Acidosis during Reoxygenation Has an Early Detrimental Effect on Neuronal Metabolic Activity

Abstract

We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation. We hypothesized that the detrimental effect of acidosis during reoxygenation was due to a negative effect on mitochondrial function. Human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and then reoxygenated for 0, 1, 4, 9, or 21 h. The detrimental effect of acidotic reoxygenation on metabolic activity was evident already after 1 h of reoxygenation, when MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction (percentage of normoxic controls) was significantly higher in cells reoxygenated with neutral compared with acidotic medium both after acidotic hypoxia (83 Ϯ 26% versus 67 Ϯ 27%, p ϭ 0.006) and after neutral hypoxia (51 Ϯ 12% versus 41 Ϯ 7%, p ϭ 0.005). Hypoxanthine, a marker of cellular energy failure, increased more with acidotic compared with neutral reoxygenation both after acidotic hypoxia (after 21 h: 7.7 Ϯ 2.7 versus 3.1 Ϯ 1.9 M, p Ͻ 0.001) and after neutral hypoxia (10.4 Ϯ 2.6 versus 7.9 Ϯ 2.8 M, p ϭ 0.001). During hypoxia and reoxygenation, there was an earlier reduction in the activity of complex IV compared with complexes IIϩIII, and the ratio between these complexes fell during the first hour of reoxygenation. The reduction in complex IV activity was alleviated with acidotic hypoxia. Acidosis during reoxygenation, however, had no effect on the activity of either complex IV or complexes IIϩIII. We conclude that acidosis during hypoxia increases neuronal survival and preserves complex IV activity. Acidosis during reoxygenation has an early detrimental effect on metabolic activity, but this is not mediated through an effect on the mitochondrial complexes IV or IIϩIII. We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation in a human NT2-N neuronal culture (1). Several mechanisms with opposing effects may be involved. In our hypoxia model, cell death is mediated through NMDA and non-NMDA receptors, and pretreatment with MK-801 offers almost complete protection (1-4). Whereas the protective effect of acidosis during hypoxia is probably to a large extent mediated through an inhibitory effect on glutaminergic NMDA receptors (1,5), the mechanisms behind the detrimental effect of acidosis during reoxygenation are less clear. Acidosis causes a 50% increase in free radical formation in the current model (1). However, as we have previously found a rather limited role for free radicals in hypoxic cell death of NT2-N neurons, this may not sufficiently explain the deleterious effect of acidosis during reoxygenation (3).Abnormalities in mitochondrial function and mitochondrial (mt) DNA have been reported in several neurodegenerative diseases (6,7), and mitochondria are important for both apoptotic and necrotic cell death (8). In perinatal hypoxic-ischemic insults, a delayed secondary energy failure may occur after an initial normalization of the energy status of the brain (9

Mild Acidosis Protects Neurons during Oxygen-Glucose Deprivation by Reducing Loss of Mitochondrial Respiration

“…Ischemic injury can cause the activities of mitochondrial complexes to decrease. ,, Previous studies have shown that acidosis can increase the protein levels of several complexes during hypoxia or increase the activity of complex IV during OGD. These studies and our results, which show that mild acidosis attenuates OGD-induced reductions in the activities of mitochondrial complexes I and II, demonstrate that acidosis protected mitochondrial function during OGD by targeting electron transport chain complexes.…”

Antioxidant dysfunction: potential risk for neurotoxicity in ethylmalonic aciduria

“…Enzyme assays were performed at 30°C in 200 µl final volume using a SPECTRAmax PLUS 384 Micro plate Spectrophotometer (Molecular Devices). Succinate-cytochrome c reductase (complex II+III) activity was measured as ferricytochrome c reduction, whereas cytochrome c oxidase (complex IV) activity was determined as the initial rate of ferrocytochrome c oxidation (Froyland et al 2005). All samples were measured three times, and enzyme activities were expressed as nanomole per minute per milligram fibroblast protein.…”