Am J Physiol Cell Physiol 291: C1225–C1231, 2006.
First published August 2, 2006; doi:10.1152/ajpcell.00307.2006.
Nitric oxide regulation of mitochondrial oxygen consumption I:
cellular physiology
Cecilia Giulivi,1 Kazunobu Kato,1 and Christopher Eric Cooper2
1
University of California, Department of Molecular Biosciences, Davis, California; and
University of Essex, Department of Biological Sciences, Colchester, United Kingdom
2
Submitted 2 June 2006; accepted in final form 28 July 2006
is the change in therapeutic strategies of diseases related to
mitochondrial oxidative/nitrative stress, such as amyotrophic
lateral sclerosis and Alzheimer’s. These strategies may have to
be modified when several new factors have to be considered:
oxygen consumption and availability, RONS, mitochondrial
metabolic state, and antioxidant defenses.
NO production in mitochondria is tightly regulated by temporospatial mechanisms. Calcium signal is central to temporal
regulation of NO production in mitochondria. Upon stimulation of calcium release from endoplasmic reticulum (ER),
calcium enters mitochondria via an electrogenic uniporter.
Calcium activates mtNOS releasing NO. In turn, NO inhibits
mitochondrial cellular respiration via binding to cytochrome c
oxidase. This effect has been observed in a variety of biological settings (13, 19, 70; Giulivi and Cooper, unpublished
observations). Spatial regulatory mechanisms of NO production allow a localized production of NO close to its targets.
Controlled NO production sites have the advantage of minimizing secondary reactions (e.g., peroxynitrite production);
however, at this point it is not clear how a highly diffusible
(and hydrophobic) molecule as NO will be confined to a certain
subcellular compartment. In the next section, we will discuss
various aspects of the tight control of the temporal and spatial
production of NO in mitochondria.
TEMPORAL REGULATION OF NO PRODUCTION
THE PRESENCE OF A CONSTITUTIVE nitric oxide synthase (NOS) in
mitochondria (mtNOS; 4, 5, 29, 31, 33, 34, 44, 83) has
provided the grounds to review several aspects of mitochondrial physiology.
Mitochondria are considered the main source of intracellular
reactive oxygen species (ROS) under normal, physiological
conditions via complex I and/or complex III (33). With the
presence of NO, this situation had to be reviewed to include
nitrogen species [reactive oxygen and nitrogen species
(RONS)]. This reexamination is not a simple nomenclature
issue, for the biochemistry (i.e., reactivity, production, and
localization, among others) changes the potential final biological effects. In this category we include RONS-mediated signal
transduction pathways (12, 60, 71, 80) and damaging processes
to important biomolecules including nitration of mitochondrial
proteins (3, 8, 12, 15, 23, 62, 63). One of the main implications
In vivo, two opposite processes regulate the steady-state
concentration of NO: production and consumption of NO.
The main consumption pathways of NO include its reaction
with oxymyoglobin or oxyhemoglobin (yielding nitrate) (22,
52), with superoxide anion to produce the highly reactive
species peroxynitrite (8, 62), and with oxygen (53). Although
the third pathway, namely the reaction between NO and oxygen, is slow (53), it may represent a critical clearance mechanism in hydrophobic milieu (28). As a result, the concentration
of NO can be adequately balanced by its production and
consumption allowing NO to operate as a signaling molecule.
In mammalians, NO is generated by nitric oxide synthase
(NOS). In other organisms, alternative pathways had been
found for the production of NO, e.g., nitrate reductase (59,
97–99). NO production can be regulated by expression of NOS
and/or modulation of NOS activity by calcium. For instance,
inducible NOS (iNOS) is overexpressed in response to infectious pathogens through transcription regulation (26, 27, 58,
90). Calcium signaling plays a crucial role at modulating
constitutive NOS activity such as endothelial NOS (eNOS),
Address for reprint requests and other correspondence: C. Giulivi, Univ. of
California, Dept. Molecular Biosciences, 1311 Haring Hall, Davis, CA 95616
(e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
nitric oxide synthase; cytochrome c oxidase
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Giulivi, Cecilia, Kazunobu Kato, and Christopher Eric Cooper.
Nitric oxide regulation of mitochondrial oxygen consumption I: cellular
physiology. Am J Physiol Cell Physiol 291: C1225–C1231, 2006. First
published August 2, 2006; doi:10.1152/ajpcell.00307.2006.—Mitochondrial biochemistry is complex, expanding from oxygen consumption,
oxidative phosphorylation, lipid catabolism, heme biosynthesis, to apoptosis, calcium homeostasis, and production of reactive oxygen species,
including nitric oxide (NO). The latter molecule is produced by a
mitochondrial NO synthase (mtNOS). The rates of consumption and
production determine the steady-state concentration of NO at subcellular
levels, leading to regulation of mitochondrial events. Temporospatial
processes tightly regulate production of NO in mitochondria to maximize
target effects and minimize deleterious reactions. Temporal regulatory
mechanisms of mtNOS include activation by calcium signaling and
transcriptional/translational regulations. Calcium-activated mtNOS inhibits mitochondrial respiration, resulting in a decrease of the oxygen
consumption. This negative regulation antagonizes the effects of calcium
on calcium-dependent dehydrogenases in the citric acid cycle, preventing
the formation of anoxic foci. Temporal regulation of NO production by
intracellular calcium signaling is a complex process, considering the
heterogeneous intracellular calcium response and distribution. NO production in mitochondria is spatially regulated by mechanisms that determine subcellular localization of mtNOS, likely acylation and proteinprotein interactions, in addition to transcriptional regulation as neuronal
NOS. Because NO rapidly decays in mitochondria, subcellular localization of mtNOS is crucial for NO to function as a signal molecule. These
temporospatial processes are biologically important to allow NO to act as
an effective signal molecule to regulate mitochondrial events such as
oxygen consumption and reactive oxygen species production.
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Fig. 1. Dynamics of calcium release by endoplasmic reticulum (ER) stores,
nitric oxide (NO) production, and oxygen gradients. Upon stimulation, the ER
stores release calcium. A: calcium stimulates the mitochondrial calciumdependent dehydrogenases, increasing the rate of oxygen consumption. As a
consequence, oxygen diffuses toward these mitochondria from other subcellular compartments. B: mitochondria located closer to ER calcium stores,
activate not only the calcium-dependent dehydrogenases but also NO synthase.
As a consequence, these mitochondria do not consume oxygen as fast as in A,
and less oxygen needs to diffuse to these areas to sustain oxidative phosphorylation, preventing formation of anoxic foci.
AJP-Cell Physiol • VOL
calcium efflux antiporter different from the uniporter, evidences the complexity of calcium homeostasis in mitochondria, and difficulty in delineating the temporal regulatory
mechanism of how calcium influences NO production and its
inhibitory effect on cellular respiration.
One clue for better understanding about the correlation
between calcium and NO-regulated mitochondrial respiration
is the information about cytosolic and intramitochondrial calcium concentrations that affect mitochondrial motility and
activity of the enzymes. Mitochondria have higher motility at
resting level of cytosolic calcium; when calcium rises, mitochondria motility is brought to a halt (100), facilitating the
uptake of calcium as well as providing ATP required for the
active calcium uptake by ER calcium-ATPases (94). The EC50
of inhibitory effects of calcium on mitochondrial motility has
been reported as 0.4 ␮M; cytosolic calcium concentration
([Ca2⫹]cyt) (100), corresponding to ⬃1.2 ␮M as free intramitochondrial calcium concentration ([Ca2⫹]mt). At this concentration of calcium, half of the mitochondria motility is halted,
whereas the three calcium-dependent dehydrogenases are almost fully activated (86% for ␣-ketoglutarate K0.5 ⫽ 0.2 ␮M
[Ca2⫹]mt and mtNOS is 55% activated K0.5 ⫽ 1.0 ␮M
[Ca2⫹]mt; see Ref. 84). Under these conditions, NO generated
from mtNOS may inhibit ⬃27% of the mitochondrial respiration via cytochrome c oxidase, resulting in a similar decrease
of the rate of oxidative phosphorylation. Thus mitochondria are
still able to produce ATP at a significant rate, preventing a
local point of anoxia. These effects were calculated at 220 ␮M
oxygen, concentrations considered hyperoxic for mitochondria; if hypoxic (or normoxic for mitochondria) conditions are
considered (10 –20 ␮M oxygen), these effects could be exacerbated (78). If cytosolic calcium concentrations increase further (e.g., 1 ␮M [Ca2⫹]cyt, equivalent to 2.3 ␮M [Ca2⫹]mt) as
mitochondria are located closer to microdomains with higher
calcium concentration, 71% of the mitochondria motility may
become arrested. Simultaneously, the activity of dehydrogenases reaches almost maximal activation (92%), and mtNOS is
70% activated, resulting in a 35% decrease of the oxygen
consumption. Therefore, activated mtNOS antagonizes the
effects of dehydrogenases on mitochondrial respiration, implying that mtNOS functions as a negative regulator for mitochondrial respiration especially at higher calcium concentrations
(84). Owed to this effect, mitochondria can still produce
enough ATP while avoiding the occurrence of anoxic foci.
This system allows oxygen to diffuse to mitochondria located
away from calcium microdomains, and these mitochondria
(exposed to lower calcium) have the potential to produce ATP
via the activation of calcium-dependent dehydrogenases without activating mtNOS (Fig. 1). In this model, not all mitochondria operate in synchrony as an energy source, but rather they
respond to the microenvironment changes. NO-induced inhibitory effect on mitochondrial respiration contributes to these
mechanisms and acts as one of the important regulatory factors
to control intracellular oxygen consumption.
Despite the clear evidences for the inhibitory role of NO on
mitochondrial respiration and its susceptibility to calcium signaling, there is still an unresolved question about the correlation among calcium concentration, mitochondrial motility, and
the temporal regulation for NO production. Provided higher
calcium concentrations stop mitochondria motility (100), the
density of mitochondria should become higher at ER sites.
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neuronal NOS (nNOS), and mtNOS (1, 31, 83). In addition,
these enzymes are also regulated at the transcriptional or
translational levels (10, 35, 42, 86).
The calcium-dependent mtNOS activity becomes relevant
considering the critical role of mitochondria at maintaining
calcium homeostasis. Calcium can regulate mitochondrial
functions by activating key metabolic enzymes (pyruvate dehydrogenase, NAD⫹-dependent isocitrate dehydrogenase, and
2-oxoglutarate dehydrogenase or ␣-ketoglutarate dehydrogenase) and mtNOS. The activation of calcium-dependent dehydrogenases leads to increased oxygen consumption, whereas
that of mtNOS results in a decreased oxygen uptake (Fig. 1).
The activation of the dehydrogenases or mtNOS is achieved at
different calcium concentrations (31) being the former activated at concentrations as low as 0.2 ␮M, whereas the latter
occurs at 1 ␮M (83, 84). Thus, ultimately the same signal,
calcium, regulates opposing effects on mitochondrial oxygen
consumption by means of different thresholds.
It is known that there is a heterogeneous distribution of
calcium in the cell (56), and how this distribution may affect
NO production is not well understood. Upon cell activation,
mitochondria are exposed to different calcium concentrations;
those close to endoplasmic reticulum (ER) calcium stores face
microdomains of higher calcium concentrations through the
opening of inositol trisphosphate-gated channel or ryanodine
receptor (57, 65, 66). In addition, it has been indicated that
ryanodine receptor isoform 1 is localized to the inner membrane of mitochondria, which may be one of the mechanisms
for calcium influx into mitochondria (7). This, in addition to a
REGULATION OF MITOCHONDRIAL OXYGEN CONSUMPTION BY NO
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lated nNOS expression (89). These findings imply that living
organisms have tightly regulated transcriptional/translational
mechanisms for controlling nNOS expression in response to
milieu changes. As an extension, it is possible that expression
of mtNOS is tightly regulated at the transcription/translation
steps.
SPATIAL REGULATION OF NO
NO is a small, hydrophobic species with a diffusion coefficient similar to that of oxygen (92). Applying Ficks’ law and
assuming a half-life for NO of ⬃1 s (43), its diffusion distance
would be ⬃140 ␮m. Bearing in mind that eukaryotic cells are
10 to 100 ␮m, then NO would have the potential to diffuse to
neighboring cells. In support of this concept, NO produced
from nNOS plays an important role as a diffusible neurotransmitter at synapse (50, 68, 69, 93). Analogously, it could be
hypothesized that NO produced by NOSs other than mtNOS
could diffuse to other cells, affecting the cellular respiration at
target cells. A few in vitro experiments endorse this hypothesis.
NO produced from endothelial cells or macrophages can diffuse to co-cultured smooth muscle cells (48), and non-activated
macrophages or fibroblasts (14), decreasing their oxygen consumption.
However, arguments based on biological observations may
favor a localized NO effect through a variety of mechanisms.
Some of these are the consumption of NO by mitochondria and
the localization or targeting of NOSs to specific subcellular
compartments.
NO consumption by mitochondria can influence how NO
exerts its biological effects. The consumption of NO is increased by 5 times in the presence of mitochondria (2 mg
protein/ml; 75). With the use of an unique microsensor tip that
can directly detect NO, withdrawing the tip (in 1-␮m increment) from a single mitochondrion proved that NO generated
by a mitochondrion could diffused only ⬍10 ␮m (41). These
experiments indicate that most of NO decays within the organelle and that extramitochondrial effects by NO endogenously produced from the organelles may be considered
minimum. This entails NO production system (mtNOS) in the
organelle, and the subcellular location of NOS would be
critical for NO to have any biological effects on mitochondrial
respiration.
Localization or targeting of NOS to specific subcellular
compartments may assure a confined effect. For mtNOS, this
could be achieved by means of posttranslational modifications
and protein-protein interactions.
Co- or posttranslational modifications (myristoylation and
palmitoylation) are essential for the proper localization of, for
example, eNOS at caveolae (73, 79). Caveolae are specialized
invaginations of the plasma membrane, found to be basic
structures for NO-triggered signaling by eNOS (2, 20, 30).
mtNOS has been found to be E-myristoylated (24), and based
on the biological functions of acylation, E-myristoylation probably contributes to trafficking of mtNOS to mitochondria or to
anchoring the enzyme to mitochondrial membranes.
Protein-protein interactions are another factor to determine
NOS localization at the subcellular level. nNOS-␣ has a PDZ
domain important for protein scaffolding for signal transduction (9, 25, 47, 74). In this regard, nNOS can associate with
postsynaptic density (PSD)-95, PSD-93 or skeletal muscle
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However, no evidence for these settings has been provided (11,
100).
Factors other than calcium may regulate the recruitment (or
derecruitment) of mitochondria to specific compartments in the
cell; for example protein-protein interactions or docking/anchoring points. Obviously, calcium-regulated NO production
in mitochondria will be affected by these mechanisms.
One of the key issues to resolve these questions is to
consider the intracellular transport system for mitochondria.
Docking of mitochondria to cytoskeletal structure, such as
intermediate filaments or microtubes, is important for intracellular transport of mitochondria, which determines intracellular
mitochondrial distribution (6, 54, 85). However, it is uncertain
how docking of mitochondria may alter physiological processes, among them NO production and consumption.
Another significant issue that might affect mitochondrial NO
production and its regulation of the oxygen consumption is
mitochondrial morphology. The typical representation of mitochondrion is a solitary, kidney-bean shaped organelle. A
predominant percentage of mitochondria are identified as single, isolated particles (85). However, a growing amount of
evidence has revealed that mitochondria can also have filamentous forms of long snake-like tube with branched reticulum,
composing extended network throughout the cytosol (6, 91,
95). Mitochondrial fusion may contribute to the formation of
this interconnected mitochondrial network in the cell (17, 37,
91, 95, 96). Importantly, the formation of an extensive mitochondrial network has functional importance (18, 77). For
example, the network might be essential for intracellular delivery of energy to specific cell compartments of the cell (77).
Using mammalian cells with mitochondria unable to fuse,
Chen et al. (18) showed that morphological defects could lead
to dysfunctional mitochondrial respiration. Mitochondrial fragmentation has been associated with increased ROS production
during hyperglycemia (101). These data suggest that morphological changes (fusion and formation of the network) could be
relevant to control mitochondrial respiration. Nonetheless,
there have been few evidences on how calcium-regulated NO
production is influenced by the dynamics of mitochondrial
morphology. To this end, considering the calcium dependence
of mtNOS and the three dehydrogenases, it will be of significance to elucidate how intracellular transportation and morphology of mitochondria affects the cross-talk between calcium and NO signaling in mitochondria.
In addition to calcium-mediated regulatory mechanisms of
NO production in mitochondria, expression of mtNOS can be
regulated at the transcriptional and translational levels (42).
Considering the identification of mtNOS as an nNOS isoform
(24), it could be safely assumed that regulatory mechanisms for
nNOS expression are operative for mtNOS. Several alternative
spliced variants have been found for nNOS, and the expression
of the variants is controlled in developmental stage-specific- or
tissue-specific-manners (51, 67, 76). These variants have different biological roles: for example distinct morphine tolerance
(45) or unique function for myotube fusion in differentiated
skeletal muscle (76). Some of nNOS variants lacking PDZ
domain such as nNOS-␤ and -␥ may have distinctive functions
owed to defective protein-protein interactions (9). Moreover, a
complex use of alternative promoters of nNOS (87) can affect
transcription patterns and translational efficiencies (49). In this
regard, a recent study indicated that hypoxia rapidly upregu-
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gen consumption, thus allowing other enzymes with a higher
Km for oxygen to be active (32, 38).
An example of the effect of oxygen redistribution, resulting
from the inhibition of the cellular respiration with NO, on the
destabilization of the hypoxia-inducible transcription factor
(HIF)-1␣ has been reported (36) (Fig. 2). The potential redistribution of oxygen allowed Pro hydroxylase to hydroxylate
HIF-1␣ (destabilization), preventing the upregulation of genes
related to glucose transport, glycolysis and erythropoiesis (21,
72). Intriguingly, it is not clear why such effect could be
observed in cultured cells when oxygen diffusion is expected to
be homogenous and not rate limiting. In addition, several
studies have shown that HIF-1␣ expression/stabilization is not
necessarily correlated with oxygen concentration in vivo (21).
The issue of oxygen gradient redistribution mediated by NO
becomes more puzzling when cells in culture are utilized. For
most culture cells, the energy budget is unavailable, meaning
whether main energy sources are from oxidative phosphorylation or from anaerobic glycolysis. Since Warburg’s earliest
research (88), it has been known that cancer cells preferably
utilize anaerobic glycolysis over oxidative phosphorylation.
Fig. 2. Effect of NO on the stabilization of hypoxia-inducible factor-1␣ (HIF-1␣). When oxygen is available, it can be
utilized as a substrate by the iron-dependent Pro hydroxylases and Asn hydroxylases. These enzymes hydroxylate the
HIF-1␣. Pro-hydroxylase introduces a hydroxyl group
within HIF-1␣ oxygen-dependent degradation domain
(ODDD; white domain) whereas Asn-hydroxylase hydroxylates the HIF-1␣ COOH-terminus end (gray domain). These
modifications cause the recruitment of the von HippelLindau tumor-suppressor protein (pVHL), which targets
HIF-1␣ for proteosomal degradation. The proteolysis of
HIF-1␣ prevents the transcriptional activity of this factor.
Oxygen may become available to the hydroxylases when NO
is inhibiting cytochrome c oxidase, promoting the diffusion
of oxygen to other subcellular sites. Conversely, when oxygen is not available, i.e., hypoxia or no NO available (16),
several pathways become activated (e.g., MAPK) that promotes the phosphorylation of HIF-1␣ (55, 64). Phosphorylation increases the transcriptional activity of HIF-1␣ via the
dimerization with HIF-1␤. The heterodimer activates the
transcription of several factors like vascular endothelial
growth factor (VEGF), erythropoietin (39), glucose transporters (GLUT1 and GLUT3), and several glycolytic genes.
HO, heme oxygenase; PI-3K, phosphatidylinositol 3-kinase;
UQ, ubiquinone.
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␣1-syntrophin via this domain (9, 46, 74), allowing a proper
target to neuronal postsynaptic densities (9).
In terms of mtNOS, protein-protein interaction via PDZ
motif has been recently observed (61; V. Haynes, N. N.
Sinitsyna, and C. Giulivi, unpublished observations). A physical interaction via PDZ domain has been demonstrated between subunit Va of cytochrome c oxidase and mtNOS, using
immunoelectron microscopy and immunoprecipitation experiments. Intriguingly, the interaction between cytochrome c
oxidase and mtNOS depends on calcium (V. Haynes, N. N.
Sinitsyna, and C. Giulivi, unpublished observations). This
implies that the spatial control might be affected also by
temporal control, specifically calcium signaling.
These temporospatial regulations of NO production in mitochondria may lead to important biological events. One of the
downstream outcomes is intracellular oxygen redistribution, as
previously discussed. Cells have intracellular gradients of oxygen (81, 82) from the plasma membrane to mitochondria,
where oxygen is consumed at a high rate by coupling electron
transport to oxidative phosphorylation. NO endogenously produced inhibits cytochrome c oxidase activity, decreasing oxy-
REGULATION OF MITOCHONDRIAL OXYGEN CONSUMPTION BY NO
GRANTS
This research was supported by National Institutes of Health Grants
ES-012691 and ES-005707 (to C. Giulivi) and Wellcome Trust (to C. E.
Cooper).
REFERENCES
1. Alderton WK, Cooper CE, and Knowles RG. Nitric oxide synthases:
structure, function and inhibition. Biochem J 357: 593– 615, 2001.
2. Anderson RG. The caveolae membrane system. Annu Rev Biochem 67:
199 –225, 1998.
3. Aulak KS, Koeck T, Crabb JW, and Stuehr DJ. Dynamics of protein
nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol 286:
H30 –H38, 2004.
4. Bates TE, Loesch A, Burnstock G, and Clark JB. Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain
and liver. Biochem Biophys Res Commun 213: 896 –900, 1995.
5. Bates TE, Loesch A, Burnstock G, and Clark JB. Mitochondrial nitric
oxide synthase: a ubiquitous regulator of oxidative phosphorylation?
Biochem Biophys Res Commun 218: 40 – 44, 1996.
6. Bereiter-Hahn J and Voth M. Dynamics of mitochondria in living
cells: shape changes, dislocations, fusion, and fission of mitochondria.
Microsc Res Tech 27: 198 –219, 1994.
7. Beutner G, Sharma VK, Giovannucci DR, Yule DI, and Sheu SS.
Identification of a ryanodine receptor in rat heart mitochondria. J Biol
Chem 276: 21482–21488, 2001.
8. Boczkowski J, Lisdero CL, Lanone S, Carreras MC, Aubier M, and
Poderoso JJ. Peroxynitrite-mediated mitochondrial dysfunction. Biol
Signals Recept 10: 66 – 80, 2001.
9. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano
DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, and Bredt DS.
Interaction of nitric oxide synthase with the postsynaptic density protein
PSD-95 and ␣1-syntrophin mediated by PDZ domains. Cell 84: 757–767,
1996.
10. Brenman JE, Xia HH, Chao DS, Black SM, and Bredt DS. Regulation
of neuronal nitric oxide synthase through alternative transcripts. Dev
Neurosci 19: 224 –231, 1997.
11. Brough D, Schell MJ, and Irvine RF. Agonist-induced regulation of
mitochondrial and endoplasmic reticulum motility. Biochem J 392:
291–297, 2005.
12. Brown GC and Borutaite V. Inhibition of mitochondrial respiratory
complex I by nitric oxide, peroxynitrite and S-nitrosothiols. Biochim
Biophys Acta 1658: 44 – 49, 2004.
13. Brown GC and Cooper CE. Nanomolar concentrations of nitric oxide
reversibly inhibit synaptosomal respiration by competing with oxygen at
cytochrome oxidase. FEBS Lett 356: 295–298, 1994.
AJP-Cell Physiol • VOL
14. Brown GC, Foxwell N, and Moncada S. Transcellular regulation of cell
respiration by nitric oxide generated by activated macrophages. FEBS
Lett 439: 321–324, 1998.
15. Cassina AM, Hodara R, Souza JM, Thomson L, Castro L, Ischiropoulos H, Freeman BA, and Radi R. Cytochrome c nitration by
peroxynitrite. J Biol Chem 275: 21409 –21415, 2000.
16. Chandel NS and Schumacker PT. Cellular oxygen sensing by mitochondria: old questions, new insight. J Appl Physiol 88: 1880 –1889,
2000.
17. Chen H and Chan DC. Emerging functions of mammalian mitochondrial fusion and fission. Hum Mol Genet 14: R283–289, 2005.
18. Chen H, Chomyn A, and Chan DC. Disruption of fusion results in
mitochondrial heterogeneity and dysfunction. J Biol Chem 280: 26185–
26192, 2005.
19. Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, and
Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide.
Implications for neurodegenerative diseases. FEBS Lett 345: 50 –54,
1994.
20. Couet J, Li S, Okamoto T, Ikezu T, and Lisanti MP. Identification of
peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
J Biol Chem 272: 6525– 6533, 1997.
21. Dery MAC, Michaud MD, and Richard DE. Hypoxia-inducible factor
1: regulation by hypoxic and non-hypoxic activators. Int J Biochem Cell
Biol 37: 535–540, 2005.
22. Eich RF, Li T, Lemon DD, Doherty DH, Curry SR, Aitken JF,
Mathews AJ, Johnson KA, Smith RD, Phillips GN Jr, and Olson JS.
Mechanism of NO-induced oxidation of myoglobin and hemoglobin.
Biochemistry 35: 6976 – 6983, 1996.
23. Elfering SL, Haynes VL, Traaseth NJ, Ettl A, and Giulivi C. Aspects,
mechanism, and biological relevance of mitochondrial protein nitration
sustained by mitochondrial nitric oxide synthase. Am J Physiol Heart
Circ Physiol 286: H22–H29, 2004.
24. Elfering SL, Sarkela TM, and Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem 277: 38079 –38086, 2002.
25. Fan JS and Zhang M. Signaling complex organization by PDZ domain
proteins. Neurosignals 11: 315–321, 2002.
26. Fang FC. Antimicrobial reactive oxygen and nitrogen species: concepts
and controversies. Nat Rev Microbiol 2: 820 – 832, 2004.
27. Fang FC. Perspectives series: host/pathogen interactions. Mechanisms
of nitric oxide-related antimicrobial activity. J Clin Invest 99: 2818 –
2825, 1997.
28. Ford PC, Wink DA, and Stanbury DM. Autoxidation kinetics of
aqueous nitric-oxide. FEBS Lett 326: 1–3, 1993.
29. Frandsen U, Lopez-Figueroa M, and Hellsten Y. Localization of nitric
oxide synthase in human skeletal muscle. Biochem Biophys Res Commun
227: 88 –93, 1996.
30. Frank PG, Woodman SE, Park DS, and Lisanti MP. Caveolin,
caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol
23: 1161–1168, 2003.
31. Ghafourifar P and Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 418: 291–296, 1997.
32. Giulivi C. Characterization and function of mitochondrial nitric-oxide
synthase. Free Radic Biol Med 34: 397– 408, 2003.
33. Giulivi C. Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism. Biochem J 332: 673– 679, 1998.
34. Giulivi C, Poderoso JJ, and Boveris A. Production of nitric oxide by
mitochondria. J Biol Chem 273: 11038 –11043, 1998.
35. Govers R and Rabelink TJ. Cellular regulation of endothelial nitric
oxide synthase. Am J Physiol Renal Physiol 280: F193–F206, 2001.
36. Hagen T, Taylor CT, Lam F, and Moncada S. Redistribution of
intracellular oxygen in hypoxia by nitric oxide: effect on HIF1␣. Science
302: 1975–1978, 2003.
37. Hales KG and Fuller MT. Developmentally regulated mitochondrial
fusion mediated by a conserved, novel, predicted GTPase. Cell 90:
121–129, 1997.
38. Haynes V, Elfering S, Traaseth N, and Giulivi C. Mitochondrial
nitric-oxide synthase: enzyme expression, characterization, and regulation. J Bioenerg Biomembr 36: 341–346, 2004.
39. Henry Y, Lepoivre M, Drapier JC, Ducrocq C, Boucher JL, and
Guissani A. EPR characterization of molecular targets for NO in mammalian cells and organelles. FASEB J 7: 1124 –1134, 1993.
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For example, myxothiazol, a specific inhibitor of the respiratory chain, can lower ATP production by ⬍25% in HeLa cells,
and substantial suppression (⬎50%) on cellular respiration can
be done with the combination of myxothiazol with a specific
inhibitor for glycolysis like 2-deoxyglucose (40). Therefore, to
ascertain effects of NO on cellular gradients, it is imperative to
evaluate the contribution of anaerobic glycolysis and oxidative
phosphorylation. In vitro experimental findings from cell culture system do not necessarily represent the actual biological
events in vivo. This raises a concern about validity of cell
culture experiments; it is a real challenge to recruit validated
experimental models to explore the in vivo events in mitochondria. Needless to say, this scenario applies for studies on
isolated organelles performed under hyperoxic conditions, and
thus more studies are required to comprehend the mechanism
for spatiotemporal regulation of NO production.
The implications of the modulation of mitochondrial respiration by NO via cytochrome c oxidase activity are a subject of
controversial discussions and views, which raise challenging
questions concerning the regulatory mechanisms and physiological relevance of in vivo modulation of mitochondrial oxygen consumption by NO.
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AJP-Cell Physiol • VOL
63. Radi R, Rodriguez M, Castro L, and Telleri R. Inhibition of mitochondrial electron transport by peroxynitrite. Arch Biochem Biophys 308:
89 –95, 1994.
64. Richard DE, Berra E, Gothie E, Roux D, and Pouyssegur J. p42/p44
mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1␣ (HIF-1␣) and enhance the transcriptional activity of HIF-1. J Biol
Chem 274: 32631–32637, 1999.
65. Rizzuto R, Pinton P, Brini M, Chiesa A, Filippin L, and Pozzan T.
Mitochondria as biosensors of calcium microdomains. Cell Calcium 26:
193–199, 1999.
66. Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz
LM, Tuft RA, and Pozzan T. Close contacts with the endoplasmic
reticulum as determinants of mitochondrial Ca2⫹ responses. Science 280:
1763–1766, 1998.
67. Saur D, Paehge H, Schusdziarra V, and Allescher HD. Distinct
expression of splice variants of neuronal nitric oxide synthase in the
human gastrointestinal tract. Gastroenterology 118: 849 – 858, 2000.
68. Schuman EM and Madison DV. Locally distributed synaptic potentiation in the hippocampus. Science 263: 532–536, 1994.
69. Schuman EM and Madison DV. Nitric oxide and synaptic function.
Annu Rev Neurosci 17: 153–183, 1994.
70. Schweizer M and Richter C. Nitric oxide potently and reversibly
deenergizes mitochondria at low oxygen tension. Biochem Biophys Res
Commun 204: 169 –175, 1994.
71. Schweizer M and Richter C. Peroxynitrite stimulates the pyridine
nucleotide-linked Ca2⫹ release from intact rat liver mitochondria. Biochemistry 35: 4524 – 4528, 1996.
72. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem
Pharmacol 64: 993–998, 2002.
73. Shaul PW. Regulation of endothelial nitric oxide synthase: location,
location, location. Annu Rev Physiol 64: 749 –774, 2002.
74. Sheng M and Sala C. PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24: 1–29, 2001.
75. Shiva S, Brookes PS, Patel RP, Anderson PG, and Darley-Usmar
VM. Nitric oxide partitioning into mitochondrial membranes and the
control of respiration at cytochrome c oxidase. Proc Natl Acad Sci USA
98: 7212–7217, 2001.
76. Silvagno F, Xia H, and Bredt DS. Neuronal nitric-oxide synthase-␮, an
alternatively spliced isoform expressed in differentiated skeletal muscle.
J Biol Chem 271: 11204 –11208, 1996.
77. Skulachev VP. Mitochondrial filaments and clusters as intracellular
power-transmitting cables. Trends Biochem Sci 26: 23–29, 2001.
78. Solien J, Haynes V, and Giulivi C. Differential requirements of calcium
for oxoglutarate dehydrogenase and mitochondrial nitric-oxide synthase
under hypoxia: impact on the regulation of mitochondrial oxygen consumption. Comp Biochem Physiol A 142: 111–117, 2005.
79. Sullivan JC and Pollock JS. NOS 3 subcellular localization in the
regulation of nitric oxide production. Acta Physiol Scand 179: 115–122,
2003.
80. Szabo C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett
140 –141: 105–112, 2003.
81. Takahashi E and Asano K. Mitochondrial respiratory control can
compensate for intracellular O2 gradients in cardiomyocytes at low PO2.
Am J Physiol Heart Circ Physiol 283: H871–H878, 2002.
82. Takahashi E, Endoh H, and Doi K. Intracellular gradients of O2 supply
to mitochondria in actively respiring single cardiomyocyte of rats. Am J
Physiol Heart Circ Physiol 276: H718 –H724, 1999.
83. Tatoyan A and Giulivi C. Purification and characterization of a nitricoxide synthase from rat liver mitochondria. J Biol Chem 273: 11044 –
11048, 1998.
84. Traaseth N, Elfering S, Solien J, Haynes V, and Giulivi C. Role of
calcium signaling in the activation of mitochondrial nitric oxide synthase
and citric acid cycle. Biochim Biophys Acta 1658: 64 –71, 2004.
85. Wagner OI, Lifshitz J, Janmey PA, Linden M, McIntosh TK, and
Leterrier JF. Mechanisms of mitochondria-neurofilament interactions.
J Neurosci 23: 9046 –9058, 2003.
86. Wang Y, Newton DC, and Marsden PA. Neuronal NOS: gene structure, mRNA diversity, and functional relevance. Crit Rev Neurobiol 13:
21– 43, 1999.
87. Wang Y, Newton DC, Robb GB, Kau CL, Miller TL, Cheung AH,
Hall AV, VanDamme S, Wilcox JN, and Marsden PA. RNA diversity
has profound effects on the translation of neuronal nitric oxide synthase.
Proc Natl Acad Sci USA 96: 12150 –12155, 1999.
291 • DECEMBER 2006 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
40. Izyumov DS, Avetisyan AV, Pletjushkina OY, Sakharov DV, Wirtz
KW, Chernyak BV, and Skulachev VP. “Wages of fear”: transient
threefold decrease in intracellular ATP level imposes apoptosis. Biochim
Biophys Acta 1658: 141–147, 2004.
41. Kanai AJ, Pearce LL, Clemens PR, Birder LA, VanBibber MM,
Choi SY, de Groat WC, and Peterson J. Identification of a neuronal
nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc Natl Acad Sci USA 98: 14126 –14131, 2001.
42. Kato K and Giulivi C. Critical overview of mitochondrial nitric-oxide
synthase. Front Biosci 11: 2725–2738, 2006.
43. Kelm M and Schrader J. Nitric-oxide release from the isolated guineapig heart. Eur J Pharmacol 155: 317–321, 1988.
44. Kobzik L, Stringer B, Balligand JL, Reid MB, and Stamler JS.
Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem Biophys Res Commun 211: 375–381,
1995.
45. Kolesnikov YA, Pan YX, Babey AM, Jain S, Wilson R, and Pasternak GW. Functionally differentiating two neuronal nitric oxide synthase
isoforms through antisense mapping: evidence for opposing NO actions
on morphine analgesia and tolerance. Proc Natl Acad Sci USA 94:
8220 – 8225, 1997.
46. Kone BC. Protein-protein interactions controlling nitric oxide synthases.
Acta Physiol Scand 168: 27–31, 2000.
47. Kone BC, Kuncewicz T, Zhang W, and Yu ZY. Protein interactions
with nitric oxide synthases: controlling the right time, the right place, and
the right amount of nitric oxide. Am J Physiol Renal Physiol 285:
F178 –F190, 2003.
48. Lancaster JR Jr. Simulation of the diffusion and reaction of endogeneously produced nitric oxide. Proc Natl Acad Sci USA 91: 8137– 8141,
1994.
49. Landry JR, Mager DL, and Wilhelm BT. Complex controls: the role
of alternative promoters in mammalian genomes. Trends Genet 19:
640 – 648, 2003.
50. Ledo A, Frade J, Barbosa RM, and Laranjinha J. Nitric oxide in
brain: diffusion, targets and concentration dynamics in hippocampal
subregions. Mol Aspects Med 25: 75– 89, 2004.
51. Lee MA, Cai L, Hubner N, Lee YA, and Lindpaintner K. Tissue- and
development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase. J Clin Invest 100: 1507–
1512, 1997.
52. Liu X, Miller MJ, Joshi MS, Sadowska-Krowicka H, Clark DA, and
Lancaster JR Jr. Diffusion-limited reaction of free nitric oxide with
erythrocytes. J Biol Chem 273: 18709 –18713, 1998.
53. Liu X, Miller MJ, Joshi MS, Thomas DD, and Lancaster JR Jr.
Accelerated reaction of nitric oxide with O2 within the hydrophobic
interior of biological membranes. Proc Natl Acad Sci USA 95: 2175–
2179, 1998.
54. Miller KE and Sheetz MP. Direct evidence for coherent low velocity
axonal transport of mitochondria. J Cell Biol 173: 373–381, 2006.
55. Minet E, Michel G, Mottet D, Raes M, and Michiels C. Transduction
pathways involved in hypoxia-inducible factor-1 phosphorylation and
activation. Free Radic Biol Med 31: 847– 855, 2001.
56. Monteith GR and Blaustein MP. Heterogeneity of mitochondrial matrix free Ca2⫹: resolution of Ca2⫹ dynamics in individual mitochondria
in situ. Am J Physiol Cell Physiol 276: C1193–C1204, 1999.
57. Montero M, Alonso MT, Carnicero E, Cuchillo-Ibanez I, Albillos A,
Garcia AG, Garcia-Sancho J, and Alvarez J. Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2⫹ transients that modulate secretion. Nat Cell Biol 2: 57– 61, 2000.
58. Nathan C and Shiloh MU. Reactive oxygen and nitrogen intermediates
in the relationship between mammalian hosts and microbial pathogens.
Proc Natl Acad Sci USA 97: 8841– 8848, 2000.
59. Nohl H, Staniek K, and Kozlov AV. The existence and significance of
a mitochondrial nitrite reductase. Redox Rep 10: 281–286, 2005.
60. Packer MA and Murphy MP. Peroxynitrite causes calcium efflux from
mitochondria which is prevented by cyclosporin A. FEBS Lett 345:
237–240, 1994.
61. Persichini T, Mazzone V, Polticelli F, Moreno S, Venturini G,
Clementi E, and Colasanti M. Mitochondrial type I nitric oxide synthase physically interacts with cytochrome c oxidase. Neurosci Lett 384:
254 –259, 2005.
62. Radi R, Cassina A, Hodara R, Quijano C, and Castro L. Peroxynitrite
reactions and formation in mitochondria. Free Radic Biol Med 33:
1451–1464, 2002.
REGULATION OF MITOCHONDRIAL OXYGEN CONSUMPTION BY NO
AJP-Cell Physiol • VOL
96. Yaffe MP. The machinery of mitochondrial inheritance and behavior.
Science 283: 1493–1497, 1999.
97. Yamamoto-Katou A, Katou S, Yoshioka H, Doke N, and Kawakita
K. Nitrate reductase is responsible for elicitin-induced nitric oxide
production in Nicotiana benthamiana. Plant Cell Physiol 47: 726 –735,
2006.
98. Yamasaki H and Sakihama Y. Simultaneous production of nitric oxide
and peroxynitrite by plant nitrate reductase: in vitro evidence for the
NR-dependent formation of active nitrogen species. FEBS Lett 468:
89 –92, 2000.
99. Yamasaki H, Sakihama Y, and Takahashi S. An alternative pathway
for nitric oxide production in plants: new features of an old enzyme.
Trends Plant Sci 4: 128 –129, 1999.
100. Yi M, Weaver D, and Hajnoczky G. Control of mitochondrial motility
and distribution by the calcium signal: a homeostatic circuit. J Cell Biol
167: 661– 672, 2004.
101. Yu T, Robotham JL, and Yoon Y. Increased production of reactive
oxygen species in hyperglycemic conditions requires dynamic change of
mitochondrial morphology. Proc Natl Acad Sci USA 103: 2653–2658,
2006.
291 • DECEMBER 2006 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
88. Warburg O, Posener K, and Negelein E. On the metabolism of
carcinoma cells. Biochemische Zeitschrift 152: 309 –344, 1924.
89. Ward ME, Toporsian M, Scott JA, Teoh H, Govindaraju V, Quan A,
Wener AD, Wang G, Bevan SC, Newton DC, and Marsden PA. Hypoxia
induces a functionally significant and translationally efficient neuronal NO
synthase mRNA variant. J Clin Invest 115: 3128 –3139, 2005.
90. Weinberg J. Human mononuclear phagocyte nitric oxide production and
inducible nitric oxide synthase expression. In: Nitric Oxide and Infection,
edited by Fang FC. New York: Kluwer Academic/Plenum, 1999, p. 95–150.
91. Westermann B. Merging mitochondria matters: cellular role and molecular machinery of mitochondrial fusion. EMBO Rep 3: 527–531, 2002.
92. Wise DL and Houghton G. Solubilities and diffusivities of oxygen in
hemolyzed human blood solutions. Biophys J 9: 36 –53, 1969.
93. Wood J and Garthwaite J. Models of the diffusional spread of nitric
oxide: implications for neural nitric oxide signalling and its pharmacological properties. Neuropharmacology 33: 1235–1244, 1994.
94. Xu W, Liu L, Charles IG, and Moncada S. Nitric oxide induces
coupling of mitochondrial signalling with the endoplasmic reticulum
stress response. Nat Cell Biol 6: 1129 –1134, 2004.
95. Yaffe MP. Dynamic mitochondria. Nat Cell Biol 1: E149 –150, 1999.
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