Histochemical Journal 27, 770-776 (1995)

R E V I E W

Nitric oxide synthase in invertebrates

A L F R E D O M A R T I N E Z *

Department of Cytology and Histology, University of Navarra, 31080 Pamplona, Spain

Received 11 April 1994 and in revised form 6 June 1994

Summary

The gas nitric oxide is now recognized as an important signalling molecule that is synthesized from L-arginine by the enzyme nitric oxide synthase. This enzyme can be localized by different methods, including immunocytochemistry and the histochemical reaction for NADPH diaphorase. It has been demonstrated in various vertebrate cells and tissues, and recently several studies dealing with the production of nitric oxide in invertebrates have been published. Diploblastic animals, flatworms and nematodes seem to lack NADPH diaphorase activity but it has been found in the rest of the phyla studied. The most frequently reported sites for the production of nitric oxide are the central and peripheral nervous systems and, in primitive molluscs, the muscle cells. In insects, it has also been described in the Malpighian tubules. The roles of nitric oxide in invertebrates are closely related to the physiological actions described in vertebrates, namely, neurotransmission, defence, and salt and water balance. The recent cloning of the first nitric oxide synthase from an invertebrate source could open interesting avenues for further studies.

Introduction

The L-arginine:nitric oxide pathway is a newly discovered regulatory system involved in a variety of biological functions (Moncada, 1992). Nitric oxide (NO) is synthe- sized from L-arginine by a group of enzymes, the NO synthases. These enzymes require NADPH as a cofactor and are inhibited by some analogues of L-arginine. At present, two broad types of NO synthases have been identified, i.e. constitutive and inducible (Moncada et al., 1991; F6rstermann et aI., 1991). In mammals, constitutive NO synthases are present in endothelium, brain, adrenal glands and platelets (Moncada et al., 1991). The inducible enzyme, which is synthesized in response to cytokines and inflammatory mediators, has been identified in macro- phages, hepatocytes, vascular smooth muscle, endothelial cells and neutrophils (Moncada et al., 1991; Rimele et al., 1991).

NO synthases were originally identified in mammalian tissues. More recently, however, they have been found in all the groups of non-mammalian vertebrates (Li et al., 1992, 1993; Powers & Giusti, 1993; Earle et al., 1993; Kurenny et al., 1993; Kalamkarov et al., 1993; and personal unpublished data), mainly in the central and peripheral nervous systems (Fig. 1).

*Present address: NCI, DCPC, BPRB, 9610 Medical Center Drive, Rockville, MD 20850, USA.

'0018-2214 �9 1995 Chapman & Hall

Subsequent to these findings in vertebrates, several studies have been initiated in invertebrates. Most of them are preliminary and have been published as abstracts of specialized meetings, but the growing accumulation of data and the potential transcendency of the subject encouraged the writing of this short review.

Methods used to detect nitric oxide production

Several techniques have been used to localize the sites of NO synthesis. These include immunocytochemistry, histochemistry, and biochemical methods.

Different antibodies raised against the various NO synthase enzymes are available: polyclonal sera against the whole neuronal constitutive enzyme (Bredt et al., 1990; Springall et al., 1992a; Charles et aL, I993), monoclonal antibodies raised against the constitutive endothelial iso- form (Pollock et al., 1993), or antibodies raised against synthetic peptifles selected from the deduced sequences of rat neural (Riveros-Moreno et aL, 1993) or murine inducible (Harold et al., 1993) NO synthases. The com- parison of the results obtained using antibodies against different isoforms can characterize the NO synthases present in invertebrate species (Martlnez et aL, 1994) but we must always be careful interpreting the immunocyto- chemical results obtained in invertebrates with antibodies raised in other species (for a review, see Pollock's paper in this issue). A parallel approach to the study of the

Nitric oxide synthase in invertebrates 771

Fig. 1. Myenteric plexus of the chicken proventriculus immunocytochemically stained with an antiserum raised against a synthetic peptide of the mammalian neural nitric oxide synthase. Several neurons and fibres are labelled, m = muscle, x 600.

Fig. 2. Paraffin section of the pyloric stomach of the starfish, Marthas~erias glaciatis, stained with the antiserum used in Fig. 1. Two slender epithelial neuroendocrine cells and the basiepithelial plexus (arrows) appear clearly stained, x 600.

Fig. 3. Cryostat section through the head of the fruit fly, Drosophila melanogaster, showing two cerebral ganglia (arrows) containing numerous neurons positive for the NADPH diaphorase reaction, e=eye, x375.

Fig. 4. Unfixed Malpighian tubules of the fruit fly, Drosophila melanogaster, after the application in toto of the NADPH diaphorase technique. Three positive cells are present (Courtesy of Dr M. Garayoa, University of Navarra.) x 600.

biology of N O is the immunocytochemical localization of the guanylyl cyclase, the target enzyme for N O (Elphick et al., 1993c).

N O synthases have NADPH diaphorase activity and, consequently, histochemicaI techniques for NADPH diaphorase can be used to locate the enzymes producing NO. In mammals it has been proposed that both activities have identical distribution (Hope el aI., 1991), but when

careful studies of co-localization of both activities were performed in the rat brain, a more complex pattern emerged with some sites having NADPH diaphorase but not N O synthase (Rodrigo et aI., 1994; Spessert et al., 1994). This complexity could be due to the presence of other NADPH-requiring enzymes. It has been observed that, by increasing the concentration of paraformaldehyde in the fixative up to 4%, the N O synthase-related NADPH

772 MARTiNEZ

diaphorase staining becomes more specific (Matsumoto et al., 1993). The NADPH diaphorase staining method in invertebrates has been validated by purification of the locust NO synthase and demonstration of co-elution for both NO synthase and NADPH diaphorase activities (Elphick et al., 1994).

NO' synthases produce equimolar citrulline with NO, from molecular oxygen and L-arginine. Therefore, measure- ment of the conversion of [14C]arginine to [14C]citrulline can be used as a method for assaying the production of NO (Knowles et aI., 1989). This technique has been

successfully used in insects (Elphick eta]., 1993a, I994) and molluscs (Elofsson et al., 1993; Elphick et aI., 1993b).

A complete characterization of the NO-synthases present in invertebrates is needed to design more precise methods for the localization of these enzymes. The first steps in this process have been taken recently with the cloning of an invertebrate NO synthase from a Drosophila genomic library. The deduced sequence of the protein has only a 40% homology with its mammalian counterparts but contains all the identified functional domains of NO synthase enzymes: heme-, calmodulin-, FMN-, FAD-,

Table 1. Compilation of studies showing the distribution of nitric oxide-producing systems in invertebrates

Animals Techniques Localization Reference

Coelenterata Hydra oligactis HC Aiptasia sp HC

Plathelminthes Dugesia gonocephala HC

Nemathelminthes Caenorhabditis elegans HC

Annelida Lumbricus terrestris HC Haemopis sanguisuga HC

Mollusca Lepidopteurus asellus HC Littorina ]ittorea HC Helix aspersa HC Helix pomatia HC Cepaea nemoralis HC Limax maximus HC Lymnaea stagnalis ICC + HC Lymnaea ovata HC Physa fontinalis HC Planorbarius corneus HC Planorbis sp HC Aplysia californica HC Balla sp HC Phylaplysia sp HC Helisoma trivolvis HC Biomphalaria sp ICC + HC

Arthropoda Limulus polyphemus Biochemistry Cambarellus montezumae HC Schistocerca gregaria HC + Biochemistry Locusta migratoria HC + Biochemistry Stagmatoptera biocelta Physiology Drosophila melanogaster HC + Biochemistry

Echinodermata Marthasterias glacialis ICC + HC

Urochordata Ascidietla aspersa HC

No staining 1 No staining 1

No staining 1

No staining 1

CNS, muscle, other 1 CNS, muscle, other 1

Buccal muscles CNS CNS, CNS, CNS CNS CNS CNS CNS CNS CNS CNS CNS CNS CNS CNS,

skin, other skin, other skin, other skin, other osphradium, skin, other osphradium, skin, other osphradium, skin, other osphradium, skin, other osphradium, skin, other PNS, salivary gland, osphradium PNS, salivary gland, osphradium PNS, salivary gland, osphradium PNS, salivary gland, osphradium PNS, salivary gland, osphradium

1 1 1,2 1 1 1 1,3,4,5,6,7 5,6,7 5,6,7 5,6,7 5,6,7 8 8 8 6,8 6

Haemocytes 9 CNS 10 CNS 3,11 CNS 3,11 CNS 12 Malpighian tubules 13

CNS, PNS 14

Endostyle 1

CNS: central nervous system; PNS = peripheral nervous system; HC = histochemistry for NADPH diaphorase; ICC = immunocytochemistry. 1 = Elofsson et al., 1993; 2 = Sd~nchez-Alvarez et al., 1993; 3 = Elphick et al., 1993b; 4 = Moroz & Roylance, 1993; 5 = Moroz et aI., 1993a; 6 = Moroz et al., 1993b; 7 = Winlow et al., 1993; 8 = Lukowiak et al., 1993; 9 = Radomski et al., 1991; 10 = Martinez-Lorenzana et al., 1993; 11 = Elphick et al., 1993c; 12 = D'Alessio eta]., 1982; 13 = Dowet al., 1994; 14 = Martinez et al., 1994.

Nitric oxide synthase in invertebrates 773

and NADPH-binding sites (Regulski & Tully, 1993). The tow homology with the vertebrate enzymes could explain why, in some cases, NO can be identified by biochemical or histochemical methods but the enzyme cannot be located by immunocytochemistry, possibly because the epitopes against which the antibodies were raised are different in the invertebrate molecule.

Distribution of nitric oxide-producing systems in invertebrates

Table 1 contains a summary of the results so far obtained in several invertebrate species. The methods used are different so that comparison is not straightforward; however, general patterns can be observed.

In the lower phyla no specific staining for NADPH diaphorase has been found, implying either that NO- related systems are not present in these primitive animals or that the production of NO occurs via a different syn- thetic pathway. From annelids upwards the presence of NO synthase-like enzymes is widespread, particularly in the central and peripheral nervous systems.

An interesting observation is the apparent evolution of NO production in control of muscle cell physiology. This has been specially analysed in molluscs, the phylum in which more species have been studied. The more primitive groups (for instance the polyplacophora, represented by Lepidopleurus asellus in Table 1) present the diaphorase reaction only in the buccal muscle cells, but as we move upwards through the phylogeny the positivity appears in the neurons innervating these muscles. This could repre- sent a switching in the site of NO-mediated control of muscle motility. The primitive animals seem to have an autocrine NO-dependent muscle regulation while the more evolved organisms entrust this task to the nervous system (L. L. Moroz, personal communication).

An obvious conclusion from Table I is that only the nervous system has been systematically studied so far and that further studies in the cardiovascular and defensive systems are needed to complete a preliminary survey of NO production in invertebrates.

Possible roles for nitric oxide in invertebrates

The structural similarity of the Drosophila NO synthase gene to its mammalian homologues (Regulski & Tully, 1993) suggests that the functions of the enzyme, and thus of NO, have been highly conserved through evolution. The homology with the vertebrate enzymes has also been shown by biochemical studies, demonstrating that the same activators and inhibitors act in invertebrate species (Elphick et al., 1993a). Several functions have been proposed for NO through an analysis of the distribution of the NO synthases. In all cases NO appears to be a multifunctional trans-cellular messenger. These functions include neurotransmission, defence, and salt and water balance.

In most of the animals studied NO synthase appears in a specific subpopulation of neurons and nerve tracts, in both the central and peripheral nervous systems (Figs 1-3). This distribution suggests a clear involvement of NO in neurotransmission, as is the case in mammals (Bredt et al., 1990).

Specific studies in molluscs and insects, where the neurons are well characterized, point to a predominantly sensory function for NO. The involvement of NO syn- thase in the olfactory processing of the slugs has been recently demonstrated (Gelperin et al., 1993; Gelperin, 1994) and the same enzyme has been identified in the antenal lobes of the locust brain, indicating some involve- ment of NO in insect olfaction as well (Elphick et aI., 1994). Interestingly, the olfactory bulb in the rat brain is also rich in NO synthase (Bredt & Snyder, 1992; Spessert et al., 1994) and this molecule is thought to participate in neural processing of olfactory (Breer & Shepherd, 1993) and visual (Kurenny et al., 1993; Kalamkarov et al., 1993) sensory input in vertebrates.

Pharmacological approaches, such as the use of NO donors and inhibitors of NO synthase, have generally been used to confirm the presumptive involvement of NO as a mediator in different physiological preparations. In these experiments S-nitrocysteine has been employed as a NO-generating agent, and NC-methyl-L-arginine (NMA) and other derivatives of L-arginine as potent inhibitors of the NO-synthase (Moroz el al., 1993a). The results of such experiments indicate a role for NO in feeding and respiratory behaviours in molluscs (Winlow et al., 1993; Moroz & Park, 1993; Moroz et al., 1993b). The relevance of NO-producing neurons has also been suggested in the digestive system of echinoderms (Martlnez et al., 1994).

There is also evidence that NO plays an important role in the cellular processes associated with learning and memory in the mammalian brain (Bredt & Snyder, 1992). Surprisingly, D'Alessio et al. (1982) demonstrated, several years before NO was described as a biologically active molecule, that the precursor of NO, L-arginine, is required for memory consolidation in the brain of the praying mantis. These observations suggest a considerable conservation of the physiological roles of NO.

Similarities between NO signalling in mammalian and invertebrate nervous systems probably also extend to the cellular mechanisms by which its effects are mediated. This idea is supported by the recent detection of a NO-activated guanylyl cyclase in an insect brain (Elphick et al., I993a).

An important role for NO in mammals is its integration in defensive strategies. It is produced, in response to certain injuries, in neutrophils, hepatocytes, endothelial cells, etc (Moncada et aI., 1991) and could act as a bacteriostatic agent in macrophages (see Cattell & Jansen in this issue). The NO synthase located in the haemocytes of the horseshoe crab Limulus polyphemus (Radomski et al., 1991) could have a similar function. A wider study in

774 MARTINEZ

amebocytes of other phyla could provide important insights into the defensive role of NO in invertebrates.

Recently, NADPH diaphorase has been localized in cells of the Malpighian tubules of some insects (Dow & Maddrell, 1993; D o w e t al., 1994; Fig. 4). This tubular system is responsible for regulation of salt and water balance in insects. In a similar way, NO synthase has been found in the juxtaglomerular complex of the mammalian kidney (Springall et al., 1992b) suggesting that NO is also involved in the regulation of the excretory system.

New perspectives

The recent cloning of an invertebrate's NO synthase (Regulski & Tully, 1993) opens the possibility of obtain- ing specific invertebrate antibodies for immunocytochem- istry and probes for in situ hybridization, thus overcoming the problems generated by heterologous reagents and allowing a complete mapping of NO-production sites.

To date, most physiological studies of NO function have been carried out in mammals, and the studies in invertebrates present interesting corollaries to these reports. However, some invertebrate nervous systems are very well described and their simplicity allows a thorough knowledge of all the neurons and their functions. These invertebrate models could therefore be indispensable in the characterization of the neural roles of NO.

Acknowledgements

I gratefully acknowledge the comments and friendly advice of Professor J. Rodrigo, Dr M. R. Elphick, Dr L. L. Moroz, Dr J. A. T. Dow and Dr M. Garayoa during the writing of this manuscript.

Note added in proof Since this paper was accepted for publication, several articles have appeared that further the characterization of NO synthases in invertebrates. Some of them broaden the study on groups previously known to produce NO, as is the case with some new articles on molluscs (Moroz et al., 1994; Jacklet & Gruhn, 1994; Sfinchez-Alvarez et al., 1994) and insects (MLiller, 1994; M/iller & Buchner, 1994), while others describe NO production in new animals like cephalopods (Chichery & Chichery, 1994), arachnids (Meyer, 1994) and other species of crayfish (Johansson & Carlberg, 1994). More evidence on the defensive role of NO synthase in invertebrates has been produced by Ottaviani et al. (1994) which demonstrate the bacterio- cidal effect of the NO produced by molluscan immuno- cytes. In addition, a new function for NO synthase in invertebrates has been discovered by Ribeiro & Nussenzveig (1993): The hematophagous insect Rhodnius prolixus presents NO synthase activity in the salivary glands. When the insect is searching for a blood meal, it injects NO-containing saliva into the host, resulting in

blood vessels relaxation and facilitation of blood intake. This interesting behaviour illustrates how animals can adapt previously existing regulatory systems to new circumstances.

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