The International Journal of Biochemistry & Cell Biology 34 (2002) 348–357 Melatonin increases the activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria Miguel Mart´ ın, Manuel Mac´ ıas, Josefa León, Germaine Escames, Houda Khaldy, Dar´ ıos Acuña-Castroviejo ∗ Departamento de Fisiolog´ ıa, Instituto de Biotecnolog´ ıa, Universidad de Granada, E-18012 Granada, Spain Received 4 July 2001; accepted 17 October 2001 Abstract We recently showed that melatonin counteracted mitochondrial oxidative stress and increased the activity of the mitochon- drial oxidative phosphorylation (OXPHOS) enzymes both in vivo and in vitro. To further clarify these effects, we studied here the activity of OXPHOSenzymes and the synthesis of ATP in rat liver and brain mitochondria in vitro. In sub-mitochondrial par- ticles, melatonin increases the activity of the complexes I and IV dose-dependently, the effect being significant between 1 and 10 nM. Blue native-PAGE followed by histochemical analysis of the OXPHOS enzymes further showed the melatonin-induced increase of complex I activity. Titration studies show that melatonin counteracts the partial inhibition of complex IV induced by 5 M potassium cyanide. However, melatonin (up to 5mM) was unable to recover the activity of complex IV when it was completely blocked by 100 M cyanide. These data suggest that the indoleamine could stimulate the activity of the non-inhibited part of the complex IV. Melatonin also increases the production of ATP in control mitochondria and counteracts the cyanide-induced inhibition of ATP synthesis. These results provide new hormonal mechanism regulating mitochondrial homeostasis and may explain, at least in part, the anti-aging and neuroprotective properties of melatonin. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Melatonin; Mitochondria; ATP; Electron chain transport; Complex I; Complex IV; Oxidative stress 1. Introduction A deleterious consequence of oxygen consumption and ATP production by mitochondria is the produc- tion of free radicals such as the superoxide anion Abbreviations: BN-PAGE, blue-native polyacrylamide gel elec- trophoresis; OXPHOS, oxidative phosphorylation; CN, cyanide; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; ETC, electron transport chain ∗ Corresponding author. Tel.: +34-958-243520; fax: +34-958-246295. E-mail address: dacuna@ugr.es (D. Acuña-Castroviejo). and hydroperoxides. These radicals may impair the efficiency of oxidative phosphorylation (OXPHOS) enzymes, leading to energy depletion and cell death. It is classically considered that the main intramitochon- drial antioxidant system, i.e. superoxide dismutase and glutathione, maintain a good mitochondrial per- formance protecting them against attack from reactive oxygen species [1]. Indirect experimental evidence points to a role of melatonin on mitochondria. Melatonin counteracts cyanide-induced seizures [2], reduces the suppression of mitochondrial respiration caused by peroxynitrites 1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S1357-2725(01)00138-8