In the present study we show the existence of a functional nitric oxide synthase (NOS) in rat liver mitochondria. The enzyme uses L-arginine (L-arg) to produce nitric oxide (NO) and L-citrulline, and is Ca2+-dependent. L-Arg analogues, N(omega)monomethyl-L-arg and N(omega)-nitro-L-arg, inhibit the enzyme, and D-arginine is not a substrate for it. We found mitochondrial NOS (mtNOS) activity associated with the inner mitochondrial membrane but not with the matrix fraction. In intact, succinate-energized mitochondria, the enzyme is constitutively active and exerts substantial control over mitochondrial respiration and membrane potential. The activity is further stimulated when Ca2+ is taken up by mitochondria. We suggest that the existence of mtNOS and its Ca2+ dependence are highly relevant for mitochondrial functioning.

Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.

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Alpha-synuclein is a neuron-specific protein that contributes to the pathology of Parkinson's disease via mitochondria-related mechanisms. The present study investigated possible interaction of alpha-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing alpha-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that alpha-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of alpha-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by alpha-synuclein.

In the present study we show that N-acetylsphingosine (C 2 -ceramide), N-hexanoylsphingosine (C 6 -ceramide), and, to a much lesser extent, C 2 -dihydroceramide induce cytochrome c (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nM) of Bcl-2. The release takes place when cyto c is oxidized but not when it is reduced. Upon cyto c loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (⌬⌿), and Ca 2؉ retention are diminished. Incubation with Bcl-2 prevents, and addition of cyto c reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ⌬⌿, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cyto c. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cyto c followed by its release and that the redox state of cyto c influences its detachment by ceramide from the inner mitochondrial membrane.

). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca 2؉ by mitochondria triggers mt-NOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mt-NOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca 2؉ -induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca 2؉ levels induce apoptosis.

Tamoxifen is an anticancer drug that induces oxidative stress and apoptosis via mitochondria-dependent and nitric oxide (NO)-dependent pathways. The present report shows that tamoxifen increases intramitochondrial ionized Ca 2+ concentration and stimulates mitochondrial NO synthase (mtNOS) activity in the mitochondria from rat liver and human breast cancer MCF-7 cells. By stimulating mtNOS, tamoxifen hampers mitochondrial respiration, releases cytochrome c, elevates mitochondrial lipid peroxidation, increases protein tyrosine nitration of certain mitochondrial proteins, decreases the catalytic activity of succinyl-CoA:3-oxoacid CoA-transferase, and induces aggregation of mitochondria. The present report suggests a critical role for mtNOS in apoptosis induced by tamoxifen. [Cancer Res 2007;67(3):1282-90]

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