Nitric oxide biogenesis, signalling and roles in molluscs: The Sepia officinalis paradigm

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Nitric oxide biogenesis, signalling and roles in molluscs: The Sepia officinalis paradigm Anna Palumbo1, and Marco d’Ischia2 1

Zoological Station Anton Dohrn, Villa Comunale, 80121 Naples, Italy Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Via Cinthia 4, 80126 Naples, Italy 2

Abstract. The past decade has witnessed a burst of interest in the biological roles of nitric oxide (NO) and its signalling pathway in molluscs. Several roles of NO have been demonstrated in different functions often related to specific behaviours such as olfaction, feeding, learning, defence, development and movement. The complex roles of NO in the ink gland and nervous system of the cuttlefish Sepia officinalis are paradigmatic in this respect. Stimulation of NO production via the N-methyl-D-aspartate (NMDA) receptor and cyclic guanosine monophosphate (cGMP) signal transduction pathway induces a series of events that, though apparently unrelated, come together to control overall regulation of the ink defence system. It is the aim of this chapter to provide a brief overview of the biogenesis and roles of NO in molluscs with specials reference to studies carried out in the authors’ laboratories on the ink system of S. officinalis. A prospective analysis of future advances in the field is also offered. Keywords: dopa; dopamine; ink gland; invertebrates; melanin; molluscs; nitration; nitric oxide; nitric oxide synthase; S. officinalis; a-tubulin; tyrosinase; ink; ink sac; catecholamine; melanosome; NMDA receptor; glutamate; cuttlefish; tyrosine.

Introduction The discovery of nitric oxide (NO) as a unique endogenous regulator of blood flow, a neurotransmitter in the central and peripheral nervous system, and a pathophysiological mediator of inflammation and host defence in humans and mammals has stimulated during the past decades intense and extensive research aimed at unravelling its origin and functions into nearly every living organism (Bruckdorfer, 2005; Moncada et al., 1991). The enzyme NO synthase (NOS) occurs in mammals in at least three distinct isoforms, neuronal NOS or NOS1/NOS I (nNOS), inducible NOS or NOS2/NOS II (iNOS) and endothelial NOS or NOS3/NOS III (eNOS). All NOS isoforms are homodimers with subunits of 130–160 kDa. All have binding sites for NADPH, FAD and FMN near the carboxyl terminus (the reductase domain), and binding sites for tetrahydrobiopterin Corresponding author: Tel.: 39-81-5833276(293). Fax: 39-81-7641355. E-mail: [email protected] (A. Palumbo). ADVANCES IN EXPERIMENTAL BIOLOGY VOLUME 01 ISSN 1872-2423 DOI: 10.1016/S1872-2423(07)01002-2

r 2007 ELSEVIER B.V. ALL RIGHTS RESERVED

46 (BH4) and heme near the amino terminus (the oxygenase domain). The reductase and oxygenase domains are linked by a calmodulin (CaM) binding site, which is believed to promote electron transfer from the cofactors in the reductase domain to heme, leading to NO production (Alderton et al., 2001; Ghosh and Salerno, 2003; Li and Poulos, 2005; Stuehr, 2004). Parallel to the studies on NOS and NO signalling in humans and mammals, interest in the biogenesis, signalling and roles of NO in invertebrates has grown steadily, following demonstration of the involvement of NO in diverse physiological processes and in a variety of organisms (Bicker, 2001; Davies, 2000; Enikolopov et al., 1999; Moroz, 2000; Moroz and Gillette, 1995; Palumbo, 2005; Torreilles, 2001; Trimmer et al., 2004; Walker et al., 1999). This chapter focuses on NO synthesis, regulation and signalling in molluscs, with special reference to the cuttlefish Sepia officinalis. It aims to update the interested reader on the latest advances in this rapidly unravelling area of NO research, largely as a result of continuing efforts in the authors’ laboratory and other research centres. It also seeks to stimulate new studies directed at gaining a more complete understanding of the many roles NO plays in invertebrates as compared to mammals. Molluscs An overall view of the roles and functions of NO in molluscs is schematically illustrated in Fig. 1. Gastropoda Among the Gastropoda Pulmonata, NO has been reported in Biomphalaria glabrata, Helisoma trivolvis, Achatina fulica, in different species of Helix and Limax, in Lymnaea stagnalis and in Planorbarius corneus. The snail B. glabrata is intermediate host for the transmission of the trematode parasite Schistosoma mansoni, the causative agent of the human tropical disease schistosomiasis. It has been reported that NO is involved in killing Schistosoma mansoni sporocysts by hemocytes from resistant snails (Bayne et al., 2001; Hahn et al., 2001). In the pond snail H. trivolvis NO regulates early embryonic behaviour, stimulating ciliary beating of embryos within the egg capsule during development (Cole et al., 2002; Doran et al., 2003). It is also a signalling molecule in neuronal development (Trimm and Rehder, 2004; Welshhans and Rehder, 2005). In the snails A. fulica and Helix aspersa NO modulates heart

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Fig. 1. Biological roles of NO in molluscs.

activity (White et al., 2004). In the common snail Helix lucorum NO is involved in neural transmission to intestinal muscles, and its production is blocked during dormancy (Roszer et al., 2004). In the snail Helix pomatia NO is involved in food-attraction conditioning (Teyke, 1996). Blocking NOS prior to conditioning significantly affects acquisition of memory, impairing the food-finding ability, whereas memory recall and olfactory orientation are not disturbed, as revealed by the ability of the snail to locate the conditioned food. The effect of NO on ion currents in snail neurons has been studied and it has been found that NO increases excitability by depressing a calcium-activated potassium current (Zsombok et al., 2000). Interestingly, a new class of NOS enzymes has been identified in H. pomatia. From an expression library, a cDNA was isolated which

48 codes for a 60 kDa protein recognised by an antibody to human neuronal NOS (Huang et al., 1997). This protein does not contain consensus binding sites for NADPH, FAD, arginine or CaM and it is similar to GTPase proteins found in bacteria, insects, mammals and plants (Zemojtel et al., 2004). In the terrestrial slug Limax marginatus NO is involved through the cyclic guanosine monophosphate (cGMP) pathway in olfactory processing, modulating electrical oscillations in the procerebral lobes (Fujie et al., 2002, 2005). In the terrestrial mollusc Limax maximus NO, together with carbon monoxide, modulates oscillations of olfactory interneurons (Gelperin et al., 1996, 2000). In the terrestrial slug Limax valentianus NO plays a crucial role in fine olfactory discrimination (Sakura et al., 2004). In the pond snail L. stagnalis NO is involved in long-term memory formation (Kemenes et al., 2002; Korneev et al., 2005), embryonic development (Serfozo and Elekes, 2002), feeding (Elphick et al., 1995; Kobayashi et al., 2000a,b; Korneev et al., 2002; Moroz, 2000), and the cardiorespiratory response to hypoxia (Taylor et al., 2003). Important insights into the mechanisms of NOS regulation have been gained in L. stagnalis: in addition to the full-length mRNA encoding for NOS, two pseudo-NOS transcripts, originated by DNA inversion, have been identified. These regulate NOS expression at the translational level by a natural antisense mechanism and at a post-translational level by the formation of nonfunctional heterodimers (Korneev and O’Shea, 2002; Korneev et al., 1998, 1999). Differential regulation of the activity of this group of related genes occurs during long-term memory formation and in the neural network involved in this process (Korneev et al., 2005). Moreover, gene silencing experiments have demonstrated that the expression of NOS is essential for normal feeding behaviour (Korneev et al., 2002). In the freshwater snail P. corneus NO acts as a messenger molecule in neuron–microglia communication in the central nervous system (CNS) (Peruzzi et al., 2004). Among the Gastropoda Prosobranchia, it has been reported that in the marine snail Ilyanassa obsoleta NO acts as an endogenous inhibitor of metamorphosis (Bishop and Brandhorst, 2003; Leise et al., 2004), and in Viviparus ater NO is used as bactericidal substance in the defence mechanism (Franchini et al., 1995; Ottaviani et al., 1993). Regarding the Gastropoda Opistobranchia, NO has been reported in Aplysia californica, Clione limacina, Melibe leonina and Pleurobranchaea californica. The sea slug A. californica is characterised by large identifiable neurons that allow identification of the neurons involved in specific

49 behaviours, and thus related to biochemical events in individual cells and specific functions. In particular, the serotonergic feeding neural circuit, the metacerebral cell, has received a good deal of attention. It has been reported that NO, via cGMP, mediates the membrane responses of metacerebral neurons (Koh and Jacklet, 1999), and that NO, serotonin and histamine all depolarise and increase the excitability of these neurons by diverse mechanisms (Jacklet and Tieman, 2004; Jacklet et al., 2004). NO has also been shown to modulate acetylcholine release in the buccal and the abdominal ganglia (Meulemans et al., 1995) and to be involved in chemosensory processes, as revealed by localisation of putative nitrergic neurons in peripheral chemosensory areas (Moroz, 2006). Learning experiments have revealed that during training in Aplysia, NO signalling plays a critical role in the formation of multiple memory processes (Katzoff et al., 2002). Furthermore, NO is involved in the regulation of feeding (Lovell et al., 2000). The NOS from the mollusc A. californica has been cloned (NCBI Accession ID AF288780 – Sandreyev et al., 2000) and biochemically characterised (Bodnarova et al., 2005). In the opistobranchia C. limacina NO is involved in feeding and locomotion (Moroz et al., 2000). In the nudibranch M. leonina NO is used in the CNS to modulate swimming (Newcomb and Watson, III, 2002). In P. californica NO is involved in feeding behaviour (Hatcher et al., 2006; Moroz, 2000).

Bivalva Among the Bivalva, NO has been reported in Mercenaria mercenaria, Mytilus edulis, Mytilus galloprovincialis, Ruditapes decussatus, Tapes philippinarum and Venus verrucosa. In the clam M. mercenaria the potentiation of gill muscle is mediated by a NO–cGMP–PKG (protein kinase G) signalling pathway (Gainey and Greenberg, 2003). In the bivalves M. edulis and M. galloprovincialis NO is involved in defence mechanisms: NO is produced by the hemocytes to kill pathogens (Arumugam et al., 2000; Franchini et al., 1995; Ottaviani et al., 1993). The same mechanism is also used by the carpet shell clam R. decussatus (Tafalla et al., 2003). In the mussel M. edulis NO also modulates microglial activation and the physiological control of ciliary activity (Cadet, 2004; Stefano et al., 2004). In T. philippinarum NO has a regulatory role in mucus secretion (Calabro et al., 2005), and in V. verrucosa it is involved in the control of reproduction (Barbin et al., 2003).

50 Cephalopoda NO is involved in the complex mechanisms implicated in the initiation and maintenance of symbiont infection of the light organ of the Hawaiian bobtail squid, Euprymna scolopes, by the bacterium Vibrio fischeri (Davidson et al., 2004). The production of NO begins during light organ embryogenesis, and reaches its highest levels in the light organ of newly hatched animals. Interestingly, it has been suggested that NO regulates the number of bacteria. Indeed, NO is released into the mucus secreted by the light organ, where the symbiotic bacteria aggregate before migrating into the final sites of colonisation. In Octopus vulgaris NO is reported to be involved in both visual and tactile learning (Robertson et al., 1994, 1996). Intramuscular injections of the NOS inhibitor NGnitro-L-arginine methyl ester (L-NAME) block both types of learning. These studies underscored the importance of molluscs as model systems for investigating NO biogenesis, signalling and roles in different functions, often underlying specific behaviours. A noticeable example is provided by the cephalopod Sepia officinalis, in which NO has been implicated in manipulative behaviour, modulation of statocyst activity, regulation of blood flow and blood pressure, and the complex processes and neural pathways associated with the ink defence system. Very recently, S. officinalis NOS has been cloned, allowing the first insight into the structure of a cephalopod NOS. A summary of current knowledge of the NO signalling pathway in S. officinalis is provided below. Sepia officinalis The first evidence for the presence of NOS activity in a cephalopod came in 1994 from a study by Chichery and Chichery (1994). These authors showed that NADPH-diaphorase activity was selectively localised in the CNS of S. officinalis. In particular, only the neuropils of the spines of the peduncle and posterior anterior basal lobes were found to exhibit an intense positive staining, which by contrast was completely absent from the cell bodies. These two regions of the brain are thought to constitute cerebellar analogues (Hobbs and Young, 1973; Messenger, 1967a,b). In light of the structural analogies of the peduncle and anterior basal lobes with the cerebellum, these findings were taken to suggest that NO may be involved as a signal molecule in learned motor skills. Some years later, our group reported for the first time the occurrence of a Ca2+/CaM-dependent NOS activity in the ink gland of S. officinalis (Palumbo et al., 1997) by measuring the formation of radiolabelled

51 citrulline from [14C] L-arginine. Moreover, immunohistochemical analyses showed the presence in the ink gland of NMDAR1 (N-methyl-Daspartate receptor 1) glutamate receptors, raising the possibility that the glutamate–NO neurotransmission pathway played a regulatory role in melanin synthesis and related processes associated with the inking mechanism. Inking is a characteristic behaviour adopted by nearly all coleoid cephalopods, i.e., S. officinalis, Loligo vulgaris and Octopus vulgaris, to confuse predators and alert conspecifics to danger while retreating. It represents the final event of the defence behaviour which initially consists of the development of a series of chromatophore patterns involving mantle spots and strips, followed by body blanching and darkening, jet propulsion movements, erratic jetting and finally inking (Hanlon and Messenger, 1996). The ink is a suspension of black melanin granules produced by the ink gland. The ink gland is composed of two distinct zones possessing different histological and biochemical characteristics: the inner glandular epithelium and the outer glandular epithelium (Fig. 2). In the inner glandular epithelium the cells are immature, do not produce melanin and are considered young cells. They gradually mature and migrate towards the external portion where they differentiate,

Fig. 2. The cuttlefish S. officinalis. The central nervous system, comprising the brain and optic lobes, is shown together with the ink-producing system. Note the ink gland and the ink sac. Inset: microscopic structure of the ink gland. (See Colour Plate Section in this book).

52 acquire the ability to produce melanin, and form the outer glandular epithelium. Melanin formation within mature cells shares many features in common with the process of melanogenesis in epidermal melanocytes (Hearing, 1999, 2005). The initial step of the process is the tyrosinasecatalysed oxidation of L-tyrosine to an unstable ortho-quinone, which is rapidly converted to melanin within specific organelles called melanosomes. An overview of the biochemical events leading to melanin formation in S. officinalis has recently been provided (Palumbo, 2003). In addition to melanin, ink gland cells are able to synthesise 3,4dihydroxyphenylalanine (DOPA) and dopamine (DA) through the tyrosine hydroxylase and DOPA decarboxylase route (Fiore et al., 2004) (Fig. 3).

Fig. 3. Tyrosine metabolism in a mature ink gland cell. The ultrastructure

of a mature ink gland cell shows the nucleus (N), a highly developed rough endoplasmic reticulum (RER), a mitochondrion (M), several melanosomes and catecholamine-containing vesicles. The two biochemical pathways leading to melanin production and 3,4-dihydroxyphenylalanine (DOPA) and dopamine (DA) biosynthesis are highlighted.

53 Within mature cells, DA and melanin are present in separate cellular compartments: whereas melanin is confined to the melanosomes, DA appears to be segregated within electron-dense vesicles resembling the catecholamine-containing vesicles reported in other systems. When maturation of the cells is complete, the melanin and other cellular components are shed into the lumen of the gland by a poorly defined mechanism. They are then transferred to the ink sac, which serves as reservoir for the black ink that the animal ejects when frightened (Fig. 2). The biochemical mechanisms involved in the regulation of tyrosinase activity and ink production are still little understood. The discovery in 1997 of the presence of NOS and NMDAR1 glutamate receptors in the ink gland (Palumbo et al., 1997) raised the possibility that the glutamate–NO transmitter pathway is involved in the specific signalling system that keeps the biochemical machinery for ink production active to ensure that sufficient amounts of melanin are available when necessary. To test this hypothesis, ink glands were incubated in the presence of various compounds with the aim of stimulating or inhibiting the endogenous production of NO, and the effects on the activity of tyrosinase were determined (Palumbo et al., 2000). Glutamate and NMDA were found to cause a dramatic increase in tyrosinase activity, whereas the NOS inhibitor L-NAME suppressed the NMDA-induced stimulation of tyrosinase. The role of NO in glutamate-induced activation of tyrosinase was confirmed by incubation experiments with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO), which elicited an increase in tyrosinase activity. 8-Bromo-cyclic GMP, a permeable and nonhydrolysable analogue of cGMP, resulted in a substantial increase of tyrosinase activity, suggesting that cGMP mediates the stimulating effect of NMDA. This conclusion was corroborated by the finding that stimulation of ink glands with NMDA resulted in a more than six-fold increase of cGMP levels compared to basal levels. Immunohistochemical evidence indicated that enhanced cGMP production is localised largely in the mature part of the ink gland. Tyrosinase activation by NO apparently involved phosphorylation through the action of PKG, without de novo synthesis of the enzyme. NMDA receptor stimulation and exposure to NO in the presence of DOPA, an excellent substrate of tyrosinase, resulted in a marked increase in the melanin content of the ink gland. These results demonstrate that the excitatory neurotransmitter L-glutamate promotes activation of tyrosinase at the appropriate stage of maturation and maintains the enzyme in an active state, acting via NMDA receptors, as schematically outlined in Fig. 4. Activation of the NMDA glutamate receptor causes an influx of calcium, which binds to CaM and activates

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Fig. 4. NMDA–NO–cGMP signalling pathway in mature ink gland cells: tyrosi-

nase activation, melanin synthesis and secretion of ink constituents.

the NOS to produce NO. NO targets guanylyl cyclase to produce higher levels of cGMP, which activates tyrosinase by phosphorylation and promotes melanin formation. Besides acting on melanin production, NO was found to affect the secretion of ink constituents into the lumen (Fiore et al., 2004). NMDA stimulation or treatment with a NO donor of ink glands exposed to [14C] tyrosine resulted in a marked decrease in the levels of radioactive DOPA and DA along with a consistent loss of DA immunoreactivity in mature cells. This loss was due to release of radiolabelled DOPA and DA in the incubation waters. The involvement of cGMP in the ink constituents’ release was shown by incubation experiments with [14C] tyrosine in the presence of 8-bromo-cyclic GMP or a guanylyl cyclase inhibitor. Thus, DOPA and DA appear to be released from the ink glands by processes controlled through the NMDA–NO–cGMP signalling pathway (Fig. 4). Enzymatic assays have revealed that a Ca2+-dependent NOS is also found in the protein extracts of brain and optic lobes (Palumbo et al., 1999). Moreover, a NOS-like immunoreactivity and operation of the glutamate–NO–cGMP signalling pathway have also been demonstrated

55 in the cuttlefish nervous system, both at the central and peripheral levels. Cephalopods have arguably the largest and most complex nervous systems amongst the invertebrates, but despite the squid giant axon being one of the best-studied nerve cells in neuroscience, and the availability of much information on the morphology of some cephalopod brains, there is surprisingly little known about the operation of the neural networks that underlie the sophisticated range of behaviour these animals display. The brain of S. officinalis is formed by the complex of supraoesophageal mass and suboesophageal mass, as distinct from the optic lobes. It is essentially organised hierarchically: motor programmes, usually originating in the optic lobes, are executed via higher motor centres, where specific motor commands are generated and directed to the appropriate sets of motoneurons in the lower centres (Messenger, 1983). Immunohistochemical mapping of S. officinalis CNS showed specific localisation of NOS-like and NMDAR2/3-like immunoreactivities in the regions and nervous fibres controlling the inking system, i.e., the latero-ventral palliovisceral lobe, the visceral lobe, the pallial and visceral nerves, as well as in the sphincters and wall of the ink sac (Palumbo et al., 1999). Parallel studies revealed that NOS is also present in other regions of the CNS involved in a variety of functions, including feeding, motor, learning, visual and olfactory systems (Di Cosmo et al., 2000). In addition to the cGMP-dependent signalling pathway, in S. officinalis brain the NO signal can also be transduced through cGMP-independent processes, such as protein nitration (Schopfer et al., 2003). Interestingly, a major target of nitration proved to be a-tubulin (Palumbo et al., 2002). After activation of endogenous NOS by treating optic lobes with NMDA, an approximately 50% decrease of the a-tubulin band at 30 min was observed, with respect to the control, followed by a partial recovery (75%) at 4 h. The 3-nitrotyrosine immunopositivity shows an opposite trend, with an increase after 30 min stimulation followed by a substantial decrease. Overall these results point to the existence of an a-tubulin-mediated signalling in the optic lobes (Fig. 5). After NOS activation, the nitrated a-tubulin is formed, and as soon as the nitration process exceeds a threshold value, a specific mechanism becomes operative which not only efficiently degrades the modified protein but also acts to restore basal levels of functionally active a-tubulin by protein synthesis. On this basis, a-tubulin nitration may represent a natural mechanism of cytoskeletal protein turnover. Structural insight into cuttlefish NOS has recently been gained by cloning of the enzyme (Scheinker et al., 2005). Two splicing variants, Sepia NOSa and NOSb, differing by 18 nucleotides, were found in the

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Fig. 5. a-Tubulin nitration and turnover in S. officinalis nervous system. NRS, nitrogen-reactive species.

nervous system and in the ink gland. RT-PCR (reverse transcriptase polymerase chain reaction) of S. officinalis NOS mRNA showed expression of both forms in the optic lobes and preferential expression of Sepia NOSb in the ink gland, suggesting specific biological functions for these isoforms. Phylogenetic analysis of NOS sequences in various species confirms the conserved nature of S. officinalis NOS, and particularly conservation of the various cofactor-binding domains: heme, CaM, FMN, FAD and NADPH (Fig. 6). The identity of individual NOSs with that of S. officinalis NOSs at the level of the cofactor-binding sites is high. The cuttlefish enzyme, together with the enzyme of another mollusc, Lymnaea, has a shorter N-terminal region than mammalian nNOS. In situ hybridisation experiments with a S. officinalis NOS antisense probe able to recognise both splicing forms of S. officinalis NOS revealed that the protein is expressed in the CNS and ink gland. In the CNS, the analysis has been restricted to those regions that are related to the ink defence system, such as the palliovisceral lobe. From this lobe arises the visceral nerve, from which derives a branch, the ink sac nerve, which innervates the ink sac. S. officinalis NOS mRNA is expressed in the small inner neurons of both the latero-ventral palliovisceral and central palliovisceral lobes of S. officinalis. In the ink gland NOS is expressed both in immature and mature cells. In addition to being involved in defence and neurotransmission, NO plays other roles in cuttlefish. It regulates blood flow and blood pressure, acting as a vasodilatatory mediator as in mammals (Schipp and Gebauer, 1999). NO donors induce vasodilatation of S. officinalis cephalic aorta, although NOS is not present in the endothelium but seems to be located within the nerve tissue supplying the vessel. NO is also involved in the manipulative behaviour of S. officinalis (Halm et al., 2003). Manipulative behaviour requires extensive chemo-tactile

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Fig. 6. Amino acid alignment of Sepia NOSa and Sepia NOSb with other known NOS sequences. Numbers indicate the percentage sequence identity of individual NOSs to Sepia NOSs at the level of the conserved domains. Mus, Mus musculus. NOS from Mus musculus and Homo sapiens refer to neuronal forms.

sensory processing, fine motor control and probably motor learning processes. Cuttlefish actively explored crabs with their arms and lips, which are NADPH-diaphorase positive, and received information about the toughness of the carapace or the progress of paralysis of the crab. Injection of the NOS inhibitor L-NAME into the vena cava of the cuttlefish resulted in an increase in the latency of prey paralysis, suggesting that NO could play an important role in the transmission of chemical and/or tactile information. NO is also involved in modulating the resting activity of crista afferent fibres in the statocyst (the equilibrium receptor organ) of S. officinalis (Tu and Budelmann, 2000).

58 Conclusions and perspectives Following publication of the seminal papers on the gastropod L. stagnalis (Korneev et al., 1998, 2002, 2005), the presence of NOS has also been demonstrated in other gastropods, bivalves and cephalopods. As a result of intense efforts, molluscs stand today among the most useful model systems in which to investigate NO biogenesis, signalling and roles in different functions, often related to specific behaviours, as highlighted in Fig. 1. These studies on molluscs allowed the demonstration of an association between NO and olfaction, feeding, learning, defence, development and movement, suggesting that the NO signalling pathway has great versatility and has evolved to play a variety of roles in widely varying biological settings and cellular environments. The complex roles of NO in S. officinalis ink gland and nervous systems are paradigmatic in this respect. Stimulation of NO production via the NMDA receptor and the cGMP signal transduction pathway has been shown to induce a series of events that, though apparently unrelated, come together to control overall regulation of the ink defence system. Useful insights are coming from investigations of the anatomical distribution and functional significance of NOS in various areas and regions of the CNS of S. officinalis as well as other molluscs, mainly in the context of behaviour and neuronal plasticity. Additional advances that appear to be within our grasp will hopefully unravel the complex mechanisms controlling the maturation and differentiation of ink gland cells. Another fascinating area for investigation concerns the role of post-translational protein modification by NO-derived species in the overall regulatory mechanisms mediated by NO. Relevant in this respect is the demonstration of temporary patterns of a-tubulin nitration in S. officinalis brain (Palumbo et al., 2002). Thus, what we have discussed so far is probably only the tip of an iceberg, and it is likely that in the near future new regulatory pathways and functional roles will be uncovered.

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Plate 1. The cuttlefish S. officinalis. The central nervous system, comprising the

brain and optic lobes, is shown together with the ink-producing system. Note the ink gland and the ink sac. Inset: microscopic structure of the ink gland. (For Black and White version, see page 51).