Study On Bivalve Nitric Oxide System And Its Mechanisms Of Effects Within The Neuroendocrine-immune Regulatory Network

Nitric oxide (NO) system is indispensable for the mediation of vertebrateneuroendocrine-immune regulatory network and the sustaining of homeostasis. Notonly is it involved in the removing of pathogens as part of immune effects, but also itcan mediate the immune responses through the interaction effects within theneuroendocrine-immune regulatory network as an important section of nervoussystem. There have been reports about the irreplaceable position of invertebrate NOsystem for its broad involvements in invertebrate growth, development, learning,memory, behaviors and immunity. However, few researches have been investigatedupon the constitution characters of invertebrate NO system, and its effects within theneuroendocrine-immune regulatory network. In the present study, the constitution andbasic features of NO system, and the reciprocity patterns between the NO andneuroendocrine systems were explored in bivalve for better understanding of theimmunomodulation of NO system under control of the neuroendocrine system.The enzyme NOS was cloned and characterized from the scallop Chlamys farreri(CfNOS). Its cDNA sequence consisted of a complete cDNA sequence of5023bpand an open reading frame (ORF) of4461bp encoding a polypeptide of1486aminoacids. CfNOS contained PDZ, oxygenase and reductase domains, and the latter twowere connected by the binding site for CaM. Based on the bioinformatic analysis ofthe genome sequences of the oyster Crassostrea gigas, only one sequence of CgNOSgene was identified. Based on the cDNA library of oyster haemocytes after0,12and24hr of stimulation by LPS and TNF-α, the cDNA sequence of the N-, C-terminaland medial domains of CgNOS were amplified and only one band in each sample wasobtained. The CfNOS transcripts could be detected in all scallop embryos and larvae except for oocytes and fertilized eggs, and were constitutively expressed in thehaemocytes, muscle, gill, kidney, hepatopancreas, mantle and gonad while theCfNOS protein was expressed both in the cytoplasm and the cell membrane ofhaemocytes. Besides, the domain-specific antibody OxFMN-Ab and PDZ-Ab wereused for the detection of CgNOS expression in the cytoplasm and cell membraneproteins, and2-3protein forms of CgNOS were discovered. Immunoprecipitated byOxFMN-Ab, the native CfNOS protein was qualified to catalyze the synthesis of NO(30.31±0.26U mgprot-1), while the inhibitors of nNOS and iNOS—Spd and SMTinhibited the catalytic activity of CfNOS significantly.The CfNOS mRNA transcripts were significantly augmented when stimulated byPAMPs (LPS、PGN and β-glucan) and TNF-α. Furthermore, co-stimulation of LPSand SMT decreased the LPS-induced NO concentration, NOS activity, phagocytosisability, anti-bacterial activity and the ROS content of scallop haemolymph, increasedthe apoptosis rate, GSH concentration and the SOD activity, and restored theactivities of lysozyme and PO. On the other hand, CgNOS, including thephosphorylated part, would translocate from the cell membrane to the cytoplasmwithin the oyster haemocytes stimulated by LPS and TNF-α. Before that, the restingCgNOS was binded to PSD-95protein, which was anchored to the membrane.The concentration of NO and norepinephrine (NE) were elevated after thestimulation of LPS in the primary scallop haemocytes. And the NE responded morerapidly and lasting. In concomitant stimulation with LPS, NE and the α-AR agonistdecreased the LPS-induced NO content significantly, while the β-AR agonistincreased it significantly. The agonists of α-and β-AR could also decrease the cAMPlevel lower than the concentration with single stimulation of LPS, and the β-ARagonist promoted the LPS-induced Ca2+production. Besides, the concentration of NEwas elevated by SNP, the donor of NO. Meanwhile, the expression of CgNF-κB andCgSTAT mRNA transcripts were increased, while that of CgAP-1decreasedsignificantly after12hr of stimulation by LPS and TNF-α. Cytoplasm CgNF-κB1wasless phosphorylated and part of it translocated to the nuclei of oyster haemocytes after12-24hr of stimulation. There was no significant change upon the expression of CgAP-1proten while it existed in both cytoplasm and the nucleus. The EMSAassays revealed the interactions between native CgNF-κB1, CgSTAT proteins, whileonly purified CgAP-1, and the corresponding binding sites within the promoter ofCgNOS.The above results suggested that the mollusc NO system played as important rolesas it was in the vertebrates, except that its constitutions were specific anddistinguished. The multi-isoform constitution of NOS family did not exist in mollusc,whereas, the mollusc NOS was structurally similar to nNOS, biochemically similar toboth nNOS and iNOS, and immunologically similar to iNOS. Moreover, there wasonly one mRNA transcript of mollusc NOS, while there were several expressionforms of NOS proteins. During the process of immune defense, the catecholamine(CA) mediated the responses of NO system through the pathway ofNE-α/β-AR-cAMP/Ca2+, and through the regulation of NOS transcription via NF-κBand STAT. The resting NOS was anchored to the inner membrane of haemocytes byinteracting with PSD-95, and translocated to the cytoplasm responded against immunestimulations, and then catalyzed the release of NO. The synthesized NO finallyregulated the innate immune responses of molluscs.