Effects of Partial Inhibition of Respiratory Complex I on H2O2 Production by Isolated Brain Mitochondria in Different Respiratory States

Abstract

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Methodology of Petri networks for simultaneous evaluation of the impact of different modifiers on the fluorescence of nucleotides from electron transport chain in isolated mitochondria and on the process of swelling

“…2). This result was in agreement with the data obtained from the lung tissue (Staniszewski et al, 2013), brain neurons (Shuttleworth et al, 2010), and isolated brain and myocardium mitochondria (Michelini et al, 2014;Agarwal et al, 2014). However, rotenone did not affect the FAD fluorescence (Staniszewski et al, 2013).…”

Age-related diseases as vicious cycles

“…(1) Spreading αS oligomers inhibit mitochondrial Complex I 271,272 (2) The inhibition of Complex I leads to an increase in ROS 273 (3) ROS modify αS and dopamine, promoting αS-dopamine covalent binding [274][275][276][277] (4) Dopamine adducts promote the conversion of αS into toxic oligomers/protofibrils but prevent the formation of mature fibrils [274][275][276][277] This mitochondrial cycle is supported by the involvement of parkin 278 , PINK-1 279 and DJ-1 [280][281][282] in mitochondrial maintenance and antioxidant activity, by deficient Complex I activity in Parkinson's disease patients 271,283,284 , as well as by the induction of parkinsonism by mitochondrial Complex I inhibitors and ROS inducers [285][286][287][288][289] . Crucially, toxin-induced parkinsonism is progressive 290,291 and mediated by αS 292 .…”

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

“…FAD emits at 530-540 nm on excitation at 450 nm [21,22]. The amount of data on changes in fluorescent signal of NADH under influence of ETC effectors in isolated mitochondria [5,12,20,23] is limited, and there is no data on changes in FAD signal. There is also no information on the corresponding studies on smooth muscle cell and mitochondria.…”