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The production of NO ⅐ by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio ‫؍‬ 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-Fe II produced a signal ascribed to the NO ⅐ spin adduct (g ‫؍‬ 2.04; a N ‫؍‬ 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor N G -monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO ⅐ (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO ⅐ ؋ (min⅐mg protein)؊1 , respectively, with a K m for L-Arg of 5-7 M. Comparison of the rates of NO ⅐ production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO ⅐ , the production of which may effect energy metabolism, O 2 consumption, and O 2 free radical formation. Nitric oxide (NO ⅐ )1 is a free radical generated in biological systems by nitric oxide synthases (NOS). Because of its effect on neurotransmission, vasodilation, and immune response (1-3), NO ⅐ plays an important role in physiology, pathology, and pharmacology.Studies with brain tissue and macrophage lysates have shown that NOS is localized exclusively in the soluble fraction (3-6), and recent studies have indicated that the majority (Ͼ80%) of bovine endothelial NOS activity is bound to the particulate fraction of cell homogenates (7,8). Because the particulate fraction used in the studies was expected to contain plasma membranes, as well as microsomes, and, possibly, intracellular organelles, the actual subcellular location of the activity remained to be determined. Other lines of evidence have indicated the presence of NOS in the perinuclear region, in discrete regions of the plasma membrane of cultured endothelial cells, and in intact blood vessels (9, 10); immunocytochemical studies have revealed the presence of a NOS, or an antigenically related protein, in mitochondria isolated from different tissues (11-13). The predominant association of this mtNOS with the mitochondrial membrane (11, 12), and its co-localization with succinate dehydrogenase, a mitochondrial marker of the inner membrane (13), suggested that this enzyme has a particulate localization.These studies as well as the presence of substrates and cofactors in mitochondria required for NOS activity such as L-arginine (L-Arg), L-Arg transporters, Ca 2ϩ , calmodulin, NADPH, and the availability of O 2 , led us to postulate mitochondria as a potential source of NO ⅐ production.Fol...

Nitric oxide ('NO) release, oxygen uptake and hydrogen peroxide (H202) production elicited by increasing phorbol12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 pg PMA/ml CNO) and O.OOl~.Ol pg PMA/ml (H,OJ. At saturated PMA concentrations, 'NO production, oxygen uptake and H,O, release were 0.56 ? 0.04, 3.32 f 0.52 and 1.19 f 0.17 nmol min-' 1 O6 cells-'. 'NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.00-0.01 pg PMA/ml. Preincubation with A'"-monomethyl-L-arginine (L-NMMA) decreased 0, uptake and 'NO release but increased H,Oz production, while superoxide dismutase (SOD) increased 'NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that 'NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of-NO and O;-release favour the formation of other oxidants like peroxynitrite.

Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O₂, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O₂ utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis.