
xanthine oxidase) and the mitochondrial
complex III at the Q(o) site.