Nitric oxide and its congeners in mitochondria: implications for apoptosis.

Apoptosis is an evolutionarily conserved form of physiologic cell death important for tissue development and homeostasis. The causes and execution mechanisms of apoptosis are not completely understood. Nitric oxide (NO) and its congeners, oxidative stress, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. Recent findings strongly suggest that mitochondria contain a factor or factors that upon release from the destabilized organelles, induce apoptosis. We have found that oxidative stress-induced release of Ca2+ from mitochondria followed by Ca2+ reuptake (Ca2+ cycling) causes destabilization of mitochondria and apoptosis. The protein product of the protooncogene bcl-2 protects mitochondria and thereby prevents apoptosis. We have also found that NO and its congeners can induce Ca2+ release from mitochondria. Thus, nitrogen monoxide (.NO) binds to cytochrome oxidase, blocks respiration, and thereby causes mitochondrial deenergization and Ca2+ release. Peroxynitrite (ONOO-), on the other hand, causes Ca2+ release from mitochondria by stimulating a specific Ca2+ release pathway. This pathway requires oxidized nicotinamide adenine dinucleotide (NAD+) hydrolysis to adenosine diphosphate ribose and nicotinamide. NAD+ hydrolysis is only possible when some vicinal thiols are cross-linked. ONOO- is able to oxidize them. Our findings suggest that NO and its congeners can induce apoptosis by destabilizing mitochondria via deenergization and/or by inducing a specific Ca2+ release followed by Ca2+ cycling.

cytotoxic events leading to formation of reactive oxygen species (ROS) and necrosis or apoptosis. These events include overactivation of protein kinase C, Ca2+/ calmodulin-dependent protein kinase II, phospholipases, proteases, protein phosphatases, xanthine oxidase, endonucleases, and nitric oxide synthase (NOS).
Although the exact role of Ca2+ in cell killing is unclear, a disturbance of mitochondrial Ca2+ handling can be fatal [for reviews see Nicotera et al. (5) and Richter (6)]. The normal Ca2+ uptake and release (cycling) across the inner mitochondrial membrane requires little energy (7).
However, when the Ca2+ release pathway is stimulated by prooxidants, cycling may become excessive and lead to loss of the mitochondrial membrane potential (DY), general leakiness of the inner mitochondrial membrane, inhibition of adenosine triphosphate (ATP) synthesis, mitochondrial damage, and cell death (8).
Several conditions, molecules, or organelles such as oxidative stress, Ca2+, proteases, nucleases, or mitochondria are considered participants of apoptosis, but at present it is not always clear whether they are required for or are the consequence of apoptosis. There is ample evidence that apoptosis is accompanied by oxidative stress [reviewed by Buttke and Sandstrom (9)]. A valuable tool used to elucidate the importance of oxidative stress is the protooncogene bcl-2, which stimulates an antioxidative response in cells and prevents apoptosis (10,11).
The requirement of Ca2+ for apoptosis is also controversial (12). Early reports suggested that a rise of the intracellular Ca2+ leads to apoptosis via endonuclease activation, and more recent work indicated that apoptosis is accompanied by shifts of Ca2+ between various intracellular pools. It is worth noting that cellular Ca2+ handling and ROS production are related. Thus, increased mitochondrial Ca2+ release followed by reuptake driven by DY (Ca2+ cycling) stimulates ROS production (13).

Mitochondria in Apoptosis
Although the causes and execution mechanisms of apoptosis are not clearly understood, oxidative stress, NO (NO indicates nitric oxide independent of its redox state, whereas NO, NO+, and NO refer to the nitrogen monoxide radical, nitrosonium ion, and nitroxyl anion, respectively) and its congeners, Ca2 , proteases, nucleases, and mitochondria are important mediators of apoptosis. Recendy, the role of mitochondria, particularly in apoptosis, was scrutinized (14,15). At present, their importance and exact role are elusive, but it is dear that mitochondria are both the target and the source of oxidative stress, NO, and Ca2 . During apoptosis DY, which is the driving force for mitochondrial ATP synthesis, declines, and maintenance of DY prevents apoptosis. Because apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand. Indeed, it was recently proposed that apoptosis induced by intracellular Ca2+ overload in neurons requires active mitochondria (16). We have proposed (15) that the cellular ATP level is an important determinant for cell death.
Another line of evidence also puts mitochondria on the center stage of apoptosis; when they are destabilized, for example, by Ca2+ cycling, they release proteins, some of which induce apoptosis. One is cytochrome c, which acts with cytosolic factors to induce nuclear apoptosis (17). The other is a 50-kDa protease that by itself suffices to cause nuclear apoptosis (18). The present knowledge suggests that mitochondria function as a cellular sensors of stress into which very different apoptosis induction pathways converge and that mitochondria act as central apoptotic executioner (19).
Presently, a popular concept in apoptosis is the so-called mitochondrial permeability transition, a phenomenon related to the opening of a putative pore in the inner mitochondrial membrane. In the author's opinion, the mitochondrial permeability transition represents the initial phase of unspecific damage to mitochondria that can, for example, be induced by mitochondrial Ca2+ cycling (20). Whether a pore opens or mitochondria are unspecifically damaged may not be relevant for apoptosis.
The crucial event appears to be the release of proapoptotic factors such as cytochrome c or proteases from mitochondria.

NO in Mitochondria
Biology ofNO NO presently receives enormous attention. It mediates beneficial responses such as maintenance of blood pressure, inhibition of platelet aggregation, tumoricidal activities, or destruction of foreign invaders in the immune response, and is probably of major importance in long-term memory.
However, when produced in excessive amounts, NO can become toxic, for example, during septic shock.
The dichotomy of NO is in part due to a broad array of redox species with distinctive properties and reactivities: NO+, 'NO, and NO-[reviewed by Stamler et al. (21) and Stamler (22)], and the ability of 'NO to combine with superoxide anion radicals (02--) to yield peroxynitrite (ONOO-) (23).
ONOO-is an efficient oxidant of thiols (24). Its in vivo formation was recently shown in a variety of cells (25). ONOOproduction is associated with the activation and expression of inducible NOS and implicated in the pathophysiology of diseases such as acute endotoxemia, inflammatory bowel disease, neurologic disorders, and atherosclerosis. Inhibition by superoxide dismutase, the 02-scavenger, of 'NO-mediated cytotoxicity suggests that ONOO-contributes to the NO-mediated biologic effects (26).
NO-is formed from 'NO by reduced superoxide dismutase (27). It is another NO congener that oxidizes thiols. NO-is, like ONOO-, neuroprotective at N-methyl-D-aspartate receptors because these compounds lead to disulfide formation at critical thiols of the redox modulator site of the receptor (28,29), which inhibits the Ca2+ entry into the cell.

NO in Mitochondria
Nitrogen Monoxide and the Regwlation of Cytochrome Oxidase. The most-cited and best-understood physiologic target of 'NO is guanyl cyclase. *NO binds to and stimulates it and thus controls cell functions via (cGMP), cGMP-gated channels, cGMPdependent protein kinases, and phosphodiesterases. However, 'NO also binds to cytochrome oxidase and reversibly inhibits respiration as seen with the isolated enzyme, submitochondrial particles, mitochondria, hepatocytes, brain nerve terminals, and astrocytes (30)(31)(32)(33)(34)(35)(36). Cytochrome oxidase inhibition is competitive with oxygen because of binding of NO to the oxygen binding site of the reduced enzyme (37,38). Why the inhibition is transient is not clear at the moment, but several findings point to consumption of NO as the underlying reason (39). Thus, cytochrome oxidase can reduce NO (40), 'NO can combine with 02 to form NON, and with 02 to form ONOO-.
Concentrations of 'NO measured in a range of biologic systems are similar to those that inhibit cytochrome oxidase and mitochondrial respiration, and inhibition of 'NO synthesis results in a stimulation of respiration in many systems. It was, therefore, proposed that 'NO exerts a good part of its physiologic and pathologic effects on cells by inhibiting cytochrome oxidase (41).
Presence ofNitric Oxide Synthase in Mitochondria. NOS is present within mitochondria (42). This offers exciting new insights into the biology of NO. For example, because the enzyme is stimulated by Ca2+ and located in the matrix or at the inner side of the inner mitochondrial membrane, this may provide a self-regulating system for mitochondrial Ca2+ homeostasis in which Ca2+ uptake by mitochondria would lead to 'NO formation. *NO could promote Ca2+ release by collapsing DY via inhibition of cytochrome oxidase.
Other Putative Targets ofNO in Mitochondria. 'NO not formed inside mitochondria may also have a profound impact on the organelles, as it is uncharged and can easily traverse membranes. For example, extramitochondrially formed *NO could combine in mitochondria with 02'and form ONOO-, which could then stimulate Ca2+ release from mitochondria with maintenance of DY. 'NO has been compared with ONOOas to its inhibitory capacity in mitochondria. Aconitase is the principal site of inhibition by ONOO-(and 02-), whereas this enzyme is resistant to 'NO (43,44).
Another possibility would be a *NOcatalyzed auto-ADP-ribosylation of mitochondrial NAD+-binding proteins, as reported for several cytosolic enzymes (45,46). Also, mitochondria are rich in glutathione and contain key sulfhydryl enzymes such as the adenine nucleotide translocator or creatine kinase that are putative targets of nitric oxide congeners.

Reactive Oxygen and Nitrogen Species as Regulators of Mitochondrial Ca2+ Homeostasis
Mitochondria and Cellular Qa2+ Homeostasis Intracellular Ca2+ regulates many processes. Its concentration is adjusted by binding to nonmembranous proteins, by mitochondria, and by membrane-bound Ca2+-ATPases located primarily in the plasma, nuclear, and endoplasmic reticular membrane [reviewed by Carafoli (7)]. Mitochondria contain Ca2+-sensitive targets regulated by moderate Ca2+ transients. These organelles are also able to take up Environmental Health Perspectives * Vol 106, Supplement 5 * October 1998 large amounts of Ca2+ and buffer the cytosolic Ca2+. They thereby act as safety devices against potentially toxic increases of cytosolic Ca2+ [reviewed by Richter and Kass (8)]. Mitochondria take up and release Ca2+ by separate routes. As a consequence, Ca2+ is cycled across their inner membranes (7).
Mitochondria are of central importance for physiologic Ca2+ handling. They act as a reservoir for Ca2+, provide much of the ATP used by Ca2+-ATPases, and Ca2+ regulates the activity of intramitochondrial dehydrogenases as well as nucleic acid and protein synthesis (47).
The importance of mitochondria as short-term modulators of cytosolic Ca2+ under physiologic conditions was until recently considered minor. However, there is now compelling evidence [reviewed by Rizzuto et al. (48) and Hajnoczky et al. (49)] that during physiologic cell stimulation, mitochondrial Ca2+ transport directly participates in the modulation and maintenance of cellular Ca2+ homeostasis. Several reports have additionally documented that physiologic cytosolic Ca2+ pulses are relayed into mitochondria of brain, liver, and Xenopous laevis oocytes (50)(51)(52).

Mitochondrial Ca2+ Release
In principle, Ca2+ can leave mitochondria in three ways: by nonspecific leakage through the inner membrane, by reversal of the influx carrier, and by an Na+-dependent or -independent release pathway (7,53). Only the latter two are physiologically relevant because they operate when DY is high. The Na+-dependent pathway predominates in mitochondria of heart, brain, skeletal muscle, adrenal cortex, brown fat, and most tumor tissue. The Na+-independent pathway is important in liver, kidney, lung, and smooth muscle mitochondria, probably exchanges Ca2+ with H+, and is linked to the redox state of mitochondrial pyridine nucleotides. Compounds causing their oxidation (and hydrolysis) promote Ca2+ release from intact mitochondria. This release has recently been reviewed (8,47,54).
Prooidant-Induced, NAD+-Linlwd C2+ Release NAD+ Hydrolysis Is Requiredfor Ca2+ Release from Intact Mitochondria. Hydrogen peroxide can stimulate a specific Ca2+ release pathway from intact mitochondria by oxidizing mitochondrial pyridine nucleotides through the activities of glutathione peroxidase, glutathione reductase, and the energy-linked transhydrogenase. Other prooxidants such as menadione, alloxan, and divicine also stimulate the specific Ca2+ release because they furnish NAD+. The specific Ca2+ release requires for its activation the hydrolysis of intramitochondrial NAD+ to ADP-ribose and nicotinamide and is prevented by inhibitors of NAD+ hydrolysis and protein mono(ADP-ribosyl)ation. Recent experiments reveal that NAD+ hydrolysis and therefore Ca2+ release is regulated by vicinal thiols in mitochondria. When reduced or alkylated, the thiols prevent hydrolysis, but when they are cross-linked, hydrolysis takes place. Cyclosporine A (CSA), which also prevents NAD+ hydrolysis, acts distal of these vicinal thiols [for recent review see Richter (55)]. NAD+  Gliotoxin, a fungal metabolite carrying a disulfide moiety, also promotes the Ca2+dependent intramitochondrial NAD+ hydrolysis and thereby the specific Ca2+ release but is inactive when its sulfurs are reduced or methylated (57). Thus, intramitochondrial, Ca2+-dependent NAD+ hydrolysis is prevented when some vicinal thiols are in the reduced SH-form, and occurs when they are connected, either by a cross-linking reagent or by oxidation to the disulfide form.
Peroynitrite Stimulates the Specfic Mitohondrial Ca2+ Release Pathway Because ONOO-oxidizes thiols and because vicinal thiols control the specific mitochondrial Ca2+ release pathway, we tested whether ONOO-is able to activate it (58). ONOO-induces Ca2+ release from rat liver mitochondria. This release occurs a) with preservation of DY, b) when mitochondrial pyridine nucleotides are oxidized but not when they are reduced, c) parallel to NAD+ hydrolysis, d) in a CSA-inhibitable manner, e) without inhibition of respi-ration, and J) without entry of extramitochondrial solutes such as sucrose into mitochondria. These findings convincingly show that ONOO-can mobilize mitochondrial Ca2+ by stimulating the specific, ADP-ribose-dependent release pathway.
.NO Inhibits Cytochrome Oxidase and Cuses Ca2+ Release from Mitochondria NO at submicromolar, physiologically relevant concentrations potently deenergizes isolated mitochondria (32). Deenergization is observed when mitochondria use respiratory substrates such as pyruvate plus malate, succinate, or ascorbate plus tetramethyl-phenylenediamine, but not when mitochondria are energized with ATP, and is due to a transient inhibition of cytochrome oxidase. The extent and duration of deenergization are determined by the concentration of NO and oxygen and the type of respiratory substrate. The 'NO-induced changes of the mitochondrial energy state are transient and are paralleled by release and reuptake of mitochondrial Ca2+. Importantly, cytochrome oxidase is particularly sensitive to NO at oxygen concentrations below 30 pM (59), i.e., at intracellular oxygen tensions. These findings reveal a direct action of NO on the mitochondrial respiratory chain and suggest that NO exerts some of its physiologic and pathologic effects by deenergizing mitochondria.
In freshly prepared hepatocytes, NO also deenergizes mitochondria (33). Deenergization is reversible at low concentrations but longer lasting at higher NO concentrations. The drop and the recovery of DY are accompanied by a rise and fall of cytosolic Ca2+ levels. NO at higher concentrations, provided by nitrosoglutathione in combination with dithiothreitol (GSNO/ DTT), kills hepatocytes. Killing is reduced when the cytosolic Ca2+ is chelated or when Ca2+ cycling by mitochondria is prevented by CSA. Apparently NO can kill cells by releasing Ca2+ from mitochondria and thereby flooding the cytosol with Ca2+.
bcl-2 link Oiddaive Stes Ca2 and the Mitochondrial Membrane Potential to Apoptosis Given that bcl-2 elicits an antioxidative response in cells, what are the biochemical mechanism(s) by which bcl-2 prevents apoptosis? It was shown (60,61) with the aid of ruthenium red, an inhibitor of the mitochondrial Ca2+ uptake, that one mechanism is the prevention of ROSinduced mitochondrial Ca2+ cycling, a process that results in a collapse of DY and in cellular ATP depletion. Thus, bcl-2 prevents disturbances of the cellular Ca2+ homeostasis and ROS production at the mitochondrial level. On the basis of these and other findings, it was suggested (6) Environmental Health Perspectives * Vol 106, Supplement 5 * October 1998 that a prooxidant-induced Ca2+ release from mitochondria, followed by Ca2+ cycling and ATP depletion, is a common cause of apoptosis. Accordingly, maintenance of DY stabilizes mitochondria and thereby prevents apoptosis. bcl-2 thus provides the link between the antioxidant defense system, Ca2 , and DY [reviewed by Bornkamm and Richter (62)]. In this context it is interesting to recall that many carcinoma cells have an increased DY (63). As prevention of apoptosis seems to contribute to carcinogenesis, it is conceivable that DY contributes to the decision between the life and death of a cell.
Whether the NO congeners cause apoptosis because of interference with mitochondrial respiration or Ca2+ handling is unclear, but it should be noted that the GSNO/DTT-induced killing of hepatocytes (33) appears to engage mitochondrial Ca2+ cycling. Other investigators have proposed that NO induces apoptosis via triggering the mitochondrial permeability transition (104).

Conclusion
An exciting new aspect in biology is the discovery that NO congeners have an enormous impact on mitochondria. NOS is active inside mitochondria. Physiologic concentrations of NO at physiologic cellular oxygen pressure inhibit cytochrome oxidase and thereby respiration. A transient inhibition of cytochrome oxidase by NO appears to be used in some forms of cell signaling. ONOO-, the product of the reaction between superoxide anion radicals and NO, can stimulate the specific calcium release pathway from mitochondria by oxidizing some vicinal thiols in mitochondria. Mounting evidence indicates that mitochondrial calcium handling and its modulation by reactive nitrogen species is important for apoptotic cell death. It appears that NO and its congeners can induce apoptosis by destabilizing mitochondria via deenergization and/or by inducing a specific Ca2+ release followed by Ca2+ cycling.