Abstract
We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.
Highlights
Mitochondria are the key players of cell energy metabolism, programmed cell death and oxidative stress
NADH and FADH2 oxidation leads to translocation of protons through complexes I, III and IV of the mitochondrial inner membrane into intermembrane space
NADH, FADH 2 has no intrinsic fluorescence and FAD has it, which allows for visualization of redox state of these nucleotides using optical methodswithout employing fluorescent probes
Summary
Mitochondria are the key players of cell energy metabolism, programmed cell death and oxidative stress. Polarography with Clark electrode is a widely used method for evaluation of functional activity of mitochondria through measurement of oxygen consumption rate by suspension of the organelles of permeabilized cells in various functional states [1,2,3,4,5] This method requires specific equipment and large quantities of experimental material, yet it does not provide information on the biochemical processes underlying changes in mitochondrial oxygen consumption. Evaluation of redox state of pyridine nucleotides is widely used in biochemical research, in particular in studies on consequences of oxidative stress [11,12,13], cell death detection [8], estimation of level of postsynaptic neuronal activation [10, 14]. Changes in redox state of pyridine nucleotides as the primary cause of disruptions in mitochondrial respiration under fluoroacetate toxicity have been studied on asciticEhrlich carcinoma and rat liver mitochondria [20]
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