Production of Nitric Oxide by Mitochondria

Abstract

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...

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

“…In fact, mitochondria is likely the preferential subcellular setting for the interaction of DOPAC with NO, a statement that may be understood in connection with several lines of evidence, namely: (a) DOPAC synthesis occurs in the mitochondrial matrix via the activity of an unspecific aldehyde dehydrogenase (Ambroziak and Pietruszko, 1991;Keung and Vallee, 1998;Tank et al, 1981); (b) the steady-state level of NO in the mitochondrial matrix is expected to be particularly high (Boveris et al, 2000) partially due to the activity of the mitochondrial nitric oxide synthase (mtNOS) (Ghafourifar and Richter, 1997;Giulivi et al, 1998) and also due to the diffusion of NO from other cellular compartments and c) mitochondrial dysfunction seems to have a major role in the pathogenesis of Parkinson's disease (Mizuno et al, 1998;Schapira et al, 1992).…”

Mechanisms involved in the pro- and anti-apoptotic role of NO in human leukemia

“…42,43 NO, either provided by an NO donor as well as by mtNOS stimulation by loading mitochondria with calcium, was found to cause mitochondrial matrix acidification and a drop in mitochondrial transmembrane potential. 44 The uptake of Ca(2ϩ) by mitochondria triggered mtNOS activity leading to the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner and to increased lipid peroxidation.…”

Nitric Oxide, Mitochondria, and Cell Death

“…There may also be a mitochondrial isoform (mtNOS), but its origin and status is still unclear (8,9). NO may also be produced nonenzymatically from nitrite at low pH (<pH 5), for example, during ischaemia.…”