[Frontiers in Bioscience 6, d1161-1172, October 1, 2001]

NITRIC OXIDE: ONE OF THE MORE CONSERVED AND WIDESPREAD SIGNALING MOLECULES

UMR 5098, Défense et Résistance des Invertébrés Marins, IFR 56 "Eugène Bataillon", Université Montpellier II, cp 80, 2
place Eugène Bataillon, 34095-Montpellier Cédex 5, France

Jean Torreilles

TABLE OF CONTETNS

1. Abstract
2. Introduction
3. Mammals
4. Other vertebrates
5. Invertebrates
6. Low eukaryotic organisms
7. Plants
8. Bacteria
9. Perspectives
10. References

1. ABSTRACT

After the discovery of the vasodilatory functions 7 years ago (3) have identical pharmacological properties
of nitric oxide (NO), many signaling mechanisms and that endothelial cells in culture released NO. Then very
involving NO were identified through experiments on intense research into the biological functions of NO in
mammals. NO activates soluble guanylyl cyclase to living organisms began and the number of publications
induce the formation of cGMP, stimulates ADP- dealing with physiological and pathophysiological roles of
ribosylation of GAPDH to alter cell energy production, NO have increased exponentially year by year.
and combines with superoxide to generate peroxynitrite.
It then became clear that NO was a major messenger 3. MAMMALS
molecule in mammals, involved in the regulation of
blood vessel dilatation, immune function and NO is a water- and lipid-soluble radical gas that can
neurotransmission in the brain and peripheral nervous be involved in redox reactions, giving rise to nitrosylated
system. The wide spectrum of physiological effects of derivatives, and it combines with other radicals or transition
NO in mammals prompted researchers to look for the metal ions (Figure 1). Moreover, NO is electrically neutral
presence of NO in vertebrates and invertebrates. Parallel and readily diffuse through membranes (4).
findings on the presence of NO signaling in vertebrates
NO is produced during the conversion of L-
and invertebrates were observed, and then NO was found
arginine to L-citrulline (5) by a family of enzymes, i.e. NO-
to be a signaling molecule widely spread throughout the
synthases (NOS) (Figure 2). In mammalian cells, three
metazoan kingdom and whose functions were highly
distinct NOS isoforms (Table 1) have been isolated and
conserved during evolution. These features were
represent the products of three different genes (Figure 3).
extended to the entire animal kingdom after the
Type I (neuronal) and type III (endothelial) isoforms
discovery of NOS activity in protozoa, yeasts and
termed constitutive NOS, are constantly expressed and
bacteria. Recently, the involvement of NO and NOS in
Ca2+-dependent. The type II (macrophagic) isoform is not
plant disease resistance to infection was documented and
typically expressed in resting cells, must be induced by
many close similarities were detected between NO-
cytokines and microbiol products such as
dependent signaling mechanisms involved in plants and
lipopolysaccharides, and is Ca2+-independent. All NOS
those identified in animals. All of these results indicated
forms are homodimers of subunits which range between
that NO is one of the earliest and most widespread
130 and 160 kDa and have a bidomain structure (Figure 4):
signaling molecules in living organisms. This short
an oxygenase domain within the amino-terminal half and a
review was aimed at marshalling recent information that
reductase domain within the carboxy-terminal half. They
led to this conclusion.
use three substrates: L-arginine, NADPH and O2 and
2. INTRODUCTION require five cofactors: FAD, FMN, calmodulin (CaM),
tetrahydrobiopterin (BH4) and heme (6, 7).
Nitric oxide (NO) was a component of the Earth's Non-enzymatic NO production was recently
primitive atmosphere, but for metazoan biologists it is a detected in humans (8, 9), it results from the chemical
young molecule that appeared only 14 years ago. Indeed, reduction of inorganic nitrites and is effective in stomach,
although NO was known to be a central player in skin surface and ischemic heart.
bacterial bioenergetics (1), a biological function of NO in
eukaryotes was observed first by Palmer et al. (2) in Three major molecular targets of NO have been
1987. These authors showed that NO and an detected: soluble guanylyl cyclase (CGs), glyceraldehyde-3
endothelium-derived relaxing factor (EDRF) discovered phosphate dehydrogenase (GAPDH) and superoxide. They

1161
Nitric oxide in living systems

Figure 1. Chemical reactivity of nitric oxide

Figure 2. Biosynthesis of nitric oxide. The nitric oxide radical is generated through five-electron oxidation of a guanidino
nitrogen of L-arginine via the NG-hydroxyl-L-arginine intermediate.

1162
Nitric oxide in living systems

Table 1. Characteristics of the three human NOS isoforms

correspond to the three fields of NO reactivity: binding control, intestinal relaxation and antipathogen defense (16-
to metal ions, nitrosylation and combination with another 25).
radical.
But how can NO also function safely as a
CGs is a heme protein which catalazes the messenger and neurotransmitter when produced at low
formation of the guanosine cyclic 3'-5'-monophosphate concentrations and becomes cytotoxic, via peroxynitrite
(cGMP). NO binding to the heme iron of GCs leads to formation, when produced at high concentrations?
activation of the enzyme by formation of a nitrosyl-heme
complex which dislocates the iron-heme complex. This This paradoxical situation was clarified by kinetic
induces a conformational change in GCs which studies (26). Indeed, the reaction rate between NO and
stimulates its catalytic activity. GCs generates cGMP, superoxide to form peroxynitrite is 3-fold faster than the
which in turn specifically regulates protein rate of superoxide dismutation by superoxide dismutase
phosphorylation, ion channel conductivity and (SOD, the enzyme that protects tissues against superoxide).
phosphodiesterase activity. NO is the most potent and When SOD concentrations remain constant, as is generally
effective activator of GCs. This NO-cGMP signal the case in healthy cells and tissues, the NO concentration
transduction system accounts for the observed role of NO is thus the main driving force for peroxynitrite formation.
in central and peripheral nervous systems as well as in NO produced as a continuous flux by unregulated Ca2+-
cardiovascular, renal and pulmonary systems (10). independent type II NOS is then oriented toward defensive
activity.
- GAPDH (in brain, red blood cells and platelets)
is a glycolytic enzyme which normally oxidizes Moreover, the three NOS isoforms present in
glyceraldehyde phosphate and transfers electrons to mammals were found in other vertebrates.
NAD+. NO alters this reaction sequence by ADP-
ribosylating the enzyme (11, 12). ADP-ribose resulting 4. OTHER VERTEBRATES
from the cleavage of NAD+ is transferred to a GAPDH
S-nitrosylated cysteine and causes permanent inhibition The L-arginine-NO pathway observed in mammals
of the enzyme. has also been demonstrated in fishes (27, 28), for example,
neuronal type I NOS activity was detected in the brain and
- Superoxide produced during the burst of spinal cord of an amphibian, the frog Rana perezi (29, 30),
phagocytes is another major molecular target of NO. The as well as in all regions of the gut of a reptile, the estuarine
combination of both radicals generates peroxynitrite crocodile (35). This showed that the gastrointestinal
(Fig.1), a potent oxidizing and nitrating agent capable of motility in reptiles is under NO control. Inducible type II
attacking and modifying proteins, lipids and DNA and NOS was characterized in fish macrophages. The NOS
depleting antioxidant defenses (13, 14). from carp phagocytes (36-38) has 57% sequence identity to
human enzyme and contains binding sites for the same
Experiments by Kamosinska et al. (15) on lung cofactors as mammalian NOS. In rainbow trout (36-38), a
epithelial cells give an example of interaction between 130 kDa Ca2+-independent NOS was found in liver and
NO metabolic pathways. They showed that activation of head kidney. In chicken, an increase in nitrite production
the whole-cell current via NO/cGMP is inhibited by the by splenic macrophages was observed during the course of
peroxynitrite generated subsequently to the cytokine- coccidiosis (39, 40). Endothelial type III NOS was found in
induced expression of type II NOS. retina Muller cells of all phylogenetic classes of vertebrates
(41, 42). In day-old chicks, Rickard et al. (43) observed
All studies have demonstrated that NO plays a significant memory loss for 40 min post-training following
crucial role in physiological and pathophysiological the intracranial administration of diphenylene iodonium
processes in mammalian bodies: neurotransmission, (DPI) which is known to specifically inhibit endothelial
neuromodulation, vasodilatation, vasoprotection, secretary type III and inducible type II NOS. On the contrary, no

1163
Nitric oxide in living systems

Figure 3. The primary structure of the three human NOS isoforms (modified from Knowles and Moncada, 1994 (118)).
amphibians (29-31), reptiles (32) and birds (33, 34).

significant effect on memory formation was observed the extensive presence of NOS in invertebrate tissues (48-
after injection of three neuronal type I NOS specific 51).
inhibitors (3-bromo-7-nitroindazole, N-propyl-L-
arginine and S-methyl-L-thiocitrulline), at least until 2 h As for vertebrates, histochemical, physiological and
post-training. The authors concluded that the role played biochemical data show that the L-arginine-NO pathway is
by endothelial type III NOS in the memory formation is involved in many physiological functions in invertebrates.
distinct from any role that may be played by neuronal Some well documented reviews (52-56) summarize the
type I NOS. knowledge about the functional roles of NO in
invertebrates. The most specific contributions of
Therefore, studies of NOS activity in lower invertebrate studies to knowledge on physiological role of
vertebrates confirmed the results obtained with NO deal with development, synaptic plasticity, learning
mammalian cells and tissues and gave insight into the and sensorial functions associated with feeding and
physiological role of NO in two new domains: memory olfaction. This is probably due to the accessibility and
formation and light adaptation of retina. This latter NO simplicity of invertebrate nervous systems, which provide
function occurs via the NO-sGC-cGMP cascade as accurate information on all neurons and their functions.
shown by Blute et al. (42, 44) in turtle retina. Some recent reports suggest that NO could play similar
roles in vertebrates (57-60).
5. INVERTEBRATES
Moreover, recent studies on Drosophila
Soon after the detection of NO with EDRF (3), development (61) have firmly established the role of NO in
Radomski et al. (45) demonstrated the role of NO and the control of the balance between cell proliferation and
cGMP in the regulation of platelet adhesion in mammals. differentiation previously suggested by works on
Widening their studies to hemocytes from an arthropod, mammalian embryonic development (62).
the horseshoe crab Limulus polyphemus (46), these
authors observed that aggregation of these hemocytes is Interestingly, the studies of Colasenti et al. (63, 64)
under control of the NO they produce. This result revealed the presence of the NO-cGMP pathway in the
provides the first clear evidence of NO production in freshwater coelenterate Hydra. Since Hydra is the most
invertebrates. Since the horseshoe crab is a living fossil primitive organism with a nervous system, these
directly related to trilobites that lived 500 million years observations support high evolutionary conservation of the
ago, it appears that the NO-cGMP signaling cascade has NO-cGMP pathway.
been strongly conserved through the evolution process.
Some NOS from invertebrates have been
Since then, a number of studies were carried out characterized (50, 55, 64, 65). They exhibited properties
on invertebrates. After the histochemical detection of similar to that described for vertebrate enzymes. i) They
NADPH-diaphorase activity by Elofsson et al. (47) in used L-arginine as substrate, ii) formed NO
tissues of annelids, mollusks, arthropods, echinoderms stoichiometrically with L-citrulline, iii) were competitively
and urochordata, several studies provided evidence of inhibited by L-arginine analogs, and iiii) had apparent

1164
Nitric oxide in living systems

Figure 4. Schematic representation of the nitric oxide synthase monomer. Nitric oxide synthases are dimers whose monomers
are all composed of two domains; they use three substrates: L-arginine, NADPH and O2, and require five cofactors: FAD,
FMN, calmodulin (CaM), tetrahydrobiopterin (BH4) and heme.

Figure 5. Comparison of the primary structure of NOS from Drosophila and rat brain (modified from Regulski and Tully,
1995 (66)).

molecular weights in the 130-150 kDa range in SDS- The involvement of NO and NOS in invertebrate
polyacrylamide gel electrophoresis. However, it is not defensive systems is not as well documented.
clear if all isoforms described in vertebrates are present
in invertebrates. Ottaviani et al. (72) showed that the bacterial
clumping activity of Mytilus edulis haemolymph stimulated
The Drosophila NOS gene cloned in 1995 by by LPS was selectively and significantly reduced by NOS
Regulski and Trulli (66) encoded a protein having 43% inhibitors. This indirectly demonstrates that hemocytes of
amino acid sequence identity to rat type I NOS (Figure 5). M. edulis make and use NO as a bactericidal agent to kill
phagocytosed organisms.
Some years later, Ogunshola et al. (67) characterized
NOS from locust Schistocerca and pond snail Lymnaea. They Torreilles and coworkers, by spectroscopic
have more than 50% amino acid sequence identity to measurements (73-75) or using an anti-nitrotyrosine
vertebrate type I NOS and are calcium dependent (68). antibody (76), reported that upon challenge with zymosan
M. galloprovincialis and Crassostrea. gigas hemocytes
Yuda et al. (69, 70) cloned and expressed a generate peroxynitrite.
Ca2+-dependent NOS present in the salivary and CNS
of Rodnius prolixus, a blood sucking arthropod vector Franchini et al. (77) reported that immunocytes
of Chagas' disease. This enzyme was found to have a from Viviparus ater exhibit NOS activity when stimulated
130 kDa apparent molecular weight, and its amino acid with E. coli.. Calcium deprivation promoted 30% inhibition
sequence lacked part of the N terminal domain when of NOS activity, suggesting that a Ca2+-independent NOS
compared to mammalian type I and II NOS. isoform is present in immunocytes next to the Ca2+-
dependent isoform.
In a comparative analysis on NOS activity
performed in several mollusks, Moroz et al. (51, 71) Recently, in two arthropods, Drosophila
reported the presence (in Pleurobranchea and Aplysia) melanogaster and D. teissiere, Nappi et al. (78) observed
of NOS activities inhibitable by trifluoroperazine, a enhancement of NO production at a time when eggs of
calmoduline inhibitor. This suggests the presence of a Leptopilina boulardi, a Drosophila parasite, were destroyed
new NOS isoform since trifluoroperazine is ineffective by hemocyte-mediated melanoic encapsulation. This
in blocking the activity of the three NOS isoforms demonstrated that NO is involved in the Drosophila
described in mammals. defense system against eukaryotic parasites.

1165
Nitric oxide in living systems

Table 2. Characterization of NOS activity in some bacteria and yeast species

Protozoa Molecular weight Mammalian antibody Ca2+ NG-nitro-arginine L-Arg
(kDa) crossreactivity dependence Ki (µM) Km (µM)
Trypanosoma cruzi - - yes 40 (NMMA IC50) -
Leishmania donovani 110 Type I yes 6.8 4.9
Plasmodium falciparum < 100 Type II and III no 3.5 3.8
Yeasts
Saccharomices cervisiae 60 Type I yes - -
Candida tropicalis - - - - -
Bacteria
Nocardia 519 - yes 14.6 8.2

The identification of NOS in mollusks, The uptake of atmospheric NO (which results from
crustaceans, arthropods and other invertebrates confirms N2O oxidation into the atmosphere, and is one of the elements
the wide distribution of the L-arginine-NO signaling of the Earth's nitrogen cycle) by plant foliage and its
system in metazoan phyla and its strong evolutionary deleterious consequences have been known for many years
conservation (79, 80). (87, 88). However, NO synthesis in plants has been
demonstrated recently (89), and studies on the physiological
6. LOW EUKARYOTIC ORGANISMS role of NO in plants have just begun (90). However,
mounting evidence suggests that, as in animals, NO is an
Soluble NOS activities were reported to be ubiquitous signal in plants.
present in lower eukaryotic organisms, i.e. protozoan
parasites and yeast. In Trypanosoma cruzi epimastigote Noritake et al. (91) observed, in potato tuber tissue,
forms (81, 82), NOS was localized on the inner surface the induction of phytoalexin accumulation by NO, while
of cell membranes and in free cytosolic clusters in the experiments performed by Gouvea et al. (92) and Riberio et
body, the flagellum and apical extreme. NMDA and al. (93) strongly suggest that NO controls root growth and
excitatory amino acids such as glutamate activated NOS development in maize.
and increased intracellular levels of cGMP as described
for mammalian neural cells. Moreover, an effect of NO on peroxidases involved in
the cell wall lignification in plants was reported by Ferrer and
Soluble NOS activity requiring NADPH was Ros Barcelo (94).
purified 2800-fold from the protozoan parasite
Leshmania donovani (83). Its Western blot analysis with Evidence of the involvement of NO and NOS in plant
NOS type I antibody suggested a strong similarity with disease resistance to infection was recently obtained in
the neuronal NOS isoform from mammals. tobacco (95-99) and soybean (100).

In the light of Durner's results and the other published

Moreover, a Ca2+-independent NOS activity was
data, Durner et al. (101) proposed a model in which reactive
detected in Plasmodium falciparum infected red blood
oxygen species and salicylic acid act synergistically with NO
cells (84).
to activate the plant defense response to pathogens and
promote host cell death.
The three enzymes showed apparent molecular
weights of < 100 kD (Table 2).
Indeed, the most powerful weapon in plants against
pathogen attacks is rapid localized cell death at the site of
NOS activities were also detected in yeast
infection (102). This cell death response, called the
Saccharomyces cerevisiae (85) and Candida tropicalis (86).
hypersensitive response, deprives pathogens of access to a
nutrient source and limits their proliferation.
The presence of NOS activities in species
phylogenetically distant from mammalian species As emphasized by Cohn et al. (103), signaling
confirms the very ancient origin of NO synthesis in pathways involved in plant disease resistance mechanisms are
living organisms and the strong evolutionary just beginning to be unraveled, but many strong similarities
conservation of the NO transduction pathway. between these mechanisms and those involved in innate
immunity in animals have already been noted (104).
7. PLANTS
8. BACTERIA
As we have previously shown, NOS are the almost
exclusive sources of NO in animals. In plants, on the As claimed by Zumft (1), the central role played by
contrary, three major origins of NO production are effective: NO in bacterial energetics and in the global N cycle, vital to
i) the activity of nitrate and nitrate reductase, ii) the non- all organisms, has been known for a long time (105). Two
enzymatic light-mediated NO2- conversion by cartenoids, pathways for NO biosynthesis have been observed:
and iii) the activity of mammalian-type NOS (Figure 6). nitrification, which corresponds to oxidation of NH4+ to NO3-,

1166
Nitric oxide in living systems

formation, regulation of the balance between cell

proliferation and differentiation, and thus control of the
shape and size of adult animal organs. NO has a key role in
many physiological processes and could thus be considered
as the most ubiquitous biological messenger in living
organisms.

- for its occurrence and common functions in

virtually all living systems. NOS isoforms, with 150-160
kDa molecular weight and large conserved amino-acid
sequences, have been identified in mammals, vertebrates
and invertebrates. NOS were also characterized in protozoa
and prokaryotic cells but with molecular weights lower
than 100 kDa and amino acid sequences differing from
those of multicellular animal organisms. The main
functional roles and transduction pathways of NO
described for mammals have been found in other
vertebrates as well as in invertebrates. Few data are
available for protozoa, bacteria and plants, but it is
probable that future research will reveal similar signaling
functions in these organisms. The diffusion of NO as a
signaling molecule in living organisms from mammals
down to bacteria and plants suggests that NO could be one
of the most widespread biological messengers in all the
Figure 6. Enzymatic and non-enzymatic sources of NO living species.
in plants
- for the high conservation of its main signaling
and denitrification, which corresponds to the reduction pathways during evolution. Comparative investigations on
of NO3- and NO2- to N2 (106-113). the signaling functions of NO in different organisms have
also provided insight into the early evolutionary roles of
More recently, a third NO biosynthesis NO. Indeed, evidence on the involvement of NO in the
pathway was also detected in bacteria. Chen et al. (114, cellular defense system of Limulus ployphemus (an
115) isolated and purified, in Nocardia bacteria, NOS arthropod which has not evolved in 500 million years), as
which required for the same cofactors as mammalian well as in the feeding response of Hydra (the most
enzymes to ensure their activity, but they were found to primitive organism possessing a nervous system), suggest
have a 51.9 kDa apparent molecular weight. that NO could be one of the earliest biological signaling
molecules.
9. PERSPECTIVES
10. REFERENCES
NO is an odd endogenous control agent:
1. Zumft, W. G.: The biological role of nitric oxide in
- for its nature. NO is basically a gas found to bacteria. Arch Microbiol 160, 253-264 (1993)
be present everywhere in animal bodies. Locally
2. Palmer, R. M. Ferrige, A. G.& Moncada, S.: Nitric oxide
produced by many types of cells, it is an unstable radical
release accounts for the biological activity of endothelium-
with a biological half-life of only few seconds. NO acts
derived relaxing factor. Nature 327, 524-526. (1987)
as a transmitter, by diffusing in the immediate vicinity of
the cells from which it has been released, and as a 3. Furchgott, R. F. Carvalho, M. H. Khan, M. T.&
hormone, by binding to transporters and entering the Matsunaga, K.: Evidence for endothelium-dependent
blood stream to remote from the release site. Moreover, vasodilation of resistance vessels by acetylcholine. Blood
when NO acts as an intercellular messenger, it does not Vessels 24, 145-149 (1987)
react as classical messengers, by noncovalent binding to
specific receptors through complementarity of shape, but 4. Stamler, J. S. Singel, D. J.& Loscalzo, J.: Biochemistry
rather by covalent binding to its targets on the basis of of nitric oxide and its redox-activated forms. Science 258,
their redox potential. 1898-1902. (1992)

- for its large number of physiological roles. 5. Wiesinger, H.: Arginine metabolism and the synthesis of
NO serves as a multifunctional messenger affecting nitric oxide in the nervous system. Prog Neurobiol. 64,
many physiological processes such as apoptosis (116), 365-391 (2001)
regulation of vascular tone, macrophage-mediated
resistance to infection (117), control of synaptic 6. Gorren, A. C.& Mayer, B.: The versatile and complex
connectivity during development and synaptic enzymology of nitric oxide synthase. Biochemistry (Mosc)
plasticity in adults, mediation of olfactory memory 63, 734-743 (1998)

1167
Nitric oxide in living systems

7. Squadrito, G. L.& Pryor, W. A.: Oxidative chemistry 21. Bredt, D. S.: Endogenous nitric oxide synthesis:
of nitric oxide: the roles of superoxide, peroxynitrite, and biological functions and pathophysiology. Free Radic Res
carbon dioxide. Free Radic Biol Med 25, 392-403 (1998) 31, 577-596. (1999)

8. Lundberg, J. O. Weitzberg, E. Lundberg, J. M.& 22. Lundberg, J. O.& Weitzberg, E.: Nasal nitric oxide in
Alving, K.: Intragastric nitric oxide production in man. Thorax 54, 947-952. (1999)
humans: measurements in expelled air. Gut 35, 1543- 23. Torreilles, F. Salman-Tabcheh, S. Guerin, M.&
1546. (1994) Torreilles, J.: Neurodegenerative disorders: the role of
peroxynitrite. Brain Res Brain Res Rev 30, 153-163. (1999)
9. Weitzberg, E.& Lundberg, J. O.: Nonenzymatic nitric
oxide production in humans. Nitric Oxide 2, 1-7 (1998) 24. Li, H.& Forstermann, U.: Nitric oxide in the
pathogenesis of vascular disease. J Pathol 190, 244-254.
10. Schmidt, H. H. Lohmann, S. M.& Walter, U.: The (2000)
nitric oxide and cGMP signal transduction system:
regulation and mechanism of action. Biochim Biophys 25. Sethi, S.& Dikshit, M.: Modulation of
Acta 1178, 153-175. (1993) polymorphonuclear leukocytes function by nitric oxide.
Thromb Res 100, 223-247. (2000)
11. Brune, B. Dimmeler, S.& Lapetina, E. G.: NADPH: a
stimulatory cofactor for nitric oxide-induced ADP- 26. Crow, J. P.& Beckman, J. S.: The importance of
ribosylation reaction. Biochem Biophys Res Commun superoxide in nitric oxide-dependent toxicity: evidence for
182, 1166-1171. (1992) peroxynitrite-mediated injury. Adv Exp Med Biol 387, 147-
161. (1996)
12. Brune, B.& Lapetina, E. G.: Nitric oxide-induced
covalent modification of glycolytic enzyme 27. Schober, A. Malz, C. R. Schober, W.& Meyer, D. L.:
glyceraldehyde-3-phosphate dehydrogenase. Methods NADPH-diaphorase in the central nervous system of the
Enzymol 269, 400-407 (1996) larval lamprey (Lampetra planeri). J Comp Neurol 345, 94-
104. (1994)
13. Beckman, J. S. Chen, J. Ischiropoulos, H.& Crow, J.
P.: Oxidative chemistry of peroxynitrite. Methods
28. Olsson, C.& Holmgren, S.: Nitric oxide in the fish gut.
Enzymol 233, 229-240 (1994)
Comp Biochem Physiol A Physiol 118, 959-964. (1997)
14. Crow, J. P.& Ischiropoulos, H.: Detection and
quantitation of nitrotyrosine residues in proteins: in vivo 29. Munoz, M. Munoz, A. Marin, O. Alonso, J. R. Arevalo,
marker of peroxynitrite. Methods Enzymol 269, 185-194 R. Porteros, A.& Gonzalez, A.: Topographical distribution
(1996) of NADPH-diaphorase activity in the central nervous
system of the frog, Rana perezi. J Comp Neurol 367, 54-69.
15. Kamosinska, B. Radomski, A. Man, S. F. Radomski, (1996)
M. W.& Duszyk, M.: Role of inducible nitric-oxide
synthase in regulation of whole-cell current in lung 30. Munoz, M. Marin, O.& Gonzalez, A.: Localization of
epithelial cells. J Pharmacol Exp Ther 295, 500-505. NADPH diaphorase/nitric oxide synthase and choline
(2000) acetyltransferase in the spinal cord of the frog, Rana perezi.
J Comp Neurol 419, 451-470. (2000)
16. Lundberg, J. O. Farkas-Szallasi, T. Weitzberg, E.
Rinder, J. Lidholm, J. Anggaard, A. Hokfelt, T. 31. Virgili, M. Poli, A. Beraudi, A. Giuliani, A.& Villani,
Lundberg, J. M.& Alving, K.: High nitric oxide L.: Regional distribution of nitric oxide synthase and
production in human paranasal sinuses. Nat Med 1, 370- NADPH-diaphorase activities in the central nervous system
373. (1995) of teleosts. Brain Res. 901, 202-207 (2001)
17. Salman-Tabcheh, S. Guerin, M. C.& Torreilles, J.:
32. Hass, C. A. Hoffman, M. A. Densmore, L. D., 3rd&
Nitration of tyrosyl-residues from extra- and intracellular
Maxson, L. R.: Crocodilian evolution: insights from
proteins in human whole blood. Free Radic Biol Med 19,
immunological data. Mol Phylogenet Evol 1, 193-201.
695-698. (1995)
(1992)
18. Torreilles, J.& Guerin, M. C.: [Does nitric oxide
stress exist?]. C R Seances Soc Biol Fil 189, 389-400 33. Van Nerom, A. E. Ducatelle, R.& Haesebrouck, F.:
(1995) Oxygen radicals and nitric oxide production by turkey
respiratory macrophages. Dev Comp Immunol 22, 407-416.
19. Brenman, J. E.& Bredt, D. S.: Synaptic signaling by (1998)
nitric oxide. Curr Opin Neurobiol 7, 374-378. (1997)
34. von Bartheld, C. S.& Schober, A.: Nitric oxide synthase
20. Kannan, M. S. Guiang, S.& Johnson, D. E.: Nitric in learning-relevant nuclei of the chick brain: morphology,
oxide: biological role and clinical uses. Indian J Pediatr distribution, and relation to transmitter phenotypes. J Comp
65, 333-345. (1998) Neurol 383, 135-152. (1997)

1168
Nitric oxide in living systems

35. Olsson, C.& Gibbins, I.: Nitric oxide synthase in the 47. Elofsson, R. Carlberg, M. Moroz, L. Nezlin, L.&
gastrointestinal tract of the estuarine crocodile, Sakharov, D.: Is nitric oxide (NO) produced by invertebrate
Crocodylus porosus. Cell Tissue Res 296, 433-437. neurones? Neuroreport 4, 279-282. (1993)
(1999)
48. Talavera, E. Martinez-Lorenzana, G. Leon-Olea, M.
36. Djamgoz, M. B. Aguilo, R. Greenstreet, E. H. Sanchez-Alvarez, M. Sanchez-Islas, E.& Pellicer, F.:
Reynolds, R.& Wilkin, G. P.: Histochemistry of Histochemical distribution of NADPH-diaphorase in the
NADPH-diaphorase--a marker for neuronal nitric oxide cerebral ganglion of the crayfish Cambarellus montezumae.
synthase--in the carp retina. Neurochem Int 28, 283-291. Neurosci Lett 187, 177-180. (1995)
(1996)
49. Bascal, Z. A. Montgomery, A. Holden-Dye, L.
37. Saeij, J. P. Stet, R. J. Groeneveld, A. Verburg-van Williams, R. G. Thorndyke, M. C.& Walker, R. J.: NADPH
Kemenade, L. B. van Muiswinkel, W. B.& Wiegertjes, diaphorase activity in peptidergic neurones of the parasitic
G. F.: Molecular and functional characterization of a fish nematode, Ascaris suum. Parasitology 112, 125-134.
inducible-type nitric oxide synthase. Immunogenetics 51, (1996)
339-346. (2000)
50. Moroz, L. L. Chen, D. Gillette, M. U.& Gillette, R.:
38. Djamgoz, M. B. Sekaran, S. Angotzi, A. R. Haamedi, Nitric oxide synthase activity in the molluscan CNS. J
S. Vallerga, S. Hirano, J.& Yamada, M.: Light-adaptive Neurochem 66, 873-876. (1996)
role of nitric oxide in the outer retina of lower
vertebrates: a brief review. Philos Trans R Soc Lond B 51. Moroz, L. L.& Gillette, R.: NADPH-diaphorase
Biol Sci 355, 1199-1203. (2000) localization in the CNS and peripheral tissues of the
predatory sea-slug Pleurobranchaea californica. J Comp
39. Toth, T. E.: Nonspecific cellular defense of the avian Neurol 367, 607-622. (1996)
respiratory system: a review. Dev Comp Immunol 24,
121-139. (2000) 52. Martinez, A.: Nitric oxide synthase in invertebrates.
Histochem J 27, 770-776. (1995)
40. Qureshi, M. A. Heggen, C. L.& Hussain, I.: Avian
macrophage: effector functions in health and disease. 53. Muller, U.: The nitric oxide system in insects. Prog
Dev Comp Immunol 24, 103-119. (2000) Neurobiol 51, 363-381. (1997)

41. Haverkamp, S. Kolb, H.& Cuenca, N.: Endothelial 54. Jacklet, J. W.: Nitric oxide signaling in invertebrates.
nitric oxide synthase (eNOS) is localized to Muller cells Invert Neurosci 3, 1-14. (1997)
in all vertebrate retinas. Vision Res 39, 2299-2303.
(1999) 55. Colasanti, M.& Venturini, G.: Nitric oxide in
invertebrates. Mol Neurobiol 17, 157-174. (1998)
42. Blute, T. A. Lee, H. K. Huffmaster, T. Haverkamp,
S.& Eldred, W. D.: Localization of natriuretic peptides 56. Hurst, W. J. Moroz, L. L. Gillette, M. U.& Gillette, R.:
and their activation of particulate guanylate cyclase and Nitric oxide synthase imunolabeling in the molluscan CNS
nitric oxide synthase in the retina. J Comp Neurol 424, and peripheral tissues. Biochem Biophys Res Commun 262,
689-700. (2000) 545-548. (1999)

43. Rickard, N. S. Gibbs, M. E.& Ng, K. T.: Inhibition of 57. Teunissen, C. E. Steinbusch, H. W. Markerink-van
the endothelial isoform of nitric oxide synthase impairs Ittersum, M. De Bruijn, C. Axer, H.& De Vente, J.: Whole
long-term memory formation in the chick. Learn Mem 6, brain spheroid cultures as a model to study the
458-466. (1999) development of nitric oxide synthase-guanylate cyclase
signal transduction. Brain Res Dev Brain Res 125, 99-115.
44. Blute, T. A. Velasco, P.& Eldred, W. D.: Functional (2000)
localization of soluble guanylate cyclase in turtle retina:
58. Uylings, H. B.: Development of the cerebral cortex in
modulation of cGMP by nitric oxide donors. Vis
rodents and man. Eur J Morphol 38, 309-312. (2000)
Neurosci 15, 485-498. (1998)
59. Stamler, J. S.& Meissner, G.: Physiology of nitric oxide
45. Radomski, M. W. Palmer, R. M.& Moncada, S.: in skeletal muscle. Physiol Rev 81, 209-237. (2001)
Comparative pharmacology of endothelium-derived
relaxing factor, nitric oxide and prostacyclin in platelets. 60. Vidwans, S. J.& Su, T. T.: Cycling through
Br J Pharmacol 92, 181-187. (1987) development in Drosophila and other metazoa. Nat Cell
Biol 3, E35-39. (2001)
46. Radomski, M. W. Martin, J. F.& Moncada, S.:
Synthesis of nitric oxide by the hemocytes of the 61. Enikolopov, G. Banerji, J.& Kuzin, B.: Nitric oxide and
American horseshoe crab (Limulus polyphemus). Phil. Drosophila development. Cell Death Differ 6, 956-963.
Trans. R. Soc. Lond. B 334, 129-133 (1991) (1999)

1169
Nitric oxide in living systems

62. Eliasson, M. J. Blackshaw, S. Schell, M. J.& Snyder, galloprovincialis haemocytes in vitro. Fish & Shellfish
S. H.: Neuronal nitric oxide synthase alternatively spliced Immunol. 9, 509-518 (1999)
forms: prominent functional localizations in the brain.
Proc Natl Acad Sci U S A 94, 3396-3401. (1997) 75. Gourdon, I. Guérin, M. C. Torreilles, J.& Roch, P.:
Nitric Oxide Generation by Hemocytes of the Mussel
63. Colasanti, M. Lauro, G. M.& Venturini, G.: NO in Mytilus galloprovincialis. Nitric Oxide 5, 1-6. (2001)
hydra feeding response. Nature 374, 505. (1995)
76. Torreilles, J.& Romestand, B.: In vitro production of
64. Colasanti, M. Venturini, G. Merante, A. Musci, G.& peroxynitrite by haemocytes from marine bivalves: C-
Lauro, G. M.: Nitric oxide involvement in Hydra ELISA determination of 3-nitrotyrosine level in plasma
vulgaris very primitive olfactory-like system. J Neurosci proteins from Mytilus galloprovincialis and Crassostrea
17, 493-499. (1997) gigas. BMC Immunol 2, 1 (2001)

65. Palumbo, A. Di Cosmo, A. Gesualdo, I.& d'Ischia, 77. Franchini, A. Conte, A.& Ottaviani, E.: Nitric oxide: an
M.: A calcium-dependent nitric oxide synthase and ancestral immunocyte effector molecule. Adv
NMDA R1 glutamate receptor in the ink gland of Sepia Neuroimmunol 5, 463-478 (1995)
officinalis: a hint to a regulatory role of nitric oxide in
78. Nappi, A. J. Vass, E. Frey, F.& Carton, Y.: Nitric oxide
melanogenesis? Biochem Biophys Res Commun 235,
involvement in Drosophila immunity. Nitric Oxide 4, 423-
429-432. (1997)
430. (2000)
66. Regulski, M.& Trully, T.: Molecular and biochemical
79. Liu, L. Hausladen, A. Zeng, M. Que, L. Heitman, J.&
characterization of dNOS: a Drosophila Ca2+ /
Stamler, J. S.: A metabolic enzyme for S-nitrosothiol
calmodulin-dependent nitric oxide synthase. Proc Natl
conserved from bacteria to humans. Nature 410, 490-494.
Acad Sci U S A 92, 9072-9076. (1995)
(2001)
67. Ogunshola, O. Picot, J. Piper, M. Korneev, S.& 80. Kimbrell, D. A.& Beutler, B.: The evolution and
O'Shea, M.: Molecular analysis of the NO-cGMP genetics of innate immunity. Nat Rev Genet 2, 256-267.
signalling pathway in insect and molluscan CNS. Soc. (2001)
Neurosci. Abst. 21, 631 (1996)
81. Paveto, C. Pereira, C. Espinosa, J. Montagna, A. E.
68. O'Shea, M.& Park, J.-H.: Nitric oxide and orthograde Farber, M. Esteva, M. Flawia, M. M.& Torres, H. N.: The
neurotransmission in the CNS of the snail Lymnea nitric oxide transduction pathway in Trypanosoma cruzi. J
stagnalis. Soc. Neurosci. Abst. 22, 363 (1996) Biol Chem 270, 16576-16579. (1995)

69. Yuda, M. Hirai, M. Miura, K. Matsumura, H. Ando, 82. Goldstein, J. Paveto, C. Lopez-Costa, J. J. Pereira, C.
K.& Chinzei, Y.: cDNA cloning, expression and Alonso, G. Torres, H. N.& Flawia, M. M.: Immuno and
characterization of nitric-oxide synthase from the cytochemical localization of Trypanosoma cruzi nitric
salivary glands of the blood-sucking insect Rhodnius oxide synthase. Biocell 24, 217-222. (2000)
prolixus. Eur J Biochem 242, 807-812. (1996)
83. Basu, N. K. Kole, L. Ghosh, A.& Das, P. K.: Isolation
70. Yuda, M. Higuchi, K. Sun, J. Kureishi, Y. Ito, M.& of a nitric oxide synthase from the protozoan parasite,
Chinzei, Y.: Expression, reconstitution and Leishmania donovani. FEMS Microbiol Lett 156, 43-47.
characterization of prolixin-S as a vasodilator--a salivary (1997)
gland nitric-oxide-binding hemoprotein of Rhodnius
prolixus. Eur J Biochem 249, 337-342. (1997) 84. Ghigo, D. Todde, R. Ginsburg, H. Costamagna, C.
Gautret, P. Bussolino, F. Ulliers, D. Giribaldi, G. Deharo,
71. Moroz, L. L.& Gillette, R.: From Polyplacophora to E. Gabrielli, G.& et al.: Erythrocyte stages of Plasmodium
Cephalopoda: comparative analysis of nitric oxide falciparum exhibit a high nitric oxide synthase (NOS)
signalling in mollusca. Acta Biol Hung 46, 169-182 activity and release an NOS-inducing soluble factor. J Exp
(1995) Med 182, 677-688. (1995)
72. Ottaviani, E. Paeman, L. R. Cadet, P.& Stefano, G.
85. Kanadia, R. N. Kuo, W. N. McNabb, M.& Botchway,
B.: Evidence for nitric oxide production and utilization as
A.: Constitutive nitric oxide synthase in Saccharomyces
a bacteriocidal agent by invertebrate immunocytes. Eur J
cerevisiae. Biochem Mol Biol Int 45, 1081-1087. (1998)
Pharmacol 248, 319-324. (1993)
73. Torreilles, J. Guérin, M. C.& Roch, P.: Peroxidase- 86. Wilken, M.& Huchzermeyer, B.: Suppression of
release associated with phagocytosis in Mytilus mycelia formation by NO produced endogenously in
galloprovincialis haemocytes. Dev Comp Immunol 21, Candida tropicalis. Eur J Cell Biol 78, 209-213. (1999)
267-275. (1997)
87. Rowland, A. Murray, A. J.& Wellburn, A. R.: Oxides
74. Torreilles, J.& Guérin, M.-C.: Production of of nitrogen and their impact upon vegetation. Rev Environ
peroxynitrite by zymosan stimulation of Mytilus Health 5, 295-342 (1985)

1170
Nitric oxide in living systems

88. Hufton, C. A. Bestford, R. T.& Wellburn, R. A.: 101. Durner, J.& Klessig, D. F.: Nitric oxide as a signal in
Effects of NO (+ NO2) pollution on growth, nitrate plants. Curr Opin Plant Biol 2, 369-374. (1999)
reductase activities and associated protein contents in
glasshouse lettuce grown hydroponically in winter with 102. Hammond-Kosack, K. E.& Jones, J. D. G.: Resistance
CO2 enrichment. New Phytol. 133, 495-501 (1996) gene-dependent plant defense responses. Plant Cell 8,
1773-1791 (1996)
89. Wildt, J. Rockel, A. Rockel, P.& Segschneider, H. J.:
Emission of NO from higher plant species. J. Geophys. 103. Cohn, J. Sessa, G.& Martin, G. B.: Innate immunity in
Research 102, 5919-5927. (1997) plants. Curr Opin Immunol 13, 55-62. (2001)

90. Corpas, F. J. Barroso, J. B.& del Rio, L. A.: 104. Wendehenne, D. Pugin, A. Klessig, D. F.& Durner, J.:
Peroxisomes as a source of reactive oxygen species and Nitric oxide: comparative synthesis and signaling in animal
nitric oxide signal molecules in plant cells. Trends Plant and plant cells. Trends Plant Sci. 6, 177-183 (2001)
Sci. 6, 145-150 (2001)
105. Renner, E. D.: Production of Nitric Oxide and Nitrous
91. Noritake, T. Kawakita, K.& Doke, N.: Nitric oxide Oxide During Denitrification by Cornybacterium
induces phytoalexin accumulation in patato tuber tissue. Nephridii. J. Bacterial. 101, 821-826 (1970)
Plant Cell Physiol. 37, 113-116 (1996)
106. Firestone, M. K. Firestone, R. B.& Tiedje, J. M.:
92. Gouvea, C. M. C. P. Souza, J. F. Magalhaes, A. C. Nitric oxide as an intermediate in denitrification: evidence
N.& Martins, I. S.: NO-releasing substances that induce from nitrogen-13 isotope exchange. Biochem Biophys Res
growth elongation in maize root segments. Plant growth Commun 91, 10-16. (1979)
regulation 21, 182-187 (1997)
107. Goretski, J.& Hollocher, T. C.: Trapping of nitric
93. Ribeiro, E. A. Cunha, F. Q. Tamashiro, W. M.& oxide produced during denitrification by extracellular
Martins, I. S.: Growth phase-dependent subcellular hemoglobin. J Biol Chem 263, 2316-2323. (1988)
localization of nitric oxide synthase in maize cells. FEBS
Lett 445, 283-286. (1999) 108. Papen, H. von Berg, R. Hinkel, I. Thoene, B.&
Rennenberg, H.: Heterotrophic nitrification by Alcaligenes
- -
94. Ferrer, M. A.& Ros Barcelo, A.: Differential effect of faecalis: NO2 , NO3 , N2O, and NO production in
nitric oxide on peroxidase and H2O2 production by the exponentially growing cultures. Appl Environ Microbiol
55, 2068-2072. (1989)
xylem of Zinnia elengs. Plant cell and environment 22,
891-897 (1999) 109. Goretski, J.& Hollocher, T. C.: The kinetic and
isotopic competence of nitric oxide as an intermediate in
95. Durner, J. Wendehenne, D.& Klessig, D. F.: Defense denitrification. J Biol Chem 265, 889-895. (1990)
gene induction in tobacco by nitric oxide, cyclic GMP,
and cyclic ADP-ribose. Proc Natl Acad Sci U S A 95,
110. Goretski, J. Zafiriou, O. C.& Hollocher, T. C.: Steady-
10328-10333. (1998)
state nitric oxide concentrations during denitrification. J
Biol Chem 265, 11535-11538. (1990)
96. Huang, J. S.& Knopp, J. A., Involvment of nitric
oxide in Ralstonia solanacearum-induced hypersensitive
reaction in tobacco, INRA and Springer Editions, Berlin 111. Ye, R. W. Averill, B. A.& Tiedje, J. M.:
(1998) Denitrification: production and consumption of nitric
oxide. Appl Environ Microbiol 60, 1053-1058. (1994)
97. Clark, D. Durner, J. Navarre, D. A.& Klessig, D. F.:
Nitric oxide inhibition of tobacco catalase and ascorbate 112. Daum, M. Zimmer, W. Papen, H. Kloos, K. Nawrath,
peroxidase. Mol Plant Microbe Interact 13, 1380-1384. K.& Bothe, H.: Physiological and molecular biological
(2000) characterization of ammonia oxidation of the heterotrophic
nitrifier Pseudomonas putida. Curr Microbiol 37, 281-288.
98. Klessig, D. F. Durner, J. Noad, R. Navarre, D. A. (1998)
Wendehenne, D. Kumar, D. Zhou, J. M. Shah, J. Zhang,
S. Kachroo, P. Trifa, Y. Pontier, D. Lam, E.& Silva, H.: 113. Foissner, I. Wendehenne, D. Langebartels, C.&
Nitric oxide and salicylic acid signaling in plant defense. Durner, J.: In vivo imaging of an elicitor-induced nitric
Proc Natl Acad Sci U S A 97, 8849-8855. (2000) oxide burst in tobacco. Plant J 23, 817-824. (2000)

99. Navarre, D. A. Wendehenne, D. Durner, J. Noad, 114. Chen, Y.& Rosazza, J. P.: A bacterial nitric oxide
R.& Klessig, D. F.: Nitric oxide modulates the activity of synthase from a Nocardia species. Biochem Biophys Res
tobacco aconitase. Plant Physiol 122, 573-582. (2000) Commun 203, 1251-1258. (1994)

100. Delledonne, M. Xia, Y. Dixon, R. A.& Lamb, C.: 115. Chen, Y.& Rosazza, J. P.: Purification and
Nitric oxide functions as a signal in plant disease characterization of nitric oxide synthase (NOSNoc) from a
resistance. Nature 394, 585-588. (1998) Nocardia species. J Bacteriol 177, 5122-5128. (1995)

1171
Nitric oxide in living systems

116. Kolb, J. P. Roman, V. Mentz, F. Zhao, H. Rouillard,

D. Dugas, N. Dugas, B.& Sigaux, F.: Contribution of
nitric oxide to the apoptotic process in human B cell
chronic lymphocytic leukaemia. Leuk Lymphoma 40,
243-257 (2001)

117. Linares, E. Giorgio, S. Mortara, R. A. Santos, C. X.

Yamada, A. T.& Augusto, O.: Role of peroxynitrite in
macrophage microbicidal mechanisms in vivo revealed
by protein nitration and hydroxylation. Free Radic Biol
Med. 30, 1234-1242 (2001).

118. Knowles, R. G.& Moncada, S.: Nitric oxide

synthases in mammals. Biochem. J. 298, 249-258 (1994)

Key Words: Nitric Oxide, Mammals, Invertebrates,

Plants, Bacteria, Low Eukaryotic Organisms, Evolution,
Physiological Functions, Review

Send correspondence to: Jean Torreilles UMR 5098,

Défense et Résistance des Invertébrés Marins, IFR 56
"Eugène Bataillon", Université Montpellier II, cp 80, 2
place Eugène Bataillon, 34095-Montpellier Cédex 5,
France, Tel: 33 (0)4 67 14 34 29, Fax: 33 (0)4 67 14 34
29, E-mail: jtorreil@univ-montp2.fr

1172