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Sepia succus

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Biosynthesis, signalling pathways and physiopathological roles of nitric oxide and reactive nitrogen species
Principal investigator: Anna Palumbo
Collaborators: Marco d'Ischia, Anna Di Cosmo
Post doctoral fellow: Paola Di Donato
Graduate Student: Gabriella Fiore
Undergraduate Student: Stefania Comes

Research activity
Over the past decade, nitric oxide (NO) has emerged as one of the most pervasive physiological modulators yet described. Critical roles of NO in man and non human mammals include neurotransmission in excitatory glutamate-NMDA dependent pathways via the downstream target cGMP, regulation of vasal tone, inhibition of platelet aggregation and non specific immune response. Comparatively less is known about the origin and physiological roles of NO in invertebrates although increasing lines of evidence implicate this chemical messenger in the activation of chemosensory processing including olfactory and feeding behaviour and in visual and tactile learning. In this general frame, the activity of the group is mainly being centred on the biosynthesis, signalling pathways and functional roles of NO in the cuttlefish Sepia officinalis. Particular attention is focused to the peculiar inking system that the animal utilises to confuse predators and alert conspecifics to danger while retreating. This system hinges on the metabolic activity of the ink gland, situated in the bottom of the ink sac and deputed to the production of the black melanin pigment. Once synthesised, this pigment is secreted into the lumen of the gland and accumulated into the ink sac, the typical effector organ deputed to the storage and ejection of the ink. The ink gland is a highly peculiar organ characterised by the presence of immature cells which gradually maturate and migrate toward the external portion where they diffentiate acquiring the ability to produce melanin. New insights into the basic regulatory mechanisms of the inking system have derived from a series of collaborative studies, showing that the immature ink gland cells contain the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), as well as glutamate NMDA R1 receptor subunits. Moreover, stimulation of NMDA receptors in intact ink glands has been found to cause a marked elevation of the activity of tyrosinase, the key enzyme involved in melanogenesis, and enhanced melanin synthesis through activation of the NO/cGMP pathway. Glutamate, NMDA receptors and a constitutive NOS have also been detected by biochemical and immunohistochemical techniques in different regions of Sepia nervous system and in certain neural pathways putatively relating to the ink defense mechanism. All these results concur to suggest that NO may affect and modulate at various levels the ink defense system of Sepia. In this setting, current research activity focuses on

          1) A detailed characterisation of the NO signalling pathway in Sepia and its role(s) in tyrosine metabolism in the ink gland. In this context, the effects of the NO/cGMP pathway on catecholamine biosynthesis and excretion will be a primary focus of research, in view of the intriguing role of catecholamines in the ink defence mechanisms. Metabolic activity of the gland and its modulation by NO is of particular relevance in relation to the elucidation of the nature of active principle(s) in the ink and its (their) mechanism of action.

          2) The role of NO in the differentiation and maturation of ink gland cells. For its many peculiarities, the ink gland is a unique, most convenient model to dissect the various mechanisms of cell proliferation and differentiation, and to address the role played by NO in these processes.

          3) The occurrence and significance of protein nitration in Sepia tissues. Generally viewed as a deleterious epiphenomenon of oxidative and nitrosative stress, protein nitration has been implicated in the pathology of diverse human diseases and relatively little attention has been devoted to the possible physiological significance of protein nitration under basal conditions. This specific aspect and the underlying implications are currently being addressed by an integrated chemical, biochemical and immunohistochemical approach.

Another line of research concerns the investigation of the mechanisms of NO-mediated cell damage and innovative strategies for neuroprotection. In addition to serving multiple physiological roles, NO has been increasingly implicated in a variety of pathological conditions. Putative mechanisms of neuronal damage involve interaction of NO with reactive oxygen species to afford powerful nitrating agents which can target important cellular constituents including, besides proteins, catecholamine neurotransmitters and polyunsaturated fatty acids leading to potentially neurotoxic derivatives. The modulation of NO synthesis through selective inhibition of the various NOS isoforms is receiving a good deal of attention as a promising therapeutic strategy. Salient results so far obtained in this area include the identification of a series of novel NOS inhibitors, including 6-nitrocatecholamines, putative reaction products of NO with catecholamines under oxidative stress conditions, and the antithyroid drugs, thiouracil, propylthiouracil and methylthiouracil.

Relevant publications:
Palumbo, A., A. Di Cosmo, I. Gesualdo, M. d'Ischia (1997) A calcium-dependent nitric oxide synthase and NMDA R1 glutamate receptor in the ink gland of Sepia officinalis: a hint to a regulatory role of nitric oxide in melanogenesis?. Biochem. Biophys. Res. Commun. 235, 429-432.
Palumbo, A., A. Di Cosmo, A. Poli, C. Di Cristo, M. d'Ischia (1999) A calcium/calmodulin-dependent nitric oxide synthase, NMDAR2/3 receptor subunits and glutamate in the CNS of the cuttlefish, Sepia officinalis:: localization in specific neural patterns controlling the inking system. J. Neurochem., 73, 1254-1263.
Palumbo, A., A. Napolitano, P. Barone, M. d'Ischia (1999) Nitrite-and peroxide-dependent oxidation pathways of dopamine: 6-nitrodopamine and 6-hydroxydopamine formation as potential contributory mechanisms of oxidative stress- and nitric oxide-induced neurotoxicity in neuronal degeneration. Chem Res. Toxicol. 12, 1213-1222.
Palumbo, A., A. Poli , A. Di Cosmo, M. d'Ischia (2000) N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric oxide/cGMP signal transduction pathway. J. Biol. Chem. 275, 16885-16890.
Di Cosmo, A., C. Di Cristo, A. Palumbo, M. d'Ischia, J.B. Messenger (2000) Nitric oxide synthase (NOS) in the brain of the cephalopod Sepia officinalis. J. Comp. Neurol. 428, 411-427.
Palumbo, A., M. d'Ischia, F. Cioffi (2000) 2-thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerisation. FEBS Letters 485, 109-112.
Napolitano, A., A. Palumbo, M. d'Ischia (2000) Oxidation of the neurotoxin 6-nitrodopamine and related 4-nitrocatechols under biomimetic conditions. Tetrahedron 56, 5941-5945.
Palumbo, A., G. Astarita, M. d'Ischia (2001) Inhibition of neuronal nitric oxide synthase by 6-nitrocatecholamines, putative reaction products of nitric oxide with catecholamines under oxidative stress conditions. Biochem. J. 356, 105-110.
Palumbo, A., M. d'Ischia (2001) Thiouracil antithyroid drugs as a new class of neuronal nitric oxide synthase inhibitors. Biochem. Biophys. Res. Commun. 282, 793-797.
Palumbo, A., A. Napolitano, A. Carraturo, G.L. Russo, M. d'Ischia (2001) Oxidative conversion of 6-nitrocatecholamines to nitrosating products: a possibile contributory factor in nitric oxide and catecholamine neurotoxicity associated with oxidative stress and acidosis. Chem Res. Toxicol. 14, 1296-1305.
Palumbo, A., G. Astarita, M. Picardo, M. d'Ischia (2001) Ni2+, a double-acting inhibitor of neuronal nitric oxide synthase interfering with L-arginine binding and Ca2+/calmodulin-dependent enzyme activation. Biochem. Biophys. Res. Commun. 285, 142-146.
Palumbo, A., A. Napolitano, M. d'Ischia (2002) Nitrocatechols versus nitrocatecholamines as novel competitive inhibitors of neuronal nitric oxide synthase: lack of the aminoethyl side chain determines loss of tetrahydrobiopterin-antagonizing properties. Bioorg. Med. Chem. Lett. 12, 13-16.
Palumbo, A., G. Fiore, C. Di Cristo, A. Di Cosmo, M. d'Ischia (2002) NMDA receptor stimulation induces temporary a-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis. Biochem. Biophys. Res. Commun. 293, 1536-1543.
Biochemical aspects of melanogenesis
(Principal investigator Anna Palumbo)
The activity of Anna Palumbo and co-workers is centred mainly on the oxidative metabolism of tyrosine and related metabolites in melanin producing cells with special emphasis on the ink gland of the cuttlefish Sepia officinalis, which provides a most convenient model for studies of melanogenesis. The basic aim of this research project is to elucidate analogies and differences between the pigmentary systems in Sepia and mammals and to study the evolution of the process along the phylogenetic scale. Recent biochemical studies, carried out in this laboratory, showed that melanogenesis in Sepia is a very complex process which involves a variety of enzymes intervening at various sites of the pigment pathway. In particular, the gland contains, in addition to tyrosinase, a new enzyme, called dopachrome rearranging enzyme and a peroxidase. In the course of a research project, carried out in collaboration with M. Branno and F. Aniello (Stazione Zoologica), aimed at characterizing the Sepia melanogenic enzymes, a cDNA clone encoding a protein highly homologous to other peroxidases was obtained by using a combination of cDNA library screening and 5' RACE procedure. Northern blot analysis of mRNAs from various tissues of Sepia revealed that peroxidase is specifically expressed in the ink gland. This result is of interest since it represents the first characterization of a melanogenic peroxidase.
In collaboration with V. J. Hearing (NIH, Bethesda) and A. Di Cosmo (Università "Federico II" Napoli), the question of whether and to what extent the melanogenic enzymes co-localize and are functionally interactive in the melanogenic compartments of the ink gland cells has been addressed. Using different techniques, evidence was obtained that peroxidase has a different subcellular localization pattern from tyrosinase and dopachrome-rearranging enzyme.
While peroxidase is located in the rough endoplasmic reticulum (RER) and in the matrix of premelanosomes and melanosomes, tyrosinase and dopachrome-rearranging enzyme are present in the RER-Golgi transport system, at the level of trans-Golgi cisternae (TGC), trans-Golgi network (TGN) and coated vesicles, and in melanosomes on pigmented granules. These results lead to a model of melanin formation in the ink gland highlighting the dynamic interactions of the melanogenic enzymes at molecular and ultrastructural levels. Sepia melanosomes derive from two separate cellular compartments: the RER which gives rise to premelanosomes containing active peroxidase and the TGN which originates the coated vesicles containing tyrosinase and dopachrome rearranging enzyme. Coated vesicles then fuse with the membrane of premelanosomes and are subsequently incorporated into it according to a mechanism which involves inversion and reformation of the coated vesicles inside the premelanosomes. After this process, the active sites of tyrosinase and dopachrome rearranging enzyme, originally oriented into the lumen of the coated vesicle, will be exposed to the melanosomal matrix. On this basis, tyrosine, present within the melanosomal matrix, is converted by the enzymes present on the external side of the coated vesicles membrane to dopachrome and then to the indole intermediates which are subsequently oxidized and polymerized by peroxidase within the melanosomal matrix. Following the action of peroxidase, melanin synthesis begins at around the periphery of the incorporated coated vesicles and subsequently proceeds to completely cover their surfaces resulting in the formation of particulate melanosomes.
A comparison between melanin formation in the ink gland of Sepia officinalis and mammals. N, nucleus; RER, rough endoplasmic reticulum; TGN, trans Golgi network; PM, premelanosome; ML, melanosome; L, lumen; GC, Golgi complex.
When the maturation of the epithelial cell is complete, the membranes of the melanosomes fuse with those of the apical pole of the cell and all the cellular constituents, including the melanin granules, are excreted into the lumen of the ink gland. From this scheme of melanin formation in Sepia it appears that Sepia melanosomes possess a complex organization which could represent an intermediate step in the evolution of the melanin producing system along the phylogenetic scale. The major difference between Sepia and mammalian system lies in the structure of the melanosomes, whereas Sepia possesses particulate melanosomes, typical of primitive vertebrates, in mammals melanosomes are fibrillar and are characterized by a lamellar matrix completely filled by melanin. In the course of evolution there was the transition from particulate to fibrillar melanosomes. The peculiar organization of melanin in Sepia provides a unique opportunity to investigate at subcellular level the mechanism of interaction of the melanogenic enzymes