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


Jean Torreilles


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


After the discovery of the vasodilatory functions of nitric oxide (NO), many signaling mechanisms involving NO were identified through experiments on mammals. NO activates soluble guanylyl cyclase to induce the formation of cGMP, stimulates ADP-ribosylation of GAPDH to alter cell energy production, and combines with superoxide to generate peroxynitrite. It then became clear that NO was a major messenger molecule in mammals, involved in the regulation of blood vessel dilatation, immune function and neurotransmission in the brain and peripheral nervous system. The wide spectrum of physiological effects of NO in mammals prompted researchers to look for the presence of NO in vertebrates and invertebrates. Parallel findings on the presence of NO signaling in vertebrates and invertebrates were observed, and then NO was found to be a signaling molecule widely spread throughout the metazoan kingdom and whose functions were highly conserved during evolution. These features were extended to the entire animal kingdom after the discovery of NOS activity in protozoa, yeasts and bacteria. Recently, the involvement of NO and NOS in plant disease resistance to infection was documented and many close similarities were detected between NO-dependent signaling mechanisms involved in plants and those identified in animals. All of these results indicated that NO is one of the earliest and most widespread signaling molecules in living organisms. This short review was aimed at marshalling recent information that led to this conclusion.


Nitric oxide (NO) was a component of the Earth's primitive atmosphere, but for metazoan biologists it is a young molecule that appeared only 14 years ago. Indeed, although NO was known to be a central player in bacterial bioenergetics (1), a biological function of NO in eukaryotes was observed first by Palmer et al. (2) in 1987. These authors showed that NO and an endothelium-derived relaxing factor (EDRF) discovered\ 7 years ago (3) have identical pharmacological properties and that endothelial cells in culture released NO. Then very intense research into the biological functions of NO in living organisms began and the number of publications dealing with physiological and pathophysiological roles of NO have increased exponentially year by year.


NO is a water- and lipid-soluble radical gas that can be involved in redox reactions, giving rise to nitrosylated derivatives, and it combines with other radicals or transition metal ions (Figure 1). Moreover, NO is electrically neutral and readily diffuse through membranes (4).

NO is produced during the conversion of L-arginine to L-citrulline (5) by a family of enzymes, i.e. NO-synthases (NOS) (Figure 2). In mammalian cells, three distinct NOS isoforms (Table 1) have been isolated and represent the products of three different genes (Figure 3). Type I (neuronal) and type III (endothelial) isoforms termed constitutive NOS, are constantly expressed and Ca2+-dependent. The type II (macrophagic) isoform is not typically expressed in resting cells, must be induced by cytokines and microbiol products such as lipopolysaccharides, and is Ca2+-independent. All NOS forms are homodimers of subunits which range between 130 and 160 kDa and have a bidomain structure (Figure 4): an oxygenase domain within the amino-terminal half and a reductase domain within the carboxy-terminal half. They use three substrates: L-arginine, NADPH and O2 and require five cofactors: FAD, FMN, calmodulin (CaM), tetrahydrobiopterin (BH4) and heme (6, 7).

Non-enzymatic NO production was recently detected in humans (8, 9), it results from the chemical reduction of inorganic nitrites and is effective in stomach, skin surface and ischemic heart.

Three major molecular targets of NO have been detected: soluble guanylyl cyclase (CGs), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and superoxide. They correspond to the three fields of NO reactivity: binding to metal ions, nitrosylation and combination with another radical.

CGs is a heme protein which catalazes the formation of the guanosine cyclic 3'-5'-monophosphate (cGMP). NO binding to the heme iron of GCs leads to activation of the enzyme by formation of a nitrosyl-heme complex which dislocates the iron-heme complex. This induces a conformational change in GCs which stimulates its catalytic activity. GCs generates cGMP, which in turn specifically regulates protein phosphorylation, ion channel conductivity and phosphodiesterase activity. NO is the most potent and effective activator of GCs. This NO-cGMP signal transduction system accounts for the observed role of NO in central and peripheral nervous systems as well as in cardiovascular, renal and pulmonary systems (10).

- GAPDH (in brain, red blood cells and platelets) is a glycolytic enzyme which normally oxidizes glyceraldehyde phosphate and transfers electrons to NAD+. NO alters this reaction sequence by ADP-ribosylating the enzyme (11, 12). ADP-ribose resulting from the cleavage of NAD+ is transferred to a GAPDH S-nitrosylated cysteine and causes permanent inhibition of the enzyme.

- Superoxide produced during the burst of phagocytes is another major molecular target of NO. The combination of both radicals generates peroxynitrite (Fig.1), a potent oxidizing and nitrating agent capable of attacking and modifying proteins, lipids and DNA and depleting antioxidant defenses (13, 14).

Experiments by Kamosinska et al. (15) on lung epithelial cells give an example of interaction between NO metabolic pathways. They showed that activation of the whole-cell current via NO/cGMP is inhibited by the peroxynitrite generated subsequently to the cytokine-induced expression of type II NOS.

All studies have demonstrated that NO plays a crucial role in physiological and pathophysiological processes in mammalian bodies: neurotransmission, neuromodulation, vasodilatation, vasoprotection, secretary control, intestinal relaxation and antipathogen defense (16-25).

But how can NO also function safely as a messenger and neurotransmitter when produced at low concentrations and becomes cytotoxic, via peroxynitrite formation, when produced at high concentrations?

This paradoxical situation was clarified by kinetic studies (26). Indeed, the reaction rate between NO and superoxide to form peroxynitrite is 3-fold faster than the rate of superoxide dismutation by superoxide dismutase (SOD, the enzyme that protects tissues against superoxide). When SOD concentrations remain constant, as is generally the case in healthy cells and tissues, the NO concentration is thus the main driving force for peroxynitrite formation. NO produced as a continuous flux by unregulated Ca2+-independent type II NOS is then oriented toward defensive activity.

Moreover, the three NOS isoforms present in mammals were found in other vertebrates.


The L-arginine-NO pathway observed in mammals has also been demonstrated in fishes (27, 28), for example, neuronal type I NOS activity was detected in the brain and spinal cord of an amphibian, the frog Rana perezi (29, 30), as well as in all regions of the gut of a reptile, the estuarine crocodile (35). This showed that the gastrointestinal motility in reptiles is under NO control. Inducible type II NOS was characterized in fish macrophages. The NOS from carp phagocytes (36-38) has 57% sequence identity to human enzyme and contains binding sites for the same cofactors as mammalian NOS. In rainbow trout (36-38), a 130 kDa Ca2+-independent NOS was found in liver and head kidney. In chicken, an increase in nitrite production by splenic macrophages was observed during the course of coccidiosis (39, 40). Endothelial type III NOS was found in retina Muller cells of all phylogenetic classes of vertebrates (41, 42). In day-old chicks, Rickard et al. (43) observed significant memory loss for 40 min post-training following the intracranial administration of diphenylene iodonium (DPI) which is known to specifically inhibit endothelial type III and inducible type II NOS. On the contrary, no significant effect on memory formation was observed after injection of three neuronal type I NOS specific inhibitors (3-bromo-7-nitroindazole, N-propyl-L-arginine and S-methyl-L-thiocitrulline), at least until 2 h post-training. The authors concluded that the role played by endothelial type III NOS in the memory formation is distinct from any role that may be played by neuronal type I NOS.

Therefore, studies of NOS activity in lower vertebrates confirmed the results obtained with mammalian cells and tissues and gave insight into the physiological role of NO in two new domains: memory formation and light adaptation of retina. This latter NO function occurs via the NO-sGC-cGMP cascade as shown by Blute et al. (42, 44) in turtle retina.


Soon after the detection of NO with EDRF (3), Radomski et al. (45) demonstrated the role of NO and cGMP in the regulation of platelet adhesion in mammals. Widening their studies to hemocytes from an arthropod, the horseshoe crab Limulus polyphemus (46), these authors observed that aggregation of these hemocytes is under control of the NO they produce. This result provides the first clear evidence of NO production in invertebrates. Since the horseshoe crab is a living fossil directly related to trilobites that lived 500 million years ago, it appears that the NO-cGMP signaling cascade has been strongly conserved through the evolution process.

Since then, a number of studies were carried out on invertebrates. After the histochemical detection of NADPH-diaphorase activity by Elofsson et al. (47) in tissues of annelids, mollusks, arthropods, echinoderms and urochordata, several studies provided evidence of

the extensive presence of NOS in invertebrate tissues (48-51).

As for vertebrates, histochemical, physiological and biochemical data show that the L-arginine-NO pathway is involved in many physiological functions in invertebrates. Some well documented reviews (52-56) summarize the knowledge about the functional roles of NO in invertebrates. The most specific contributions of invertebrate studies to knowledge on physiological role of NO deal with development, synaptic plasticity, learning and sensorial functions associated with feeding and olfaction. This is probably due to the accessibility and simplicity of invertebrate nervous systems, which provide accurate information on all neurons and their functions. Some recent reports suggest that NO could play similar roles in vertebrates (57-60).

Moreover, recent studies on Drosophila development (61) have firmly established the role of NO in the control of the balance between cell proliferation and differentiation previously suggested by works on mammalian embryonic development (62).

Interestingly, the studies of Colasenti et al. (63, 64) revealed the presence of the NO-cGMP pathway in the freshwater coelenterate Hydra. Since Hydra is the most primitive organism with a nervous system, these observations support high evolutionary conservation of the NO-cGMP pathway.

Some NOS from invertebrates have been characterized (50, 55, 64, 65). They exhibited properties similar to that described for vertebrate enzymes. i) They used L-arginine as substrate, ii) formed NO stoichiometrically with L-citrulline, iii) were competitively inhibited by L-arginine analogs, and iiii) had apparent molecular weights in the 130-150 kDa range in SDS-polyacrylamide gel electrophoresis. However, it is not clear if all isoforms described in vertebrates are present in invertebrates.

The Drosophila NOS gene cloned in 1995 by Regulski and Trulli (66) encoded a protein having 43% amino acid sequence identity to rat type I NOS (Figure 5).

Some years later, Ogunshola et al. (67) characterized NOS from locust Schistocerca and pond snail Lymnaea. They have more than 50% amino acid sequence identity to vertebrate type I NOS and are calcium dependent (68).

Yuda et al. (69, 70) cloned and expressed a Ca2+-dependent NOS present in the salivary and CNS of Rodnius prolixus, a blood sucking arthropod vector of Chagas' disease. This enzyme was found to have a 130 kDa apparent molecular weight, and its amino acid sequence lacked part of the N terminal domain when compared to mammalian type I and II NOS.

In a comparative analysis on NOS activity performed in several mollusks, Moroz et al. (51, 71) reported the presence (in Pleurobranchea and Aplysia) of NOS activities inhibitable by trifluoroperazine, a calmoduline inhibitor. This suggests the presence of a new NOS isoform since trifluoroperazine is ineffective in blocking the activity of the three NOS isoforms described in mammals.

The involvement of NO and NOS in invertebrate defensive systems is not as well documented.

Ottaviani et al. (72) showed that the bacterial clumping activity of Mytilus edulis haemolymph stimulated by LPS was selectively and significantly reduced by NOS inhibitors. This indirectly demonstrates that hemocytes of M. edulis make and use NO as a bactericidal agent to kill phagocytosed organisms.

Torreilles and coworkers, by spectroscopic measurements (73-75) or using an anti-nitrotyrosine antibody (76), reported that upon challenge with zymosan M. galloprovincialis and Crassostrea. gigas hemocytes generate peroxynitrite.

Franchini et al. (77) reported that immunocytes from Viviparus ater exhibit NOS activity when stimulated with E. coli.. Calcium deprivation promoted 30% inhibition of NOS activity, suggesting that a Ca2+-independent NOS isoform is present in immunocytes next to the Ca2+-dependent isoform.

Recently, in two arthropods, Drosophila melanogaster and D. teissiere, Nappi et al. (78) observed enhancement of NO production at a time when eggs of Leptopilina boulardi, a Drosophila parasite, were destroyed by hemocyte-mediated melanoic encapsulation. This demonstrated that NO is involved in the Drosophila defense system against eukaryotic parasites.

The identification of NOS in mollusks, crustaceans, arthropods and other invertebrates confirms the wide distribution of the L-arginine-NO signaling system in metazoan phyla and its strong evolutionary conservation (79, 80).


Soluble NOS activities were reported to be present in lower eukaryotic organisms, i.e. protozoan parasites and yeast. In Trypanosoma cruzi epimastigote forms (81, 82), NOS was localized on the inner surface of cell membranes and in free cytosolic clusters in the body, the flagellum and apical extreme. NMDA and excitatory amino acids such as glutamate activated NOS and increased intracellular levels of cGMP as described for mammalian neural cells.

Soluble NOS activity requiring NADPH was purified 2800-fold from the protozoan parasite Leshmania donovani (83). Its Western blot analysis with NOS type I antibody suggested a strong similarity with the neuronal NOS isoform from mammals.

Moreover, a Ca2+-independent NOS activity was detected in Plasmodium falciparum infected red blood cells (84).

The three enzymes showed apparent molecular weights of < 100 kD (Table 2).

NOS activities were also detected in yeast Saccharomyces cerevisiae (85) and Candida tropicalis (86).

The presence of NOS activities in species phylogenetically distant from mammalian species confirms the very ancient origin of NO synthesis in living organisms and the strong evolutionary conservation of the NO transduction pathway.


As we have previously shown, NOS are the almost exclusive sources of NO in animals. In plants, on the contrary, three major origins of NO production are effective: i) the activity of nitrate and nitrate reductase, ii) the non-enzymatic light-mediated NO2- conversion by cartenoids, and iii) the activity of mammalian-type NOS (Figure 6).

The uptake of atmospheric NO (which results from N2O oxidation into the atmosphere, and is one of the elements of the Earth's nitrogen cycle) by plant foliage and its deleterious consequences have been known for many years (87, 88). However, NO synthesis in plants has been demonstrated recently (89), and studies on the physiological role of NO in plants have just begun (90). However, mounting evidence suggests that, as in animals, NO is an ubiquitous signal in plants.

Noritake et al. (91) observed, in potato tuber tissue, the induction of phytoalexin accumulation by NO, while experiments performed by Gouvea et al. (92) and Riberio et al. (93) strongly suggest that NO controls root growth and development in maize.

Moreover, an effect of NO on peroxidases involved in the cell wall lignification in plants was reported by Ferrer and Ros Barcelo (94).

Evidence of the involvement of NO and NOS in plant disease resistance to infection was recently obtained in tobacco (95-99) and soybean (100).

In the light of Durner's results and the other published data, Durner et al. (101) proposed a model in which reactive oxygen species and salicylic acid act synergistically with NO to activate the plant defense response to pathogens and promote host cell death.

Indeed, the most powerful weapon in plants against pathogen attacks is rapid localized cell death at the site of infection (102). This cell death response, called the hypersensitive response, deprives pathogens of access to a nutrient source and limits their proliferation.

As emphasized by Cohn et al. (103), signaling pathways involved in plant disease resistance mechanisms are just beginning to be unraveled, but many strong similarities between these mechanisms and those involved in innate immunity in animals have already been noted (104).


As claimed by Zumft (1), the central role played by NO in bacterial energetics and in the global N cycle, vital to all organisms, has been known for a long time (105). Two pathways for NO biosynthesis have been observed: nitrification, which corresponds to oxidation of NH4+ to NO3-, and denitrification, which corresponds to the reduction of NO3- and NO2- to N2 (106-113).

More recently, a third NO biosynthesis pathway was also detected in bacteria. Chen et al. (114, 115) isolated and purified, in Nocardia bacteria, NOS which required for the same cofactors as mammalian enzymes to ensure their activity, but they were found to have a 51.9 kDa apparent molecular weight.


NO is an odd endogenous control agent:

- for its nature. NO is basically a gas found to be present everywhere in animal bodies. Locally produced by many types of cells, it is an unstable radical with a biological half-life of only few seconds. NO acts as a transmitter, by diffusing in the immediate vicinity of the cells from which it has been released, and as a hormone, by binding to transporters and entering the blood stream to remote from the release site. Moreover, when NO acts as an intercellular messenger, it does not react as classical messengers, by noncovalent binding to specific receptors through complementarity of shape, but rather by covalent binding to its targets on the basis of their redox potential.

- for its large number of physiological roles. NO serves as a multifunctional messenger affecting many physiological processes such as apoptosis (116), regulation of vascular tone, macrophage-mediated resistance to infection (117), control of synaptic connectivity during development and synaptic plasticity in adults, mediation of olfactory memory 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 living species.

- for the high conservation of its main signaling pathways during evolution. Comparative investigations on the signaling functions of NO in different organisms have also provided insight into the early evolutionary roles of NO. Indeed, evidence on the involvement of NO in the cellular defense system of Limulus ployphemus (an arthropod which has not evolved in 500 million years), as well as in the feeding response of Hydra (the most primitive organism possessing a nervous system), suggest that NO could be one of the earliest biological signaling molecules.


1. Zumft, W. G.: The biological role of nitric oxide in bacteria. Arch Microbiol 160, 253-264 (1993)

2. Palmer, R. M. Ferrige, A. G.& Moncada, S.: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526. (1987)

3. Furchgott, R. F. Carvalho, M. H. Khan, M. T.& Matsunaga, K.: Evidence for endothelium-dependent vasodilation of resistance vessels by acetylcholine. Blood Vessels 24, 145-149 (1987)

4. Stamler, J. S. Singel, D. J.& Loscalzo, J.: Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898-1902. (1992)

5. Wiesinger, H.: Arginine metabolism and the synthesis of nitric oxide in the nervous system. Prog Neurobiol. 64, 365-391 (2001)

6. Gorren, A. C.& Mayer, B.: The versatile and complex enzymology of nitric oxide synthase. Biochemistry (Mosc) 63, 734-743 (1998)

7. Squadrito, G. L.& Pryor, W. A.: Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic Biol Med 25, 392-403 (1998)

8. Lundberg, J. O. Weitzberg, E. Lundberg, J. M.& Alving, K.: Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35, 1543-1546. (1994)

9. Weitzberg, E.& Lundberg, J. O.: Nonenzymatic nitric oxide production in humans. Nitric Oxide 2, 1-7 (1998)

10. Schmidt, H. H. Lohmann, S. M.& Walter, U.: The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta 1178, 153-175. (1993)

11. Brune, B. Dimmeler, S.& Lapetina, E. G.: NADPH: a stimulatory cofactor for nitric oxide-induced ADP-ribosylation reaction. Biochem Biophys Res Commun 182, 1166-1171. (1992)

12. Brune, B.& Lapetina, E. G.: Nitric oxide-induced covalent modification of glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Methods Enzymol 269, 400-407 (1996)

13. Beckman, J. S. Chen, J. Ischiropoulos, H.& Crow, J. P.: Oxidative chemistry of peroxynitrite. Methods Enzymol 233, 229-240 (1994)

14. Crow, J. P.& Ischiropoulos, H.: Detection and quantitation of nitrotyrosine residues in proteins: in vivo marker of peroxynitrite. Methods Enzymol 269, 185-194 (1996)

15. Kamosinska, B. Radomski, A. Man, S. F. Radomski, M. W.& Duszyk, M.: Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells. J Pharmacol Exp Ther 295, 500-505. (2000)

16. Lundberg, J. O. Farkas-Szallasi, T. Weitzberg, E. Rinder, J. Lidholm, J. Anggaard, A. Hokfelt, T. Lundberg, J. M.& Alving, K.: High nitric oxide production in human paranasal sinuses. Nat Med 1, 370-373. (1995)

17. Salman-Tabcheh, S. Guerin, M. C.& Torreilles, J.: Nitration of tyrosyl-residues from extra- and intracellular proteins in human whole blood. Free Radic Biol Med 19, 695-698. (1995)

18. Torreilles, J.& Guerin, M. C.: [Does nitric oxide stress exist?]. C R Seances Soc Biol Fil 189, 389-400 (1995)

19. Brenman, J. E.& Bredt, D. S.: Synaptic signaling by nitric oxide. Curr Opin Neurobiol 7, 374-378. (1997)

20. Kannan, M. S. Guiang, S.& Johnson, D. E.: Nitric oxide: biological role and clinical uses. Indian J Pediatr 65, 333-345. (1998)

21. Bredt, D. S.: Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res 31, 577-596. (1999)

22. Lundberg, J. O.& Weitzberg, E.: Nasal nitric oxide in man. Thorax 54, 947-952. (1999)

23. Torreilles, F. Salman-Tabcheh, S. Guerin, M.& Torreilles, J.: Neurodegenerative disorders: the role of peroxynitrite. Brain Res Brain Res Rev 30, 153-163. (1999)

24. Li, H.& Forstermann, U.: Nitric oxide in the pathogenesis of vascular disease. J Pathol 190, 244-254. (2000)

25. Sethi, S.& Dikshit, M.: Modulation of polymorphonuclear leukocytes function by nitric oxide. Thromb Res 100, 223-247. (2000)

26. Crow, J. P.& Beckman, J. S.: The importance of superoxide in nitric oxide-dependent toxicity: evidence for peroxynitrite-mediated injury. Adv Exp Med Biol 387, 147-161. (1996)

27. Schober, A. Malz, C. R. Schober, W.& Meyer, D. L.: NADPH-diaphorase in the central nervous system of the larval lamprey (Lampetra planeri). J Comp Neurol 345, 94-104. (1994)

28. Olsson, C.& Holmgren, S.: Nitric oxide in the fish gut. Comp Biochem Physiol A Physiol 118, 959-964. (1997)

29. Munoz, M. Munoz, A. Marin, O. Alonso, J. R. Arevalo, R. Porteros, A.& Gonzalez, A.: Topographical distribution of NADPH-diaphorase activity in the central nervous system of the frog, Rana perezi. J Comp Neurol 367, 54-69. (1996)

30. Munoz, M. Marin, O.& Gonzalez, A.: Localization of NADPH diaphorase/nitric oxide synthase and choline acetyltransferase in the spinal cord of the frog, Rana perezi. J Comp Neurol 419, 451-470. (2000)

31. Virgili, M. Poli, A. Beraudi, A. Giuliani, A.& Villani, L.: Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts. Brain Res. 901, 202-207 (2001)

32. Hass, C. A. Hoffman, M. A. Densmore, L. D., 3rd& Maxson, L. R.: Crocodilian evolution: insights from immunological data. Mol Phylogenet Evol 1, 193-201. (1992)

33. Van Nerom, A. E. Ducatelle, R.& Haesebrouck, F.: Oxygen radicals and nitric oxide production by turkey respiratory macrophages. Dev Comp Immunol 22, 407-416. (1998)

34. von Bartheld, C. S.& Schober, A.: Nitric oxide synthase in learning-relevant nuclei of the chick brain: morphology, distribution, and relation to transmitter phenotypes. J Comp Neurol 383, 135-152. (1997)

35. Olsson, C.& Gibbins, I.: Nitric oxide synthase in the gastrointestinal tract of the estuarine crocodile, Crocodylus porosus. Cell Tissue Res 296, 433-437. (1999)

36. Djamgoz, M. B. Aguilo, R. Greenstreet, E. H. Reynolds, R.& Wilkin, G. P.: Histochemistry of NADPH-diaphorase--a marker for neuronal nitric oxide synthase--in the carp retina. Neurochem Int 28, 283-291. (1996)

37. Saeij, J. P. Stet, R. J. Groeneveld, A. Verburg-van Kemenade, L. B. van Muiswinkel, W. B.& Wiegertjes, G. F.: Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics 51, 339-346. (2000)

38. Djamgoz, M. B. Sekaran, S. Angotzi, A. R. Haamedi, S. Vallerga, S. Hirano, J.& Yamada, M.: Light-adaptive role of nitric oxide in the outer retina of lower vertebrates: a brief review. Philos Trans R Soc Lond B Biol Sci 355, 1199-1203. (2000)

39. Toth, T. E.: Nonspecific cellular defense of the avian respiratory system: a review. Dev Comp Immunol 24, 121-139. (2000)

40. Qureshi, M. A. Heggen, C. L.& Hussain, I.: Avian macrophage: effector functions in health and disease. Dev Comp Immunol 24, 103-119. (2000)

41. Haverkamp, S. Kolb, H.& Cuenca, N.: Endothelial nitric oxide synthase (eNOS) is localized to Muller cells in all vertebrate retinas. Vision Res 39, 2299-2303. (1999)

42. Blute, T. A. Lee, H. K. Huffmaster, T. Haverkamp, S.& Eldred, W. D.: Localization of natriuretic peptides and their activation of particulate guanylate cyclase and nitric oxide synthase in the retina. J Comp Neurol 424, 689-700. (2000)

43. Rickard, N. S. Gibbs, M. E.& Ng, K. T.: Inhibition of the endothelial isoform of nitric oxide synthase impairs long-term memory formation in the chick. Learn Mem 6, 458-466. (1999)

44. Blute, T. A. Velasco, P.& Eldred, W. D.: Functional localization of soluble guanylate cyclase in turtle retina: modulation of cGMP by nitric oxide donors. Vis Neurosci 15, 485-498. (1998)

45. Radomski, M. W. Palmer, R. M.& Moncada, S.: Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol 92, 181-187. (1987)

46. Radomski, M. W. Martin, J. F.& Moncada, S.: Synthesis of nitric oxide by the hemocytes of the American horseshoe crab (Limulus polyphemus). Phil. Trans. R. Soc. Lond. B 334, 129-133 (1991)

47. Elofsson, R. Carlberg, M. Moroz, L. Nezlin, L.& Sakharov, D.: Is nitric oxide (NO) produced by invertebrate neurones? Neuroreport 4, 279-282. (1993)

48. Talavera, E. Martinez-Lorenzana, G. Leon-Olea, M. Sanchez-Alvarez, M. Sanchez-Islas, E.& Pellicer, F.: Histochemical distribution of NADPH-diaphorase in the cerebral ganglion of the crayfish Cambarellus montezumae. Neurosci Lett 187, 177-180. (1995)

49. Bascal, Z. A. Montgomery, A. Holden-Dye, L. Williams, R. G. Thorndyke, M. C.& Walker, R. J.: NADPH diaphorase activity in peptidergic neurones of the parasitic nematode, Ascaris suum. Parasitology 112, 125-134. (1996)

50. Moroz, L. L. Chen, D. Gillette, M. U.& Gillette, R.: Nitric oxide synthase activity in the molluscan CNS. J Neurochem 66, 873-876. (1996)

51. Moroz, L. L.& Gillette, R.: NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica. J Comp Neurol 367, 607-622. (1996)

52. Martinez, A.: Nitric oxide synthase in invertebrates. Histochem J 27, 770-776. (1995)

53. Muller, U.: The nitric oxide system in insects. Prog Neurobiol 51, 363-381. (1997)

54. Jacklet, J. W.: Nitric oxide signaling in invertebrates. Invert Neurosci 3, 1-14. (1997)

55. Colasanti, M.& Venturini, G.: Nitric oxide in invertebrates. Mol Neurobiol 17, 157-174. (1998)

56. Hurst, W. J. Moroz, L. L. Gillette, M. U.& Gillette, R.: Nitric oxide synthase imunolabeling in the molluscan CNS and peripheral tissues. Biochem Biophys Res Commun 262, 545-548. (1999)

57. Teunissen, C. E. Steinbusch, H. W. Markerink-van Ittersum, M. De Bruijn, C. Axer, H.& De Vente, J.: Whole brain spheroid cultures as a model to study the development of nitric oxide synthase-guanylate cyclase signal transduction. Brain Res Dev Brain Res 125, 99-115. (2000)

58. Uylings, H. B.: Development of the cerebral cortex in rodents and man. Eur J Morphol 38, 309-312. (2000)

59. Stamler, J. S.& Meissner, G.: Physiology of nitric oxide in skeletal muscle. Physiol Rev 81, 209-237. (2001)

60. Vidwans, S. J.& Su, T. T.: Cycling through development in Drosophila and other metazoa. Nat Cell Biol 3, E35-39. (2001)

61. Enikolopov, G. Banerji, J.& Kuzin, B.: Nitric oxide and Drosophila development. Cell Death Differ 6, 956-963. (1999)

62. Eliasson, M. J. Blackshaw, S. Schell, M. J.& Snyder, S. H.: Neuronal nitric oxide synthase alternatively spliced forms: prominent functional localizations in the brain. Proc Natl Acad Sci U S A 94, 3396-3401. (1997)

63. Colasanti, M. Lauro, G. M.& Venturini, G.: NO in hydra feeding response. Nature 374, 505. (1995)

64. Colasanti, M. Venturini, G. Merante, A. Musci, G.& Lauro, G. M.: Nitric oxide involvement in Hydra vulgaris very primitive olfactory-like system. J Neurosci 17, 493-499. (1997)

65. Palumbo, A. Di Cosmo, A. Gesualdo, I.& d'Ischia, M.: A calcium-dependent nitric oxide synthase and NMDA R1 glutamate receptor in the ink gland of Sepia officinalis: a hint to a regulatory role of nitric oxide in melanogenesis? Biochem Biophys Res Commun 235, 429-432. (1997)

66. Regulski, M.& Trully, T.: Molecular and biochemical characterization of dNOS: a Drosophila Ca2+ / calmodulin-dependent nitric oxide synthase. Proc Natl Acad Sci U S A 92, 9072-9076. (1995)

67. Ogunshola, O. Picot, J. Piper, M. Korneev, S.& O'Shea, M.: Molecular analysis of the NO-cGMP signalling pathway in insect and molluscan CNS. Soc. Neurosci. Abst. 21, 631 (1996)

68. O'Shea, M.& Park, J.-H.: Nitric oxide and orthograde neurotransmission in the CNS of the snail Lymnea stagnalis. Soc. Neurosci. Abst. 22, 363 (1996)

69. Yuda, M. Hirai, M. Miura, K. Matsumura, H. Ando, K.& Chinzei, Y.: cDNA cloning, expression and characterization of nitric-oxide synthase from the salivary glands of the blood-sucking insect Rhodnius prolixus. Eur J Biochem 242, 807-812. (1996)

70. Yuda, M. Higuchi, K. Sun, J. Kureishi, Y. Ito, M.& Chinzei, Y.: Expression, reconstitution and characterization of prolixin-S as a vasodilator--a salivary gland nitric-oxide-binding hemoprotein of Rhodnius prolixus. Eur J Biochem 249, 337-342. (1997)

71. Moroz, L. L.& Gillette, R.: From Polyplacophora to Cephalopoda: comparative analysis of nitric oxide signalling in mollusca. Acta Biol Hung 46, 169-182 (1995)

72. Ottaviani, E. Paeman, L. R. Cadet, P.& Stefano, G. B.: Evidence for nitric oxide production and utilization as a bacteriocidal agent by invertebrate immunocytes. Eur J Pharmacol 248, 319-324. (1993)

73. Torreilles, J. Guérin, M. C.& Roch, P.: Peroxidase-release associated with phagocytosis in Mytilus galloprovincialis haemocytes. Dev Comp Immunol 21, 267-275. (1997)

74. Torreilles, J.& Guérin, M.-C.: Production of peroxynitrite by zymosan stimulation of Mytilus galloprovincialis haemocytes in vitro. Fish & Shellfish Immunol. 9, 509-518 (1999)

75. Gourdon, I. Guérin, M. C. Torreilles, J.& Roch, P.: Nitric Oxide Generation by Hemocytes of the Mussel Mytilus galloprovincialis. Nitric Oxide 5, 1-6. (2001)

76. Torreilles, J.& Romestand, B.: In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas. BMC Immunol 2, 1 (2001)

77. Franchini, A. Conte, A.& Ottaviani, E.: Nitric oxide: an ancestral immunocyte effector molecule. Adv Neuroimmunol 5, 463-478 (1995)

78. Nappi, A. J. Vass, E. Frey, F.& Carton, Y.: Nitric oxide involvement in Drosophila immunity. Nitric Oxide 4, 423-430. (2000)

79. Liu, L. Hausladen, A. Zeng, M. Que, L. Heitman, J.& Stamler, J. S.: A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 410, 490-494. (2001)

80. Kimbrell, D. A.& Beutler, B.: The evolution and genetics of innate immunity. Nat Rev Genet 2, 256-267. (2001)

81. Paveto, C. Pereira, C. Espinosa, J. Montagna, A. E. Farber, M. Esteva, M. Flawia, M. M.& Torres, H. N.: The nitric oxide transduction pathway in Trypanosoma cruzi. J Biol Chem 270, 16576-16579. (1995)

82. Goldstein, J. Paveto, C. Lopez-Costa, J. J. Pereira, C. Alonso, G. Torres, H. N.& Flawia, M. M.: Immuno and cytochemical localization of Trypanosoma cruzi nitric oxide synthase. Biocell 24, 217-222. (2000)

83. Basu, N. K. Kole, L. Ghosh, A.& Das, P. K.: Isolation of a nitric oxide synthase from the protozoan parasite, Leishmania donovani. FEMS Microbiol Lett 156, 43-47. (1997)

84. Ghigo, D. Todde, R. Ginsburg, H. Costamagna, C. Gautret, P. Bussolino, F. Ulliers, D. Giribaldi, G. Deharo, E. Gabrielli, G.& et al.: Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor. J Exp Med 182, 677-688. (1995)

85. Kanadia, R. N. Kuo, W. N. McNabb, M.& Botchway, A.: Constitutive nitric oxide synthase in Saccharomyces cerevisiae. Biochem Mol Biol Int 45, 1081-1087. (1998)

86. Wilken, M.& Huchzermeyer, B.: Suppression of mycelia formation by NO produced endogenously in Candida tropicalis. Eur J Cell Biol 78, 209-213. (1999)

87. Rowland, A. Murray, A. J.& Wellburn, A. R.: Oxides of nitrogen and their impact upon vegetation. Rev Environ Health 5, 295-342 (1985)

88. Hufton, C. A. Bestford, R. T.& Wellburn, R. A.: Effects of NO (+ NO2) pollution on growth, nitrate reductase activities and associated protein contents in glasshouse lettuce grown hydroponically in winter with CO2 enrichment. New Phytol. 133, 495-501 (1996)

89. Wildt, J. Rockel, A. Rockel, P.& Segschneider, H. J.: Emission of NO from higher plant species. J. Geophys. Research 102, 5919-5927. (1997)

90. Corpas, F. J. Barroso, J. B.& del Rio, L. A.: Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 6, 145-150 (2001)

91. Noritake, T. Kawakita, K.& Doke, N.: Nitric oxide induces phytoalexin accumulation in patato tuber tissue. Plant Cell Physiol. 37, 113-116 (1996)

92. Gouvea, C. M. C. P. Souza, J. F. Magalhaes, A. C. N.& Martins, I. S.: NO-releasing substances that induce growth elongation in maize root segments. Plant growth regulation 21, 182-187 (1997)

93. Ribeiro, E. A. Cunha, F. Q. Tamashiro, W. M.& Martins, I. S.: Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells. FEBS Lett 445, 283-286. (1999)

94. Ferrer, M. A.& Ros Barcelo, A.: Differential effect of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elengs. Plant cell and environment 22, 891-897 (1999)

95. Durner, J. Wendehenne, D.& Klessig, D. F.: Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci U S A 95, 10328-10333. (1998)

96. Huang, J. S.& Knopp, J. A., Involvment of nitric oxide in Ralstonia solanacearum-induced hypersensitive reaction in tobacco, INRA and Springer Editions, Berlin (1998)

97. Clark, D. Durner, J. Navarre, D. A.& Klessig, D. F.: Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase. Mol Plant Microbe Interact 13, 1380-1384. (2000)

98. Klessig, D. F. Durner, J. Noad, R. Navarre, D. A. Wendehenne, D. Kumar, D. Zhou, J. M. Shah, J. Zhang, S. Kachroo, P. Trifa, Y. Pontier, D. Lam, E.& Silva, H.: Nitric oxide and salicylic acid signaling in plant defense. Proc Natl Acad Sci U S A 97, 8849-8855. (2000)

99. Navarre, D. A. Wendehenne, D. Durner, J. Noad, R.& Klessig, D. F.: Nitric oxide modulates the activity of tobacco aconitase. Plant Physiol 122, 573-582. (2000)

100. Delledonne, M. Xia, Y. Dixon, R. A.& Lamb, C.: Nitric oxide functions as a signal in plant disease resistance. Nature 394, 585-588. (1998)

101. Durner, J.& Klessig, D. F.: Nitric oxide as a signal in plants. Curr Opin Plant Biol 2, 369-374. (1999)

102. Hammond-Kosack, K. E.& Jones, J. D. G.: Resistance gene-dependent plant defense responses. Plant Cell 8, 1773-1791 (1996)

103. Cohn, J. Sessa, G.& Martin, G. B.: Innate immunity in plants. Curr Opin Immunol 13, 55-62. (2001)

104. Wendehenne, D. Pugin, A. Klessig, D. F.& Durner, J.: Nitric oxide: comparative synthesis and signaling in animal and plant cells. Trends Plant Sci. 6, 177-183 (2001)

105. Renner, E. D.: Production of Nitric Oxide and Nitrous Oxide During Denitrification by Cornybacterium Nephridii. J. Bacterial. 101, 821-826 (1970)

106. Firestone, M. K. Firestone, R. B.& Tiedje, J. M.: Nitric oxide as an intermediate in denitrification: evidence from nitrogen-13 isotope exchange. Biochem Biophys Res Commun 91, 10-16. (1979)

107. Goretski, J.& Hollocher, T. C.: Trapping of nitric oxide produced during denitrification by extracellular hemoglobin. J Biol Chem 263, 2316-2323. (1988)

108. Papen, H. von Berg, R. Hinkel, I. Thoene, B.& Rennenberg, H.: Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures. Appl Environ Microbiol 55, 2068-2072. (1989)

109. Goretski, J.& Hollocher, T. C.: The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification. J Biol Chem 265, 889-895. (1990)

110. Goretski, J. Zafiriou, O. C.& Hollocher, T. C.: Steady-state nitric oxide concentrations during denitrification. J Biol Chem 265, 11535-11538. (1990)

111. Ye, R. W. Averill, B. A.& Tiedje, J. M.: Denitrification: production and consumption of nitric oxide. Appl Environ Microbiol 60, 1053-1058. (1994)

112. Daum, M. Zimmer, W. Papen, H. Kloos, K. Nawrath, K.& Bothe, H.: Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida. Curr Microbiol 37, 281-288. (1998)

113. Foissner, I. Wendehenne, D. Langebartels, C.& Durner, J.: In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. Plant J 23, 817-824. (2000)

114. Chen, Y.& Rosazza, J. P.: A bacterial nitric oxide synthase from a Nocardia species. Biochem Biophys Res Commun 203, 1251-1258. (1994)

115. Chen, Y.& Rosazza, J. P.: Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. J Bacteriol 177, 5122-5128. (1995)

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