Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity.
As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.
Meyer
Laudiene Evangelista
LE
Instituto de Bioquímica Médica, Programa de Biofísica e Bioquímica Celular and Programa de Biologia Molecular e Biotecnologia.
Machado
Lilia Bender
LB
Santiago
Ana Paula S A
AP
da-Silva
Wagner Seixas
WS
De Felice
Fernanda G
FG
Holub
Oliver
O
Oliveira
Marcus F
MF
Galina
Antonio
A
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D003401:Creatine / Q000378:metabolism; D052281:Creatine Kinase, Mitochondrial Form / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006943:Hyperglycemia / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D010101:Oxygen Consumption; D010725:Phosphocreatine / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015219:Substrate Cycling
D000975:Antioxidants; D017382:Reactive Oxygen Species; D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D006861:Hydrogen Peroxide; D052281:Creatine Kinase, Mitochondrial Form; D003401:Creatine