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Mini-review
Nitric oxide signalling in insects
Shireen-A. Davies
*Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK
Received 2 March 2000; received in revised form 27 June 2000; accepted 27 June 2000
1. General features of no signal transduction
The gas, nitric oxide (NO), was first described as a second messenger molecule at least a decade ago; since then, research into mechanisms of intracellular gener-ation of NO and of the functional role of the NO signal-ling pathway has generated thousands of research papers, many reviews and a Nobel Prize. This reflects the funda-mental importance of NO and its signalling processes in biology and medicine.
The family of enzymes which form NO from l
-argi-nine are known as nitric oxide synthases (NOS). These are large complex haem-containing enzymes which con-tain binding sites for the co-factors flavin adenine dinu-cleotide (FAD), flavin mononudinu-cleotide (FMN) and nic-otinamide adenine dinucleotide phosphate (NADP), (Marletta, 1989; Bredt et al., 1991; Mayer, 1994), and also contain the co-factor tetrahydrobiopterin (Mayer, 1994). Recent work has shown that the NOS complex consists of an oxygenase (catalytic) domain and a reductase domain. The catalytic domain, which is dimerised in biologically active NOS (Stuehr, 1999), contains the haem group and tetrahydrobiopterin, and binds the substrate, l-arginine (see Fig. 1).
Ca2+/calmodulin binds at a regulatory site between the
domains to aid the process of electron transfer between them (Crane et al., 1999). NO generation occurs in the presence of NADPH in a complex two-stage reaction (via hydroxyarginine) which results in oxidation of the guanadino nitrogen group of arginine, to form citrulline (Fig. 2).
NOS enzymes are encoded by a large multigene fam-ily, and occur as several isoforms. So far, three genes have been identified in vertebrates and humans, NOS1,
NOS2andNOS3. The conservation of exon/intron struc-ture across human NOS1, NOS2 and NOS3 genes
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gests that these genes derive from a single ancestral gene (Hall et al., 1994).
NOS isoforms were first named from the tissue source of origin, i.e., eNOS (endothelial NOS), nNOS (neuronal NOS) and iNOS (inducible, macrophage NOS); these correspond to gene designations as follows: NOS1
(nNOS),NOS2(iNOS) andNOS3(eNOS). Additionally, NOS isoforms were classified into either constitutive (ecNOS and ncNOS) and inducible NOS (iNOS) and as either calcium-dependent or calcium-independent enzymes. Such classifications, however, are now some-what deceptively simple, given the present depth of understanding of NOS structure and organisation. Recent evidence clearly shows that cells and tissues can contain several isoforms of NOS, which may either be constitut-ive or inducible (Forstermann et al., 1998).
In vertebrates, one of the earliest functional roles ascribed to NO was in vasodilation as a consequence of the seminal discovery that endothelin-derived relaxing factor (EDRF) was in fact, NO (Palmer et al., 1987). As NO is important in the maintenance of vascular tone, reduced NO signalling is implicated in erectile dysfunc-tion in males (Burnett, 1995); consequently, this has driven much of the research into NO biochemistry and function, and into the pharmacology of NO-releasing compounds.
Fig. 1. Stereo view of dimerised catalytic domains of NOS with bound substrate,l-arginine. The structure of murine iNOS protein (residues 77–
496) solved by Crane et al. (1998); pdb accession 1NOD. The haem centre is shown in red with Fe in blue. The NOS complex contains tetrahydrobi-opterin (magenta), which binds very close to the active site, wherel-arginine (cyan) is bound.
Fig. 2. Reaction scheme of formation of NO by the NOS enzyme complex. NOS-catalysed generation of NO occurs using the substratel-arginine,
molecular oxygen and NADPH, to produce citrulline and NO via an intermediate compound,Nω-hydroxy-l-arginine. The substrate-binding catalytic
domain (NOSox) of the enzyme, contains haem and tetrahydrobiopterin, and is active as a dimer. The co-factors, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and NADPH bind to the reductase domain of the enzyme (NOSred). Ca2+/calmodulin acts to promote electron
transport between the NOSredand NOSox.
The main intracellular ‘receptor’ for NO is the haem moiety of soluble guanylate cyclase (Schulz et al., 1989). Activated guanylate cyclase acts to generate 3959-cyclic GMP (cGMP) which, depending on the cell-type and physiological response, will activate or inhibit several downstream targets, including cGMP-dependent protein kinases (cGKs), ion channels and cGMP-dependent phosphodiesterases (Vaandrager and de Jonge, 1996),
Fig. 3. The NO/cGMP signalling pathway. Activation of NOS to gen-erate NO occurs in response to myriad signals, depending on the cell-type and the NOS isoform. NO, as a diffusible molecule, transverses the cell membrane, where it activates soluble guanylate cyclase (sGC) by binding to the sGC haem moiety. The resultant increase in intra-cellular cGMP levels has multiple effects within the target cell, includ-ing activation of ion channels (cyclic nucleotide-gated channels, CNGs), cGMP-dependent kinases and cGMP-dependent phosphodies-terases.
2. Comparative features of NO signalling in invertebrates
Much of the recent work on NOS and NO signalling in vertebrate systems has been reviewed extensively recently, (Huang and Lo, 1998; Wang et al., 1999). Given that most of our present understanding of NOS/NO signalling derives from work carried out in vertebrate model systems, what, if anything, does work on NO signalling in invertebrates tell us?
Basic studies in comparative physiology and biochem-istry in simple organisms have provided strong evidence that NO signalling is evolutionarily and functionally conserved. NO is used as a signalling molecule in all invertebrate orders studied to date (Colasanti and Ventu-rini, 1998), and in some rather surprising contexts. For example, the use of salivary NO as a vasodilator by blood-sucking insects (Ribeiro and Nussenzveig, 1993) and the discovery of l-glutamate-stimulated NO
signal-ling in Trypanosoma cruzi epimastigotes (Paveto et al.,
1995). Furthermore, novel NO-controlled physiological functions have been uncovered by invertebrate studies. In Hydra vulgaris, for example, NO signalling contrib-utes to olfactory-like feeding behaviour (Colasanti et al., 1997) and in Sepia officinalis, a calcium-sensitive NOS is implicated in melanin production in the ink-gland (Palumbo et al., 1997). The complex area of the neuronal role of NO signalling can be investigated in organisms with simplified nervous systems. Hence, studies in developing lobsters (Scholz et al., 1998) have shown not only that NO/cGMP signalling is important in the devel-opment of lobster CNS, but also, that NO may act as a modulatory neurotransmitter in this region.
3. NO signalling in insects
In order to correlate NO signalling and function, it is necessary to undertake NO signalling studies in the con-text of identified cells. Indeed, in order to localise NO signalling components to specific cells to which a physiological function has been ascribed (identified cell biology), it is necessary to use a well-defined physiologi-cal system.
Insect systems have been particularly valuable in this regard. Larvae of the hawkmoth, Manduca sexta, for example, have been used in studies of the role of ident-ified developing neuronal networks in learning (Weeks et al., 1997), olfaction (Oland and Tolbert, 1996) and in the quest to fathom the neuronal and cellular processes that accompany metamorphosis (Truman 1992, 1996). The locusts,Locusta migratoriaandSchistocerca grega-riahave also been insects of choice for neuronal studies. These insect models, however, are not only useful to the neuroscientist; bothManducaandSchistocercahave been important models for insect physiologists. The highest gut luminal pH (pH 12!) ever recorded is in
Manduca (Dow, 1992), while goblet cells from this insect were used as a tissue source for the first isolation and purification of the vacuolar H+-ATPase, reviewed recently (Wieczorek et al., 2000).
Thus, insect model systems have been used to study precisely the kind of physiological studies in which NO signalling is thought to be involved. It is not surprising, then, that investigations of NO signalling processes in insects have been so extensively pursued. Molecular characterisation of NOS genes in insects has been under-taken; and biochemical analysis of NOS and NO/cGMP signalling has been achieved in many insects, all the more remarkable given the tiny size of some insect spec-ies.
3.1. Insect NOS genes and isoforms
1995), Anopheles stephensi (Luckhart and Rosenberg, 1999),M. sexta(Nighorn et al., 1998) andRhodnius pro-lixus (Yuda et al., 1996). The insect NOS sequences show highest similarity toNOS1(mean of 49% identity), as compared withNOS2(44% identity) andNOS3(47% identity) (Luckhart and Rosenberg, 1999). Analysis of insect NOS protein sequences (Fig. 4) shows rather strik-ingly, that there are extensive regions of 100% identity between the sequences. These regions include co-factor binding sites for calmodulin, FAD, FMN and NADPH (based on homology to vertebrate NOS isoforms). Dro-sophila NOS is the largest protein with polyglutamine repeats near the N-terminus; it is interesting to note that the N-terminal region has the least similarity between the different insect species.
Fig. 5 shows a dendrogram for the NOS isoforms drawn from presently available protein sequence infor-mation for different phyla. Clearly, the insect members of the family are closest to human nNOS (and eNOS) while iNOS isoforms cluster in a separate group. The high degree of similarity at both the gene and protein level between insect NOS and neuronal NOS over 400 million years of evolution suggests that NOS1 is the ancestral gene of the NOS gene family. One striking dif-ference, however, between vertebrate and insect nNOS is that insect isoforms lack the PDZ (post-synaptic density protein-95, discs-large, ZO-1) domain contained in the N-terminus region of vertebrate nNOS. PDZ domains are important scaffolding domains, linking proteins to intra-cellular architecture, thus creating signalling complexes. The nNOS PDZ domain acts to link the enzyme to inte-gral membrane and cytoskeletal protein in skeletal mus-cle (Brenman et al., 1996a) and to post-synaptic density proteins PSD-95 and PSD-93 in neurones (Brenman et al., 1996b). In kidney, nNOS has been shown to co-localise with PSD-93 to cytoplasmic vesicles and to the basolateral membrane (Tojo et al., 1999). Thus, PDZ domains may be particularly important in polarised epi-thelia, where localisation of proteins to either apical or basolateral membranes will critically influence cellular physiology. Given the importance of PDZ domains to the correct localisation, and hence specificity, of nNOS in vertebrates, it is remarkable that insect NOS isoforms do not contain this motif. Thus, targeting of NOS and the organisation of NOS signalling complexes in insect cells must occur by as yet, unspecified mechanisms.
The sequence conservation between different insects, and their similarity to the constitutive nNOS, implies that all members of the NOS family are regulated simi-larly. However,AnophelesNOS activity is induced upon infection (Luckhart and Rosenberg, 1999). While the only mosquito NOS gene identified so far appears to be inducible under specific conditions, it remains to be seen if further identification of insect NOS genes provides further evidence of inducible NOS activity in insects. The mechanisms of control of this infection-induced
NOS enzyme activity in Anopheles (Luckhart et al., 1998) and possibly other insects, will be particularly interesting once elucidated.
3.2. Physiological function of NOS in insects
In insects, the small quantities of tissue available for study, and until recently, the unavailability of suitable anti-NOS antibodies for insect work, has meant that many studies of NOS have relied upon the use of the NADPH-diaphorase assay as an index for NOS expression and localisation. The assay is based upon the reductase activity of NOS, associated with a cytochrome P450-like domain at the C-terminal, which can be assessed by the conversion of the electron acceptor, nitroblue tetrazolium, into a blue, reduced form in the presence of NADPH (Dawson et al., 1991). It is gener-ally accepted in vertebrates that NADPH diaphorase staining patterns do reflect NOS activity (Dawson et al., 1991; Hope et al., 1991; Pasqualotto et al., 1991).
In insects, however, a novel study using several
Orthoptheranspecies (Ott and Burrows, 1999) has dem-onstrated that NOS-related NADPH-diaphorase activity shows species-specific fixation sensitivities. In locusts and crickets (Acheta domestica), prolonged (up to overnight) fixation of CNS results in widespread diapho-rase staining, not seen after mild (2 h) fixation. However, in locusts, selective staining of identified somata and processes remains upon prolonged fixation but is abol-ished under the same conditions in crickets. This study also demonstrated that strong formaldehyde fixation induces a previously undescribed diaphorase activity in all the insects studied. This activity was determined as an increase in background staining, and as more pro-nounced staining in particular cell bodies: dorsal unpaired medial (DUM) and motor neurons in crickets and locusts; ventral association centres (VACs) in crick-ets; tracheal cells in Periplanta americana and Gryllus bimaculatus. Ott and Burrows suggest that this may be accounted for, in part, by the possibility that cyto-chrome-P450 oxidoreductase-associated haem-oxy-genase activity is associated with NOS-unrelated NADPH diaphorase activity in insect CNS. A further cautionary tale to emerge from this careful work is that the discrimination of NOS-dependent and independent NADPH activity cannot be relied upon using high (1 mM) concentrations of non-selective NOS inhibitor. It thus appears that results of NADPH diaphorase studies in insects should be interpreted carefully. However, it has been unequivocably shown that NADPH-diaphorase activity in insects is a good indicator of NOS activity (Muller, 1994), see below.
Fig. 5. Phylogenetic tree for NOS family members. Multiple sequence alignment was performed with CustalX (ftp://ftp-igbmc.ustrasbg.fr/pub/ClustalX/) using default parameters, and drawing a tree based on exclusuion of gaps, and with correction for multiple subsitu-tions. Plots were prepared using NJPlot, taking theLymnea stagnalis(T31080) NOS as an outgroup, and then formatted as a rootless tree using Treeview (Roderick Page, 1996, University of Glasgow). Major NOS groups are shaded. Full-length sequences were aligned for nNOS:Oryctolagus cuniculus (AAB68663), H. sapiens (P29475),Rattus norvegicus (P29476),Mus musculus (JN0609); eNOS:Cavia porcellus (AAD29753), M. musculus(S71424),H. sapiens(A47501),Bos taurus(A38943),Canis familiaris(AAD5216); iNOS:Cyprinus carpio(CAB60197),Gallus gallus (Q90703),Rattus norvegicus(BAA020),Canis familiaris(AAC7863),H. sapiens(A49676). Insect NOS sequences form a distinct group, most closely related to vertebrate nNOS and eNOS; the inducible iNOS family, cluster in a separate group.
ronal context in insects, evidenced by the number of recent reviews in this area (Muller, 1997; Jacklet, 1997; Bicker, 1998).
The first demonstration for a role of NO signalling in invertebrate nervous system was by purification of NOS from S. gregariabrain and demonstration of a NO-acti-vated guanylyl cyclase activity from this tissue (Elphick et al., 1993). More recently, NOS generation has been observed in defined areas of the locust brain, the mush-room bodies. The mushmush-room bodies contain previously undescribed neural structures of tubular bundles of axons enclosed by walls of NOS-expressing processes, which suggest targeting of NO to specific areas in the brain (O’Shea et al., 1998). This allows the generation of high levels of NO in a localised area of the insect brain asso-ciated with learning and memory. Also in Schistocerca, a study of neuronal networks in thoracic ganglia using the NADPH-diaphorase technique has demonstrated a possible role for NO in mechanosensory processing (Ott and Burrows, 1998). Results demonstrate strong staining in fibres in the VACs, which are known to be mechano-sensory neuropiles; stained fibres were also found in other mechanosensory neuropiles, suggesting a role for
the release of NO into these neuropiles during an early stage of tactile processing. All thoracic VACs receive NADPH-diaphorase positive innervation from the same intersegmental neurons, which are important for mediating exteroceptive events. The authors suggest that NO signalling may occur between parallel sensory chan-nels, thus integrating signals over space and time, resulting in plasticity during sensory processing. Additionally, NADPH-diaphorase staining has allowed for the morphological definition of previously unde-scribed NO-producing neurons in the thoracic nerve cord.
Also in Manduca, cGMP has been shown to be involved in the signalling mechanisms invoked by mus-carinic acetylcholine receptors (mAChRs) in CNS (Trimmer and Qazi, 1996). MAChR-expressing proleg motoneurons in larvae respond to muscarinic stimuli via increased cGMP levels and increased phosphoinositide signalling. Recent work has shown, however, that mus-carinic-induced proleg motonreuron activity is modu-lated by, but not dependent on, a NO-activated guanylate cyclase (Qazi and Trimmer, 1999). While it seems that NO may not be involved in this process, it remains poss-ible that calcium signalling mechanisms may be critical in the maintenance of neuronal activity by mAChRs, by the activation of a calcium/calmodulin-sensitive NOS.
NO has been shown to be important in vertebrate olfaction; insect studies have demonstrated that NO also contributes to this process in invertebrates. Chemosen-sory perception in insects occurs via the antennae, with processing and integration of signals occurring in the antennal lobe. In the hawkmoth,M. sexta, NOS is highly expressed in the antennae (Nighorn et al., 1998), whereas soluble guanylate cyclases are expressed in a subset of antennal lobe neurons, suggesting that com-munication between olfactory receptors and processing centres are modulated by NO signalling. NO signalling is also important in the development of antennal lobe neurons in Manduca: cGMP expression patterns corre-spond to the maturation phase of these cells during the pupal–adult transition (Schachtner et al., 1998). Further-more, a subset of local interneurons in lateral cluster I of the antennal lobe shows an elevated cGMP response to NO (Schachtner et al., 1999). Additionally, NO/cGMP signalling depends on input from the antennae and from central brain, as well as from ventral ganglia.
In other insects, NOS expression is pronounced in the olfactory interneurons of the antennal lobe of the locust,
S. gregaria (Elphick et al., 1995). Furthermore, NADPH-diaphorase studies in the honeybee,Apis melli-fera, show that NOS is expressed both in the antennae and the antennal lobe. However, in antennae, rather unexpectedly, NADPH-diaphorase staining is strongest in non-neuronal auxiliary and epithelial cells, with weak staining in the sensory cells and antennal nerve. In antennal lobe, however, strongest staining is observed in the glomeruli; inhibition of antennal lobe NOS interferes with the response to repetitive chemosensory stimuli (Muller and Hildebrandt, 1995). Hence, these studies strongly suggest that NO signalling is important in olfac-tory processing in several insect species.
Insects have also been used in the study of NO signal-ling processes in the visual system. InS. gregaria, NOS has been localised to the retina and lamina (Bicker and Schmachtenberg, 1997) and to the optic lobe (Elphick et al., 1996) using NADPH-diaphorase staining. The localisation of NOS in these tissues strongly implies that NOS is important in the visual system of insects,
although these studies do not prove a functional role for NOS/NO.
M. sextais extensively used for neuronal studies in a developmental context. Several studies have implicated the NO/cGMP signalling system in defined neuronal cir-cuits during developmental phases inManduca(Ewer et al., 1994; Wright et al., 1998). Most recently, it has been shown that cGMP is involved in the development of the peripheral neural plexus in Manduca larvae (Grueber and Truman, 1999). All neurons identified in this study showed a persistent, as opposed to transient, response to exogenous NO with associated increases in intracellular cGMP levels. However, NO-induced cGMP production in distinct neuronal subsets showed different sensitivities to external calcium concentrations, suggesting that cal-cium signalling mechanisms may regulate NO sensitivity in the plexus. Also, it is not clear that NO is the endogenous signalling molecule for these cells; it is likely that other activators of soluble guanylate cyclase may be involved. The authors of this work have also observed that persistent NO sensitivity is seen in hom-ologous cells inBombyx moriandDrosophila, and sug-gest that this feature may be a characteristic of multiden-dritic body wall neurons in holometabolous insects.
3.3. NO-transporting proteins in insects
It has long been known that hematophagous insects use salivary anti-coagulants in the feeding process, which also act to inhibit platelet aggregation and vaso-constriction in the victim (Law et al., 1992). However, it has only been discovered fairly recently that some blood-sucking insects have evolved a particularly elegant and complex mechanism involving NO, to ensure a sufficient blood meal.
The blood-sucking hemipteran, R. prolixus, has been shown to contain a salivary nitrovasodilator which con-sists of a haem protein that stores and delivers NO (Ribeiro et al., 1993). This new class of protein, the nitrophorins (Champagne et al., 1995), has since been shown to occur as several isoforms encoded by four genes, NP1–4 (Walker et al., 1999). It is also known that another species of blood-sucking insect, the bedbug
Cimex lectularius, also uses nitrophorins in its feeding strategy (Valenzuela et al., 1995).
In Rhodnius, a Ca2+/calmodulin sensitive-NOS has
been cloned from salivary glands (Yuda et al., 1996). NOS activity has also been measured in salivary gland homogenates and was shown to be Ca2+/calmodulin
complex binds the released histamine tightly and in doing so, displaces NO from its binding site thus releas-ing it into the wound. The release of NO is also pH dependent and is favoured by the pH change when the nitrophorin–NO complex is transferred from the salivary gland into the host. The physiological role of the trans-ported NO in vasodilation and in the prevention of plate-let aggregation ensures the insect derives a maximum blood meal.
4. Functional studies of NOS and NO signalling in
Drosophila
It is clear that studies of NO function in insects have revealed new insights into the role of NO signalling pathways and not least, have led to discoveries of new classes of NO-binding proteins. However, the role of the NO signalling pathway in insects has been derived larg-ely by homology to vertebrate NO signalling and by inference from immunocytochemical and/or NADPH-diaphorase expression studies.
In order to assess functional roles for NOS genes and NO signalling in vivo, it is necessary to modulate NO signalling processes selectively, via the generation of knockouts or by over- or mis-expression of transgenes. This demands a ‘genetic model’ organism, in which transgenics, targeted mutagenesis and genomic resources are available.D. melanogasteris a conspicuous exemp-lar of such an insect genetic model.
The first demonstrations of NOS expression in Droso-phila arose from NADPH-diaphorase staining studies in the brain, in particular the learning and memory centres, the mushroom bodies (Muller and Buchner, 1993). This was rapidly followed by further work to characterise the NOS in Drosophila and Apis, as part of the quest for the molecular basis for learning and memory in insects (Muller, 1994). This work showed importantly that NADPH-diaphorase activity in insects co-purified with NOS activity, suggesting that the NADPH-diaphorase assay frequently used as an index of NOS expression in insects, does indeed correctly reflect NOS expression patterns. Additionally, it was demonstrated that the NOS activity was inhibited by analogues of l-arginine and
was Ca2+/calmodulin sensitive, with a K
m of 300 nM.
At the same time, it was shown that NO signalling is important in a Drosophila peripheral tissue, the Mal-pighian tubule (Dow et al., 1994). The tubule, which acts in an osmoregulatory and excretory capacity, is now accepted as the prototypic genetic model of a fluid-trans-porting epithelium. It is possible to study ion transport and cell signalling processes in an organotypic context with the sophisticated molecular genetic tools available to Drosophila (Dow et al., 1998). It was demonstrated that NO-releasing compounds stimulated tubule fluid transport and raised intracellular cGMP levels; and that
tubules stained positively for NADPH-diaphorase (refer to Fig. 8). Furthermore, downstream elements of the NO/cGMP signalling pathway, including two forms of cGMP-dependent protein kinase G (cGK) are expressed in this tissue. There is also compelling evidence that the end-point of neuropeptide-stimulated NO/cGMP signal-ling in the tubule is the stimulation of the apical V-type ATPase, resulting in increased fluid secretion (Davies et al., 1995). This remains so far the only direct demon-stration of a role for NO signalling in the control of fluid transport by an epithelium.
4.1. Drosophila NOS
The cloning of the first insectNOS from Drosophila
(dNOS) revealed extensive similarity to nNOS/NOS1
(Regulski and Tully, 1995) at both the amino acid and DNA level. Heterologous expression of thedNOScDNA demonstrated that DNOS activity was Ca2+
/calmodulin-sensitive. Two transcripts of dNOS, one full-length and one truncated, have been detected although as yet, no function has been ascribed to the smaller transcript. Reverse-transcriptase PCR to Malpighian tubule cDNA using the dNOS sequence from Drosophila head (Regulski and Tully, 1995) showed that tubules express the full-length dNOS as in CNS (Davies et al., 1997); NOS enzyme activity is also measurable in this tissue using the 14[C]-labelled arginine–citrulline conversion
assay and is stimulated by the Drosophila neuropeptide CAP2b(Davies et al., 1997). Also, the soluble guanylate
cyclase inhibitor, methylene blue, attenuates ligand-stimulated cGMP production further, supporting the hypothesis that fluid transport is modulated by NO sig-nalling.
The use of transgenic aequorin targeted to specific tubule cell-subtypes in vivo to investigate cell-specific calcium signalling mechanisms (Rosay et al., 1997), revealed that CAP2bstimulation of tubules resulted in a
very rapid millisecond rise of cytosolic calcium levels ([Ca2+]
i) in only the Type I (principal) cells in the main,
fluid-secreting segment of the tubule. Elevation of [Ca2+]
i to levels of around 300 nM will stimulate the
activity of the Ca2+/calmodulin insect NOS, given the
Km for the reaction (Muller, 1994). We have recently
Fig. 6. Immunocytochemical localisation of Drosophila NOS (DNOS) in tubules. Immunocytochemistry performed with universal antibody to NOS (uNOS, Affinity BioReagents Inc.) on intact parafor-maldehyde-fixed tubules treated as in Rosay et al. (1997). Confocal microscopy of the tubule preparation (Rosay et al., 1997) reveals anti-NOS antibody staining in only Type 1 (principal) cells, and not in the star-shaped stellate (Type II) cells. (Unpublished data courtesy of Kate Broderick, University of Glasgow.)
4.2. NOS in insect tubules
Interestingly, while work in Drosophila tubules has so far suggested only one type of NOS in the control of fluid transport, studies of the tubules of other insect spec-ies suggests tubule expression of different forms of NOS which may correlate with functions different from fluid transport. For example, in the silkworm, B. mori, two forms of NOS have been found in Malpighian tubules (Choi et al., 1995). Both are constitutive enzymes, although one is Ca2+/calmodulin-dependent while the
other is Ca2+-independent but calmodulin dependent.
Intriguingly, both enzyme activities are reported to increase dramatically during the last larval instar, prior to spinning.
In the mosquito,A. stephensi, parasite development is limited by the inducible synthesis of nitric oxide, notably in the midgut of the animal (Luckhart et al., 1998). Per-haps surprisingly, characterisation of this inducible NOS gene suggests that this NOS is most similar to vertebrate nNOS (see Fig. 5). Furthermore, inA. gambiae, a study of six immune-responsive genes showed that expression of NOS in several tissues is increased uponPlasmodium bergheiinfection, (Dimopoulos et al., 1998). Using RT-PCR, the authors demonstrated that a 10-fold transcrip-tional induction of NOS occurred upon infection, notably the Malpighian tubules. This is the main site of NOS expression, particularly during early- and late-stage infection. It is interesting to note that, while the authors of this work did not comment on these significant find-ings, it is likely that increased NOS activity upon infec-tion fulfils a requirement for increased haemolymph clearance. This supports previous evidence for a role of NOS in epithelial fluid transport (Dow et al., 1994).
The A. gambiae study, as with the work in A. ste-phensi, demonstrated that firstly, NOS has an important function in the insect immune response and also, showed inducible NOS activity is present in insects. The only
Fig. 7. Summary of NO signalling pathway in tubule Type 1 (principal) cells. The neuropeptide, CAP2b, activates both DNOS and the nitric oxide signalling pathway (Davies et al., 1997) to stimulate the apical V-type H+-ATPase. CAP2balso stimulates a rise in intracellular calcium concentration in only principal cells in the tubule main seg-ment via calcium entry (Rosay et al., 1997). We have previously dem-onstrated that the endoplasmic reticular Ca2+-ATPase inhibitor,
thapsi-gargin, induces calcium entry in principal cells (Rosay et al., 1997). This suggests that elevation of intracellular calcium levels may occur as a consequence of activation of both plasma membrane calcium channels and store-operated calcium channels. Localisation of DNOS to principal cells demonstrates that the NO signalling pathway is com-partmentalised in this cell-type in the main, fluid-secreting segment of the tubule.
other example of inducible NOS activity comes from a study of NOS in a lepidopteran (Estigmene acrae) haemocyte cell line (Weiske and Wiesner, 1999). The authors of this work showed that NOS activity was induced up to three-fold in these cells upon infection with insect pathogenic bacteria and bacterial lipopoly-saccharide. The NOS activity (measured using the NADPH diaphorase assay, direct arginine–citrulline con-version assay and nitrite production assay) was influ-enced by the substrate, l-arginine, and inhibited by a
NOS inhibitor.
out. Given that the mosquito isoforms contain putative Ca2+/calmodulin binding sites, it will be interesting to
define possible regulatory calcium signalling processes which are implicated in activation of NOS.
Thus, it is likely that novel forms of NOS are expressed in insect epithelia and in insects in general, suggesting that genetic and functional studies of NOS in these systems is particularly valuable. For example, rec-tal pads in Drosophila stain strongly for NADPH-diaphorase activity (Fig. 8), suggesting that NO signal-ling is important in water conservation in Diptera. This novel, and possibly phylogenetically conserved, physio-logical role for NO signalling could be ideally investi-gated in Drosophila.
4.3. Developmental roles for NO signalling
Developmental roles for NOS have been investigated in several vertebrate and invertebrate systems, including neural networks in developing arthropods (Scholz et al., 1998; Truman et al., 1996), and in the developing visual systems of chicks (Wu et al., 1994). However, the well-characterised Drosophila visual system, with its repeti-tive arrangement of the compound eye (800 repeating ommatidia, with eight photoreceptor neurons, R1–R8, comprising each ommatidium which project to the optic lobes in the CNS) is a particularly good model for stud-ies of synaptic organisation. Pre-determined retinotopic connections between photoreceptors and post-synaptic elements in the optic lobe are necessary for the correct development of the visual system. Using this model sys-tem, Gibbs and Truman (1998) have elegantly demon-strated that NO signalling is essential for the correct
Fig. 8. NOS activity is present in insect epithelia: NADPH diaphor-ase staining of tubules and rectal pads in Drosophila. Left panel: tubules do not stain for diaphorase activity with nitroblue tetrazolium (NBT) in the absence of NADPH, the required co-factor for NOS activity. Centre panel: significant staining of the cells in the main seg-ment of the tubule in the presence of both NBT and NADPH. Right panel: rectal pads stain strongly for NADPH diaphorase in the presence of NBT and NADPH. (Figure reproduced with permission from Dow et al., 1994.) These data suggest that NOS has a functional role in different epithelia in insects; inDrosophila, it has been demonstrated that epithelial fluid transport is a NOS-dependent process. Furthermore, from NADPH diaphorase activity observed in the rectal pads, it is probable that NOS has an important role in water conservation.
development of the retinal projection pattern. During a 40-h period during metamorphosis, photoreceptors dis-play developmentally-regulated NO-sensitive guanylate cyclase prior to the establishment of functional connec-tions with optic lobe interneurons. Also during this per-iod, corresponding optic lobe retinal targets express NOS. Furthermore, in vitro culturing of the CNS/eye-antennal imaginal disc complexes, where there is contin-ual development of the viscontin-ual system, showed that phar-macological blockade of NOS and NO production and soluble guanylate cyclase activity results in retinal disor-ganisation and overgrowth. Rescue of this inhibition with 8-bromo-cGMP suggests that NO action is mediated solely via a soluble guanylate cyclase. Thus, it has been proposed that NO acts as an intercellular messenger in the developing visual system to stabilise or ‘arrest’ retinal axon growth during the early stages of metamorphosis prior to the establishment of permanent synaptic connections at a later stage during development. The influence of NOS signalling on the maintenance of correct retinal projection patterning has also been assessed using Drosophila mutants that show reduced, absent or apoptotically degenerating retinal innervation (Atkinson and Panni, 1999). Mutants with reduced (sine oculis) or absent (eyes absent) retinal innervation show reduced NADPH-diaphorase staining in target structures. However, this work also showed unexpectedly that pro-apoptotic (pGMR-hid) larvae, exhibited increased NADPH-diaphorase activity in laminar targets, for which there is not sufficient explanation.
InDrosophila, the organ structure in adults is determ-ined in the larval imaginal discs (Cohen, 1993). In determining the final structure of any organ, the relative timings and contributions of cell proliferation versus dif-ferentiation must be tightly controlled. Recent work in
confir-mation of the role of NOS as an anti-proliferative agent came from the use of GMR-P35 flies in this study, in which programmed cell-death is suppressed in the developing eye. Programmed cell-death is necessary for the establishment of the normal eye phenotype. If NOS is inhibited in GMR-P35 flies, there is excessive prolifer-ation in various cell types resulting in abnormal eye development. The diffusible nature of NO suggests that it can act as an important intercellular messenger to con-trol cell proliferation in development. More recent work using transgenic flies expressing the Drosophila NOS gene (dNOS) has confirmed the role of NOS as an anti-proliferative signal in developing imaginal eye discs (Kuzin et al., 2000). In the developing eye, cell cycle progression is controlled by the retinoblastoma (Rb) pathway. Utilising transgenic flies which express the Rb pathway components, Rb-like protein (RBF) and the E2F transcription factor complex components (dE2F and dDP), this work elegantly demonstrated that RBF and/or E2F are possible molecular targets for NO in the developingDrosophilaeye. The authors suggest that NO can act to modulate the entry of cells into S-phase in two ways: by suppressing the activity of a rate-limiting component of DNA synthesis (ribonucleotide reductase); and by preventing the E2F-induced transcription of genes required for the initiation of DNA synthesis.
The role of NOS in the developing Drosophila CNS has been addressed by studies of NO- and cGMP-pro-ducing neurons during embryonic, larval and pre-pupal stages (Wildemann and Bicker, 1999a). Several neuronal and glial cell types were identified as NO-producing cells using NADPH diaphorase staining. In addition, cGMP-immunoreactivity was used to detect individual motoneurons, sensory neurons and groups of interneu-rons in brain and ventral cord. Interestingly, NADPH-diaphorase positive imaginal discs containing NO-responsive sensory neurons suggests that NO can signal between epithelial and neuronal cells in a transcellular context.
This work also suggested that NO acts as a retrograde messenger from larval muscle fibres to the pre-synaptic terminals. To further investigate this possibility, a study of the role of NO/cGMP signalling at the neuromuscular junction (NMJ) was undertaken (Wildemann and Bicker, 1999b). Results from this study have shown that NO donors and cGMP analogues cause vesicle release at the NMJ in Drosophila larvae. NO-induced release at the NMJ is dependent on soluble guanylate cyclase, but does not require external Ca2+.
5. Downstream components of NO signalling in insects
Numerous studies of downstream effectors of NO sig-nalling have been undertaken in vertebrates. However,
as described below, it is clear that insects have novel forms of enzymes in the NO signalling pathway. It remains that the study of control of these novel isoforms will be particularly intriguing.
5.1. Guanylate cyclases
Signalling mediated by guanylate cyclases occurs via receptor guanylate cyclases (rGCs), soluble guanylate cyclases (sGCs) or photoreceptor rod outer segment guanylate cyclase (ROS-GC), resulting in increased intracellular cGMP concentration. The rGC group are single transmembrane proteins which act as receptors for extracellular ligands and consist of several sub-families, which have important roles in cardiac and renal function in vertebrates (Wedel and Garbers, 1997). In vertebrate photoreceptors, a separate family of guanylate cyclases, the ROS-GCs, mediate phototransduction events by operating a Ca2+/cGMP feedback loop (Pugh et al.,
1997). The ROS-GC family, comprising ROS-GC1 and ROS-GC2, are single transmembrane-spanning proteins which differ from the other membrane-bound GCs in that they are not regulated by extracellular ligands. Instead, they are regulated intracellularly by Ca2+ via
Ca2+ binding proteins. ROS-GC1 is stimulated at low
Ca2+(100 nM or below) by two cyclase activating
pro-teins, GCAP1 and GCAP2, while ROS-GC2 activity is modulated by GCAP2 (Goraczniak et al., 1998). A third activating protein, S100β (calcium-dependent GCAP, CD-GCAP) activates ROS-GC1 at Ca2+in the
micromo-lar region. Neurocalcin has also been recently found to activate ROS-GC1 (Kumar et al., 1999). Thus, in ver-tebrates, signalling via guanylate cyclases is extremely complex, and it appears that the situation is no different in invertebrates.
The recent discovery, however, of at least 29 GC sequences inC. elegans (Morton et al., 1999; Yu et al., 1997) suggest further complexity of the role of these enzymes in physiological function in both vertebrates and invertebrates. In insects, several rGCs have been cloned in Drosophila. D1, cloned using a rat rGC-A probe to a Drosophila head cDNrGC-A library (McNeil et al., 1995), is present in all stages of embryonic develop-ment, while workers from another group have found that this rGC is expressed in all adult head and body tissues studied (Liu et al., 1995). DrGC-1 has high sequence homology to the cyclase domain in rGCs but has a 430 aa non-homologous extension at the C-terminus of unknown function. Yet another rGC mRNA in Droso-phila has been shown to be developmentally regulated (Gigliotti et al., 1993).
to other diagnostic rGC domains. Furthermore, as in the
Drosophila rGCs, there is a C-terminus extension of unknown function that is not seen in other rGCs.
The main intracellular receptor for NO, however, is the sGC, a heterodimeric haemoprotein, composed of alpha (1 and 2) and beta (1 and 2) subunits (Koesling, 1999). Elegant experiments have recently demonstrated that sGC is an extremely efficient trap for NO and that previously proposed NO-storage and -transporting pro-teins are not required in the NO-cGMP cascade in ver-tebrates (Zhao et al., 1999). NO binding to the haem group forms a ferrous-nitrosyl compound via an inter-mediate in a NO-dependent fashion. sGC very rapidly binds diffusible NO, with the ensuing catalysis being dependent on NO concentration. This ensures a very sensitive NO-sensing system which allows for regulation of the activity of downstream components of the signal-ling pathway.
sGCs have been cloned in Drosophilaand have been investigated in other insect species in studies of NO action. The first sGC cloned in Drosophila (dgc1) (Yoshikawa et al., 1993) showed that the catalytic domain in Drosophila and vertebrate sGC was highly conserved. Expression studies showed that dgc1mRNA was most abundant in head, with eyes and CNS being enriched for the message. This work was supported by the observation that the alpha subunit of the enzyme, designated DGC alpha 1, is expressed preferentially in theDrosophila nervous system (Liu et al., 1995). Work published at the same time revealed a homologous alpha subunit, Dgc alpha 1, which unlike DGC alpha 1, is localised to the retina and also to the brain, suggesting a role for this sGC in vision (Shah and Hyde, 1995). The same study revealed a beta subunit, Dgc beta 1, which showed high conservation to rat beta 1 subunit, although the Drosophila protein possesses an insertion of approximately 150 amino acids at the N-terminus extension which is not present in the rat isoform. Co-expression of both subunits in a stable cell line resulted in significant guanylate cyclase activity, suggesting that these subunits associate as a heterodimer. Furthermore, this enzyme is potently stimulated by NO donors.
It is now clear that the sGC alpha subunits identified in all three studies are transcribed from the same gene, Gycα99B (Flybase, http://fly.ebi.ac.uk:7081/altviews/ topicsview.html). The gene, which has been localised to chromosome 3, 99B9-10, encodes a 676 amino acid pro-tein with an identified guanylate cyclase signature domain (http://fly.ebi.ac.uk:7081/.bin/fbidq.html?FBgn 0013972). There are identified homologues in M. sexta, C. elegans, H. sapiens and M. musculus. Four transcripts of the gene (Gycα99B+R2.4, Gycα99B+R2.5, Gycα99B+R2.8, Gycα99B+R4.1) have been described, and are expressed in the head. One transcript (Gycα99B+R2.5), however, has also been detected in embryo, pupae and in the adult optic lobe and CNS (Liu
et al., 1995). By Northern blot analysis, the other tran-scripts have also been associated with adult head, and not body (Shah and Hyde, 1995). Thus, differences in transcript selection may account for the differences in sGC alpha 1 subunit expression characterised in both the 1995 studies. It is clear, however, that signalling path-ways must be conserved in other parts of the fly apart from the head and the CNS, and that sensitive molecular techniques, for example reverse-transcriptase (RT)-PCR, are required to detect low-abundance transcripts in these peripheral tissues.
Studies of sGCs in other insects have identified new sGC genes; for example, inA. gambiae, the sequence of sGC beta subunit has been found to be highly similar to both theDrosophila and vertebrate sGC beta 1 gene (Caccone et al., 1999). Furthermore,M. sextahas proved a good model for the isolation and study of novel GCs. Nighorn et al. (1998) have shown that twoM. sextasGC subunits (MsGCalpha1 and MsGCbeta1) are present in the olfactory system of adult moths. Furthermore, the subunits form obligate heterodimers, with enzyme activity being significantly stimulated by NO.
The same group have recently isolated a novel form of sGC (Nighorn et al., 1999). This subunit, M. sexta
guanylyl cyclase beta3 (MsGC-beta3), isolated from the nervous system, shows high similarity to mammalian sGCs. However, theManducabeta subunit does not con-tain several of the amino acid residues observed in rat beta 1 subunits which contribute to haem binding or NO activation. Also, the C-terminus of the catalytic domain contains a novel sequence of 315 amino acids. Intriguingly, upon expression of MsGC-beta3 in COS-7 cells, guanylate cyclase activity is detected, suggesting that it does not require dimerisation for function. This enzyme is also only marginally activated by NO.
It is clear that the study of insect GCs is likely to provide new insights into the structure and function of this family of enzymes. The completion of the Droso-philagenome project is likely to produce more candidate GCs, with the possibility of available mutants for func-tional studies.
5.2. cGMP-dependent protein kinases
cGMP-dependent protein kinase G (cGK) is a major effector of cGMP and thus, NO signalling in many cell types (Pfeifer et al., 1999). In mammals, cGKs have been identified both biochemically and immunocytochem-ically; furthermore, two genes have been identified. cGK-I, of which there are two isoforms, alpha and beta, is preferentially expressed in smooth muscle, platelets and cerebellum, whereas cGK-II is concentrated in brain, lung and intestinal mucosa.
InDrosophila, there are two cGK genes in total,dg1
postulated to be head-specific (Kalderon and Rubin, 1989). More recent work to characterise dg1 shows localisation to optic lobes and proximal cortex (Foster et al., 1996). dg2 expression, however, was seen in the head and body of adults (Kalderon and Rubin, 1989). In fact, it has been shown that Drosophila tubules express bothdg1anddg2, suggesting that both cGKs have a role in epithelial transport (Dow et al., 1994). Both genes are characterised by transcriptional complexity, with at least three transcripts fordg1and 11 fordg2, suggesting that even for a simple organism, a variety of tissue-specific forms are functionally important. It is also likely that different cGKs are expressed in a cell-specific manner. cGK has also been implicated in Drosophila behav-iour (Osborne et al., 1997). The single gene for a nat-urally occurring polymorphism is the foraging (for) gene, associated with food-search behaviour in both lar-vae and adults. The rover allele (forR) results in the
ani-mal moving greater distances in food searches compared to those animals homozygous for forS, the sitter allele.
Both wild-type and mutant alleles of the gene map to region 24A3-5 of theDrosophilapolytene chromosome, containing dg2(de Belle et al., 1993).
P-element mutagenesis (mobilisation of the transpos-able P-element throughout the genome) is used in Droso-phila to generate mutants and gene knock-outs (Rubin and Spradling, 1982). In an elegant demonstration, Osborne et al. showed that a P-element insertion into
dg2, which resulted in a sitter phenotype, reverted to rover upon excision of the P-element. Furthermore, the sitter phenotype was rescued by overexpression of dg2. Biochemical analysis of cGK activity in fly heads of sit-ter and rover phenotypes showed that cGK activity was reduced in sitter strains. Thus, cGMP signalling, anddg2
specifically, is responsible for food-search behaviour in
Drosophila, although it has yet to be demonstrated that NO signalling is implicated in this process.
Recent work using for alleles has implicated the NO/cGMP signalling in a behavioural response of Dro-sophila larvae to hypoxia (Wingrove and O’Farrell, 1999). This work also utilised transgenic lines which overexpress iNOS (NOS2). Results suggest that under hypoxic conditions, the larval behavioural response is modulated by NO/cGMP signalling. More specifically, the response can be inhibited by pharmacological block-ade of NOS activity, and is slower in forSlarvae, which
show reduced cGK activity. However, upon expression of the NOS transgene, larvae are hypersensitive to hypoxia. Similarly, scoring of forRand forS larvae
sur-vival and recovery rates under hypoxic and post-hypoxic conditions suggested that cGK is important for survival under low oxygen conditions. Long-term developmental effects of hypoxia include the development of the tra-cheal system, which in larvae is thought to be induced in response to oxygen requirements (Manning and Kras-now, 1993). Wingrove and O’Farrell showed that
elabor-ation of tracheal tubes is reduced in forS larvae, and
when NOS is inhibited. However, expression of NOS2
enhances the development of tracheal tubes. In ver-tebrates, NOS signalling is implicated in hypoxia due to the established role of NOS in vasodilation. However, there is no known mechanism for the action of NO/cGMP signalling in either vertebrates or invert-ebrates; studies in Drosophila should go some way to addressing these issues.
6. Conclusions
The application of convergent molecular, biochemical and physiological analysis to the study of NOS and NO signalling in insects has revealed unexpected complexity in the utilisation of this pathway. Furthermore, insect studies have uncovered novel genes for components in the NO/cGMP signalling pathway, whose protein pro-ducts are regulated differently from vertebrate equiva-lents.
Amongst the estimated 30 million species of insects, all of which utilise NO as a signalling molecule, some insect species are disease vectors, yet others are econ-omic pests. It is clear that insect studies of NOS function and NO signalling will not only generate results of gen-eral significance in biology but may also result in new controls for pest species.
Acknowledgements
This work was supported by a Biotechnology and Bio-logical Sciences Research Council (BBSRC, UK) per-sonal fellowship and grants. I am grateful to Dr Martin Boocock for discussion and generation of the NOS struc-ture in Fig. 1 and to Professor Julian A.T. Dow for invaluable help and advice, and for critical reading of the manuscript.
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