Short-term impairment of energy production in isolated rat liver mitochondria by hypoxia/reoxygenation: involvement of oxidative protein modification

Abstract

The aim of the present study was to elucidate the role of mitochondria in liver impairment after ischaemia/reperfusion. It is commonly assumed that mitochondria are in part responsible for tissue damage by impaired oxidative phosphorylation as a consequence of the attack of radicals generated within the mitochondria. The principal support for this hypothesis was found by exposing isolated mitochondria to temporary hypoxia in combination with alterations of substrate supply. Rat liver mitochondria treated in this way responded with impaired ADP-stimulated respiration after reoxygenation, which decreased with time of hypoxia and reoxygenation. The decline of the activity of the NADH-cytochrome c-oxidoreductase complex found under these conditions is likely to cause the drop in active respiration. No changes in the content of respiratory chain complexes, determined by Blue Native PAGE, could be demonstrated. However, oxidative modifications of mitochondrial proteins, indicated by carbonyl formation, were found. Likewise, products of lipid peroxidation, such as lipid peroxides and malondialdehyde, were formed. Mitochondria were still able to build up a transmembrane potential and did not show drastic changes in membrane conductivity after hypoxia/reoxygenation stress. The presence of water-soluble antioxidants exhibited a beneficial effect, diminishing the decline of active respiration after 5 min of hypoxia and 10 min of reoxygenation. These observations strongly suggest that mitochondria play a pathogenic role in ischaemia/reperfusion injury, which is at least in part mediated by an oxygen-derived free-radical-linked mechanism.

Brain Mitochondria Are Primed by Moderate Ca2+ Rise upon Hypoxia/Reoxygenation for Functional Breakdown and Morphological Disintegration

“…Hypoxia/reoxygenation caused impairment of mitochondrial function in cultured astrocytes (22) and isolated mitochondria (23). Here we provide evidence that hypoxia/reoxygenation induces decrease in active respiration and changes in mitochondrial structure, which is characterized by a subpopulation of mitochondria with dented (bleb-like) outer membranes and another subpopulation of mitochondria with diminished number of cristae.…”

“…Isolated mitochondria can be maintained under conditions of almost complete anoxia (23), whereas in vivo, some oxygen may diffuse from the environment into the infarct area. Both the in vivo studies of stroke and the cell culture investigation on hypoxia/reoxygenation require a relatively long period of hypoxia to reach significant injury (2).…”

Effects of intermittent hypoxia different regimes on mitochondrial lipid peroxidation and glutathione-redox balance in stressed rats

“…It is known that hypoxia, as well as reoxygenation, can induce excessive ROS generation, resulting from the univalent reduction of molecular oxygen to O 2 • − by electrons that leak from the mitochondrial electron transport chain, mainly from complexes I and III [13,32]. The repetitive situation of short-term severe hypoxia followed by short-term reoxygenation causes a disturbance of intracellular pro-oxidant/antioxidant homeostasis, which is manifested in the intensification of lipid peroxidation [11,14,33]. The enhanced LPO in mitochondria leads to the loss of mitochondrial membrane fluidity, membrane ionic permeability, including proton permeability, which uncouples oxidative phosphorylation, as well as activity of membrane -bound enzymes [12].…”

“…Impaired electron flux through the mitochondrial respiratory chain is an important reason for oxidative stress during hypoxia and reoxygenation [14,15]. The accumulation and direct transfer of reducing equivalents within the mitochondrial respiratory chain to molecular oxygen can give rise to superoxide anion, singlet molecular oxygen, hydroxyl radical, and peroxynitrite [11].…”

Differential alterations in mitochondrial function induced by a choline-deficient diet: Understanding fatty liver disease progression

“…Carbonyl group content in both isolated mitochondria and hepatic tissue was measured spectrophotometrically by using 2,4-dinitrophenyl-hydrazine (DNPH) (Levine et al, 1990;Schild et al, 1997). Briefly, 100 mg of fresh tissue were homogenized in 1 mL of lysis buffer (250 mM sucrose, 0.5 mM EGTA and 10 mM Hepes, pH 7.4, supplemented with protease inhibitors).…”