Pedram Ghafourifar

Nitric oxide synthase activity in mitochondria.

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 ω‐monomethyl‐l‐arg and N ω‐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.

Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochromec Release from Isolated Mitochondria EVIDENCE FOR INTRAMITOCHONDRIAL PEROXYNITRITE FORMATION

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca2+ dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca2+ by mitochondria triggers mtNOS 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 mtNOS-induced cytochromec 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 Ca2+-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 Ca2+ levels induce apoptosis.

Ceramide Induces Cytochrome c Release from Isolated Mitochondria IMPORTANCE OF MITOCHONDRIAL REDOX STATE

In the present study we show thatN-acetylsphingosine (C2-ceramide),N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochromec (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 cytoc is oxidized but not when it is reduced. Upon cytoc loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨ), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cytoc reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ΔΨ, 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 cytoc. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cytoc followed by its release and that the redox state of cytoc influences its detachment by ceramide from the inner mitochondrial membrane.

Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+ release.

Mitochondria contribute to the maintenance of the intracellular Ca2+ homeostasis by taking up and releasing the cation via separate and specific pathways. The molecular details of the release pathway are elusive but its stimulation by the cross-linking of some vicinal thiols and consequently NAD+ hydrolysis are known. Thiol cross-linking and NAD+ hydrolysis can be achieved by addition of peroxynitrite (ONOO-), the product of the reaction between superoxide (O2-) and nitric oxide (nitrogen monoxide, NO*) to mitochondria. Mitochondria contain an NO synthase (mtNOS), which is stimulated by Ca2+, and are a copious source of O2-. We show here that intramitochondrially formed ONOO- stimulates the specific, NAD+-linked Ca2+ release from mitochondria. Our findings that upon Ca2+ uptake mtNOS is stimulated, that ONOO- is formed, and that Ca2+ is subsequently released from intact mitochondria suggest the existence of a feedback loop, which prevents overloading of mitochondria with Ca2+.

Peroxynitrite in the pathogenesis of Parkinson's disease and the neuroprotective role of metallothioneins

Parkinsons disease (PD) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinsons disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress has been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein (MT) against peroxynitrite-induced neurodegeneration in PD. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, alpha-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and it enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it downregulated the expression of Bcl-2 and poly(adenosine diphosphate-ribose) polymerase, but upregulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, alpha-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine (DA) to dihydroxyphenylacetaldehyde (DopaL) was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and was enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells, and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced downregulation of a mitochondrial genome and upregulation of caspase-3 as determined by reverse transcription polymerase chain reaction. These studies provide evidence that nitric oxide synthase activation and peroxynitrite ion overproduction may be involved in the etiopathogenesis of PD, and that metallothionein gene induction may provide neuroprotection.

Mammalian mitochondrial nitric oxide synthase: Characterization of a novel candidate.

Recently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development.

Determination of mitochondrial nitric oxide synthase activity.

The main biological targets of nitric oxide (NO) are hemoproteins, thiols, and superoxide anion (O2-). Mitochondria possess several hemoproteins, thiol-containing molecules, and they are one of the prime cellular producers of O2-. Thus, these organelles remain one of the main biological targets for NO. Reports on the existence of a Ca2+-sensitive mitochondrial NO synthase (mtNOS) have opened a new window in the field of NO and mitochondria research (Ghafourifar and Richter, 1997). mtNOS-derived NO reversibly decreases the activity of the mitochondrial hemoprotein, cytochrome c oxidase. This function of mtNOS regulates mitochondrial respiration and transmembrane potential (Deltapsi). The NO generated by mtNOS reacts with mitochondrial thiol-containing proteins including caspase-3. Because the S-nitrosated caspase-3 remains apoptotically silent as long as it is located within the mitochondria, this function of mtNOS portrays an anti-apoptotic property for mtNOS. mtNOS-derived NO also reacts with O2- to generate peroxynitrite. mtNOS-derived peroxynitrite induces oxidative stress and releases cytochrome c from the mitochondria, which represents a pro-apoptotic role for mtNOS. How mitochondria harmonize the reversible functions of mtNOS for mitochondrial respiration, its anti-apoptotic actions via S-nitrosation of caspase-3, versus the pro-apoptotic properties of peroxynitrite remains to be fully understood. However, intramitochondrial ionized Ca2+ concentration ([Ca2+]m) and the status of mitochondrial reducing defense barriers seem to play crucial roles in orchestrating the functions of mtNOS for mitochondria and cells (Ghafourifar and Cadenas, 2005).

Compartmentalized nitrosation and nitration in mitochondria.

A wide spectrum of the biological actions of nitric oxide and its oxidizing metabolites are mediated via mitochondria. Mitochondria are highly compartmentalized organelles consisting of three distinct compartments: the matrix, the intermembrane space, and the membranes. These compartments are different in their electrochemical properties, redox state, pH, enzymes, and ion content. Nitric oxide and its reactive species react within these compartments in distinct manners. The mitochondrial intermembrane space provides an environment that favors S-nitrosation, whereas nitration occurs largely within the matrix. This article will review some of the interactions of these species with certain mitochondrial respiratory chain complexes, apoptotic proteins, and enzymes. The reversibility and the suborganelle preference of these reactions will be discussed.

Redox Control of Mitochondrial Functions

Redox reactions and electron flow through the respiratory chain are the hallmarks of mitochondria. By supporting oxidative phosphorylation and metabolite transport, mitochondrial redox reactions are of central importance for cellular energy conversion. In the present review, we will discuss two other aspects of the mitochondrial redox state: (i) its control of mitochondrial Ca2+ homeostasis, and (ii) the intramitochondrial formation of reactive oxygen or nitrogen species that strongly influence electron flow of the respiratory chain.

Mitochondrial nitric oxide synthase.

Mitochondria produce nitric oxide (NO) through a Ca(2+)-sensitive mitochondrial NO synthase (mtNOS). The NO produced by mtNOS regulates mitochondrial oxygen consumption and transmembrane potential via a reversible reaction with cytochrome c oxidase. The reaction of this NO with superoxide anion yields peroxynitrite, which irreversibly modifies susceptible targets within mitochondria and induces oxidative and/or nitrative stress. In this article, we review the current understanding of the roles of mtNOS as a crucial biochemical regulator of mitochondrial functions and attempt to reconcile apparent discrepancies in the literature on mtNOS.