Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochrome c 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 Ca 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 Ca 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 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-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 levels induce apoptosis.

Nitric oxide (NO) 1 is a molecule of prime importance in biology. The most-cited and best understood physiological target for NO is the heme-containing protein, soluble guanylyl cyclase (1). However, at physiological concentrations, NO also binds to another hemoprotein, cytochrome oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, and thereby controls cellular functions via reversible inhibition of respiration (reviewed in Ref. 2). NO is synthesized by members of the NO synthase family (NOS, EC 1.14.13.39; reviewed in Ref. 3). In 1997, we reported for the first time on the presence of a constitutively expressed and continuously active NOS in mitochondria (mtNOS), its localization in the inner mitochon-drial membrane, its Ca 2ϩ dependence, and that the enzyme exerts substantial control over mitochondrial respiration and mitochondrial transmembrane potential (⌬⌿) (4). Soon thereafter the presence of mtNOS and its localization were confirmed, and the enzyme was enriched and shown to cross-react with antibodies directed against inducible NOS (5).
Apoptosis, also called programmed cell death, is an evolutionary conserved phenomenon which regulates normal cellular turnover. Mitochondria are essential for at least certain forms of apoptosis (reviewed in Ref. 6). For example, cytochrome c, a mitochondrial protein that is part of the respiratory chain, triggers apoptosis once it is dislocated from the organelle. It is now well accepted that many factors drive cells into apoptotic death via mitochondrial cytochrome c release.
NO reacts with O 2 Ϫ to produce the powerful oxidizing agent, peroxynitrite (ONOO Ϫ ). Recently many studies focused on the role of NO and ONOO Ϫ in apoptosis (7)(8)(9)(10). Their exact source(s) and the mechanism(s) are, however, not yet fully elucidated. An increase in cytosolic Ca 2ϩ level caused by, e.g. glutamate receptor stimulation, is apoptogenic (reviewed in Ref. 11). Many recent reports show that mitochondrial Ca 2ϩ uptake is an essential step in Ca 2ϩ -induced apoptosis (12)(13)(14). This kind of programmed cell death is accompanied by increased NOS activity (12,13) and prevented by mitochondrial superoxide dismutase (SOD), MnSOD (10,15), or by the ONOO Ϫ scavenger, urate (16). Mitochondria produce NO in a Ca 2ϩ -dependent manner (4) and are a rich source of O 2 Ϫ . Therefore, intramitochondrial Ca 2ϩ -dependent ONOO Ϫ formation seems likely. Here we show that upon Ca 2ϩ uptake by isolated mitochondria, mtNOS is stimulated and cytochrome c is released in a Bcl-2-sensitive manner. We provide evidence that the observed cytochrome c release is due to intramitochondrial ONOO Ϫ formation because it is prevented by NOS inhibitors, an SOD mimic, and an ONOO Ϫ scavenger. We suggest that Ca 2ϩ -induced apoptosis is at least partly mediated via mtNOS. Mitochondrial Preparation-Isolation of rat liver mitochondria was performed by differential centrifugation as described (17). The protein content of mitochondria and the mitochondrial supernatants were determined by the Biuret method with bovine serum albumin as standard.
Detection of Cytochrome c Release-Freshly isolated mitochondria (50 mg of protein/ml) were incubated at 4°C in 0.1 M HEPES buffer, pH 7.1, containing protease inhibitors: aprotinin, pepstatin A, phenylmethanesulfonyl fluoride, and leupeptin (2 g/ml each). NOS inhibitors, * This work was financially supported by the Kantonale Zü rcherische Krebsliga (Zurich, Switzerland) (to P. G.) and by Leica and Avina-Foundation (Switzerland) (to S.D.K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Determination of Mitochondrial Lipid Peroxidation (LPO)-Samples were prepared as described above, except that mitochondria were incubated at 2 mg/ml and in the absence of protease inhibitors. Mitochondrial LPO was determined by thiobarbituric acid assay as described (19).
Determination of mtNOS Activity-Samples were prepared as described above except that mitochondria were incubated at 10 mg/ml and in the absence of protease inhibitors. mtNOS activity was determined by measurement of the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline as described (20) and is expressed as cpm/mg of mitochondrial protein.
Determination of Mitochondrial Functions-Mitochondria (1 mg/ml) were incubated in 0.1 M HEPES, pH 7.1, at room temperature. Oxygen consumption was measured under continuous stirring with a Clarketype electrode, as described (4). Mitochondrial transmembrane potential (⌬⌿) was measured in an Aminco DW-2A spectrophotometer at 511-533 nm in the presence of 10 M safranin as described (18).

RESULTS AND DISCUSSION
mtNOS stimulation induces cytochrome c release from isolated mitochondria in a manner that is prevented by two NOS inhibitors, L-NMMA and 1400W (Fig. 1A). This panel also shows that Bcl-2 prevents mtNOS-induced cytochrome c release, which indicates that the release is not because of a general mitochondrial damage followed by a nonspecific protein release, but it is a specific phenomenon relevant to apoptosis. A considerable number of recent reports show that endogenously formed NO induces Bcl-2-sensitive apoptosis (10,13,15,(21)(22)(23)(24), which is accompanied by mitochondrial dysfunction (10, 13), increased LPO (10) and ONOO Ϫ formation (10,24,25). It is also well demonstrated that increased cytosolic Ca 2ϩinduced apoptosis requires mitochondrial Ca 2ϩ uptake (12)(13)(14), is paralleled by increased NOS activity (12,13,26), and is prevented by lowering the mitochondrial O 2 Ϫ level by MnSOD (10,15) or by scavenging ONOO Ϫ with urate (16). The reaction of NO and O 2 Ϫ with the rate constant of 1.9 ϫ 10 10 M Ϫ1 s Ϫ1 (27) is one of the fastest reactions known in biology. Mitochondria produce NO in a Ca 2ϩ -dependent fashion (4), and they are well known sources of O 2 Ϫ radicals. Intramitochondrial Ca 2ϩ -dependent ONOO Ϫ formation is, therefore, very likely. Fig. 1A shows that mtNOS-induced cytochrome c release is prevented by the SOD mimic, MnTBAP, as well as by the ONOO Ϫ scavenger, urate. This finding strongly suggests that ONOO Ϫ is indeed formed within mitochondria and that it releases mitochondrial cytochrome c. This may explain the mechanism by which an elevated Ca 2ϩ level induces apoptosis in a manner that requires mitochondrial Ca 2ϩ uptake and is prevented by inhibiting NOS activity, lowering O 2 Ϫ level, and scavenging ONOO Ϫ .
It is known that ONOO Ϫ induces LPO (10, 28, 29). Fig. 2 shows that, upon mtNOS stimulation, LPO is increased in a manner that is sensitive to L-NMMA, Bcl-2, MnTBAP, and  urate. This figure also shows that exogenously added cytochrome c prevents increased LPO. From the elegant study by Cai and Jones (30) it is known that cytochrome c release is the cause and not the consequence of reactive oxygen species formation in mitochondria. We have recently confirmed this finding by showing that the decreased mitochondrial O 2 consumption, ⌬⌿ and Ca 2ϩ retention, consequent to ceramide-induced mitochondrial cytochrome c loss, are recovered by addition of exogenous cytochrome c (18). Very recently, it has also been reported that both the shape and the volume alterations of mitochondria because of cytochrome c loss are reversible (31). Upon release of cytochrome c from its native location within the hierarchically arranged mitochondrial respiratory complexes III and IV, complex III remains mostly reduced, and therefore, electrons become available for O 2 Ϫ formation. Prevention by Bcl-2 of cytochrome c release retains the possibility for electrons to flow from complex III to cytochrome c, and from there to complex IV and, consequently, decreases the availability of electrons for the formation of O 2 Ϫ , one of the two precursors of ONOO Ϫ .
In the experiments reported above, Ca 2ϩ , L-arginine, and BH 4 were provided to mitochondria. Fig. 1B shows that Ca 2ϩ per se is sufficient to trigger cytochrome c release in an L-NMMA and Bcl-2 sensitive manner and Fig. 1C shows that the effect of Ca 2ϩ is concentration-dependent. It is not surprising that Ca 2ϩ per se is sufficient for mtNOS-induced cytochrome c release, because other substrate/cofactors seem to be available in mitochondria in adequate concentrations. Intramitochondrial concentrations of L-arginine (32) and NADPH (33) are in the mM range. FAD and FMN are components of mitochondrial respiratory complexes I and II and, therefore, present in mitochondria (34). There is also evidence for the presence of BH 4 (35) and calmodulin (36,37) in mitochondria.
To establish that the observed cytochrome c release requires the uptake of Ca 2ϩ into the mitochondria, we used specific mitochondrial release and uptake blockers. Fig. 1C shows that sequestration of Ca 2ϩ within mitochondria by CSA, a compound known to block the specific mitochondrial Ca 2ϩ release pathway (38), aggravates mtNOS-induced cytochrome c release in an L-NMMA-sensitive manner. CSA is also reported to be a closure of the nonspecific solute transport across the inner mitochondrial membrane, the mitochondrial permeability transition pore, which is considered to be the reason for many features of apoptosis including cytochrome c release (reviewed in Ref. 39). Because CSA further increases the release of cytochrome c induced by mtNOS stimulation (Fig. 1C), we conclude that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition. Blockade of mitochondrial Ca 2ϩ uptake by RR prevents Ca 2ϩ -induced cytochrome c release (Fig. 1D). This finding is compatible with reports by other investigators that RR prevents apoptosis induced by elevated cytosolic Ca 2ϩ levels (12,14,40,41). Also, when mitochondria were de-energized by antimycin A plus oligomycin or by omitting succinate and therefore did not take up Ca 2ϩ , mtNOS activity was decreased (Fig. 3A) and cytochrome c release was prevented (Fig. 1D). This finding also confirms recent reports (18,31) that cytochrome c is not detached from the mitochondrial inner membrane because of a fall in mitochondrial transmembrane potential, e.g. upon uptake of Ca 2ϩ .
To address the mechanism by which Bcl-2 prevents mtNOSinduced cytochrome c release, we measured mitochondrial Ca 2ϩ uptake (dual wavelength spectroscopy using Arsenazo III as the probe) (18) and observed that Bcl-2 does not decrease mitochondrial Ca 2ϩ uptake (not shown). Additionally, we measured mtNOS activity and observed that Bcl-2 does not decrease it (Fig. 3A). Fig. 3B shows that Ca 2ϩ -induced decreased mitochondrial O 2 consumption is prevented by L-NMMA, but not by Bcl-2. Also Fig. 3C shows that uptake of Ca 2ϩ by respiring mitochondria causes a drastic fall in ⌬⌿ that is greatly prevented by L-NMMA but not Bcl-2. These findings demonstrate that decreased O 2 consumption and ⌬⌿ induced by mitochondrial Ca 2ϩ uptake is because of NO formation by mtNOS, and not cytochrome c release, and that prevention by Bcl-2 of mtNOS-induced cytochrome c release is not because of a decreased mtNOS activity. In contrast, mitochondrial deenergization by antimycin A prevents mtNOS-induced cytochrome c release (Fig. 1D) because of a drastic decrease in mtNOS activity (Fig. 3A).
Altogether, these results show that uptake of Ca 2ϩ by mitochondria followed by mtNOS stimulation causes mitochondrial cytochrome c release and increased LPO, and provide evidence that these events are mediated via intramitochondrial ONOO Ϫ formation. We propose that mtNOS plays a hitherto undetected role in apoptosis induced by elevated cytosolic Ca 2ϩ levels.