Effects of enhancing mitochondrial oxidative phosphorylation with reducing equivalents and ubiquinone on 1-methyl-4-phenylpyridinium toxicity and complex I–IV damage in neuroblastoma cells (original) (raw)

2004, Biochemical Pharmacology

The effects of increasing mitochondrial oxidative phosphorylation (OXPHOS), by enhancing electron transport chain components, were evaluated on 1-methyl-4-phenylpyridinium (MPPþ) toxicity in brain neuroblastoma cells. Although glucose is a direct energy source, ultimately nicotinamide and flavin reducing equivalents fuel ATP produced through OXPHOS. The findings indicate that cell respiration/mitochondrial O 2 consumption (MOC) (in cells not treated with MPPþ) is not controlled by the supply of glucose, coenzyme Q 10 (Co-Q 10), NADHþ, NAD or nicotinic acid. In contrast, MOC in whole cells is highly regulated by the supply of flavins: riboflavin, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), where cell respiration reached up to 410% of controls. In isolated mitochondria, FAD and FMN drastically increased complex I rate of reaction (1300%) and (450%), respectively, having no effects on complex II or III. MPPþ reduced MOC in whole cells in a dose-dependent manner. In isolated mitochondria, MPPþ exerted mild inhibition at complex I, negligible effects on complexes II-III, and extensive inhibition of complex IV. Kinetic analysis of complex I revealed that MPPþ was competitive with NADH, and partially reversible by FAD and FMN. Co-Q 10 potentiated complex II ($200%), but not complex I or III. Despite positive influence of flavins and Co-Q 10 on complexes I-II function, neither protected against MPPþ toxicity, indicating inhibition of complex IV as the predominant target. The nicotinamides and glucose prevented MPPþ toxicity by fueling anaerobic glycolysis, evident by accumulation of lactate in the absence of MOC. The data also define a clear anomaly of neuroblastoma, indicating a preference for anaerobic conditions, and an adverse response to aerobic. An increase in CO 2 , CO 2 /O 2 ratio, mitochondrial inhibition or O 2 deprivation was not directly toxic, but activated metabolism through glycolysis prompting depletion of glucose and starvation. In conclusion, the results of this study indicate that the mechanism of action for MPPþ involves the inhibition of complexes I and IV, leading to impaired OXPHOS and MOC. Moreover, the results also indicate that flavin derivatives control the rate of complex I and more specifically complex IV, leading to impaired OXPHOS and MOC.