RESUMO
Previous studies have implicated mitochondria-derived reactive oxygen species (ROS) in both the aging process and age-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease etc. The current study, utilizing electron paramagnetic resonance (EPR) spectrometry, was designed to determine if mitochondrial respiratory stimulation, under state 4 conditions, caused extensive oxidative modifications to membrane cytoskeletal proteins and lipids in the brain. A mixed population of cortical synaptosomes and mitochondria, prepared by centrifugation techniques using rat brain cortex from adult (4-6 months) female Brown Norway rat brains, were labeled with the lipid-specific spin probe, 5-nitroxyl stearate (5-NS). Stimulation of the mitochondrial electron transport chain was accomplished using 20 mM succinate at 25 degrees C for 3 h. Mitochondrially derived free radicals, when reacted with the paramagnetic center of the spin probe, result in a loss of paramagnetism resulting in loss of intensity. A significant lowering (23%, P<0.0001) in the signal amplitude (B0) of 5-NS, indicative of generation of oxyradicals, was found. The order parameter, an inverse EPR-measure of membrane fluidity of the 5-NS spin labeled mitochondrial and synaptosomal membranes, also decreased following mitochondrial respiratory stimulation (P<0.005). Changes in the physical state of cytoskeletal and transmembrane proteins due to succinate oxidation were measured using MAL-6 (2,2,6, 6,-tetramethyl-4 maleimidopiperdin-1-oxyl), a thiol-specific nitroxide spin label. The ratio of the amplitudes of the weakly to strongly immobilized spin label reaction sites (W/S ratio) in the low-field region of the spectrum was used to determine any alteration in protein conformation. Previous studies in our laboratory have established that increased protein oxidation is associated with a decreased W/S ratio. In the current study, our results indicated significant lowering of the W/S ratio in cortex (30%, P<0.0001) upon stimulation of the mitochondria with 20 mM succinate. Thus, we conclude that respiratory stimulation of mitochondria, due to a hypermetabolic stress with succinate, caused significant oxidative modifications of cortical membrane lipids and proteins.
