|Characterization of Nitric Oxide Synthase expressed in the nervous
system of the neogasteropod Stramonita haemastoma
Mattia Toni1, Maria Carmela Bonaccorsi di Patti2, Giorgio Venturini3, Maria Vittoria Modica1, Marco Oliverio1 e Carla Cioni1
1 Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Università La Sapienza, Roma
2 Dipartimento di Scienze biochimiche, Università La Sapienza, Roma
3 Dipartimento di Biologia, Università “Roma Tre”, Roma
Present knowledge on NOS enzymes of gastropod molluscs are almost exclusively limited to opistobranchia and pulmonata (Moroz & Kohn, 2007). For these groups, biochemical evidence and cellular distribution data, as well as complete nNOS sequences from the CNS of Lymnaea stagnalis (Korneev et al., 1998, 2005), Aplysia californica (Sandreyev et al., 2008) and Lehmannia valentiana (Matsuo et al., 2008) were obtained. Data are much more limited in prosobranchia, which include most marine gastropods with over 20,000 species and extreme diversification of their trophic ecologies. In order to gain further information on the molecular evolution of NOS enzymes in the radiation of molluscs, we have characterized a new constitutive NOS isoform expressed in the central and peripheral nervous system of Stramonita haemastoma, a prosobranch neogastropod (Muricidae) which is a common species of Mediterranean coasts.
A biochemical analysis was performed by the arginine to citrulline conversion assay on homogenates of the nerve ring, the tentacle and the osphradium, that showed the presence of a calcium/calmodulin-dependent NOS activity in both soluble and particulate fractions.
The complete nucleotide sequence for Stramonita NOS (Sh-NOS) was cloned by RT-PCR and 5’ and 3’ RACE performed on RNA extracted from nerve ring. Sh-NOS is a 1517 aa protein provided with the N-terminus PDZ domain and regulatory domains similar to those found in mammalian nNOS. Typical regulatory domains allowed to identify Sh-NOS as a constitutive neuronal isoform.
The comparison of Sh-NOS with molluscan NOS sequences available in databases is consistent with the phylogenetic affinities and reveals similarities ranging from 93.5% with Ily-NOS1 (Ilyanassa obsoleta, neogastropod, partial sequence) (Hens et al., 2006), to 55.5% with Lym-NOS2 (basal pulmonata, Korneev et al., 2005). The cellular distribution of Sh-NOS was analyzed by the NADPH diaphorase (NADPHd) histochemistry and these results highlight the predominant neuronal localization of diaphorase activity both in the CNS and in peripheral structures. Most positive neurons are localized in the pedal ganglia (more than 180 per ganglion) while the number of labeled neurons is much lower in the other ganglia (1-6 per ganglion). Beyond the cell soma and axons, pedal neurons were also labeled in correspondence of small varicosities resembling basket-like synapses described in other species. These results suggest both a presynaptic and postsynaptic role for NO in pedal neurons. In peripheral structures, the NADPHd activity was intense in the osphradium, the tentacle and the foot. The basal expression of Sh-NOS mRNA was also analyzed in the main body tissues by RT-PCR. The results showed that mRNA transcripts are expressed in the CNS and peripheral structures involved in a sensory role. However, the highest expression levels were found in the nerve ring, followed by the sensory structures. Considering that NOS enzyme activity is much more intense in peripheral structures than in CNS, this finding suggests that NOS gene expression may be regulated in the CNS at the post-transcriptional level.
The distribution of NOS expression in Stramonita suggests that NO is deeply involved in the processing of sensory information from the environment and in the modulation of central neural circuits responsible for feeding and locomotion. IF/IHC studies are in progress to confirm the specificity of NADPHd distribution by means of polyclonal antibodies against mammalian nNOS. Preliminary results showed a large overlap of the two labelings in the nerve ring, osphradium, tentacle and foot.
These data have provides a solid background for further studies aimed at understanding the specific functions of NO in neogasteropods and its possible role as a biomarker of environmental stress in molluscs. Further studies are in progress to evaluate whether the constitutive expression of Sh-NOS gene may be influenced by physical and ecological parameters (i.e., water temperature, starvation, abundance of prey).
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