Cellular, biochemical, and molecular characterization of nitric oxide synthase expressed in the nervous system of the prosobranch Stramonita haemastoma (Gastropoda, Neogastropoda)

Abstract

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca(2+) /CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5' and 3' RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods.

Transcriptional activation and translocation of ancient NOS during immune response

“…NOS is the most crucial component of the NO system as a result of its exclusive role in the de novo synthesis of NO, and this enzyme synthesizes large amounts of NO when participating in or modulating the immune defenses of both vertebrates and invertebrates (10)(11)(12)41). Thus far, several NOS genes have been cloned and characterized in invertebrates (38,(49)(50)(51)(52), whereas their diversity and phylogeny remain nearly unexplored. In the present study, the molecular characterization of oyster NOS and its activation in the immune response were investigated to develop a better understanding of the phylogenetic evolution of NOS and its immunomodulation in molluscs.…”

Differential Responses of the Nitric Oxide Synthase (NOS) Gene to Immunological Challenges in Noble Scallop Chlamys nobilis

“…In mollusks, NOSs have been identified in the red‐mouthed rock shell (Cioni et al. ), great pond snail (Korneev et al. ), California sea hare (Sadreyev and colleagues, abstract), greenhouse slug (Matsuo and Ito ), common cuttlefish (Scheinker et al.…”

“…In mollusks, NOSs have been identified in gastropods, including the red‐mouthed rock shell Stramonita haemastoma (Cioni et al. ), great pond snail Lymnaea stagnalis (Korneev et al. ), California sea hare Aplysia californica (R. I. Sadreyev, Y. Panchin, P. Uvarov, A. Belyavski, and M. Matz, abstract presented at the Society for Neuroscience, 2000), and greenhouse slug Limax valentianus (Matsuo and Ito ); in a cephalopod, the common cuttlefish Sepia officinalis (Scheinker et al.…”

A Scallop Nitric Oxide Synthase (NOS) with Structure Similar to Neuronal NOS and Its Involvement in the Immune Defense

“…The highest expression level of CfNOS mRNA was observed in the gonad, indicating that NO might be able to regulate scallop gametogenesis and/or steroidogenesis [16,18]. Relative higher level of CfNOS transcripts was expressed in the mantle, which suggested the possible role of NO system in sensory process [21]. In addition, as hepatopancreas and haemocytes are most important immune tissues in mollusc, the CfNOS transcripts in these tissues indicated that CfNOS might be involved in scallop immune responses [19,20].…”

“…For example, there was only one NOS gene in Drosophila genome [14], which could be transcribed into multiple alternative RNA splicing variants [15], and a single copy of NOS ortholog was characterized from crustaceans [16–20]. In mollusc, NOSs were identified in the gastropods Stramonita haemastoma , Lymnaea stagnalis , Aplysia californica and Limax valentianus , the cephalopod Sepia officinalis , and the bivalve Crassostrea gigas and Crassostrea virginica [21–26], and all the identified NOSs belonged to one isoform which had higher similarities with nNOS than eNOS or iNOS from vertebrates. Though the composition of NOS family and the differentiation of NOS members in invertebrates were different from those in vertebrates, invertebrate NOSs had resembled broad involvements in various physiological activities.…”