| Neurotransmitters, as the vital messenger in neuro-endocrine-immune network, not only have function on signal transmission in nerve cells, but also play immunomodulatory role in immune cells. In recent decades, along with the co-evolution of nervous and immune system speculated, more attention has been paid on the immunomodulatory function of neurotransmitters in invertebrates. At present, the research on the immunomodulatory role of monoamines, neuropeptides and cholines has been reported, however, little has been mentioned as for the fucntion of amino acid neurotransmitters in immune system of invertebrates. In the present study, the dynamic changes of amino acid neurotransmitters, glutamate (Glu) and y-aminobutyric acid (GABA), were investigated after immune stimulation, the distribution and expression patterns of glutamic acid decarboxylase (GAD) were identified, and the immunomodulatory role of Glu and GABA was further explored in Pacific oyster Crassostrea gigas for better understanding of the immunomodulation of amino acid neurotransmitters in invertebrates.After LPS stimulation, the concentration of Glu in serum increased at 6 h and then decreased at 48 h, conversely, the concentration of GABA decreased at 6 h and increased at 48 h, leaving an antagonistic effect of of Glu and GABA. According to the genome information of oyster C. gigas, one rate-limiting enzyme GAD (designed as CgGAD) catalyzing the reaction from Glu to GABA was identified. The full length cDNA of CgGAD was of 1689 bp, encoding a polypeptide of 562 ami- o acids containing a conserved pyridoxal-dependent decarboxylase domain. After transfected into HEK293 cells, CgGAD could promote the production of GABA. In oyster C. gigas, CgGAD could be detected in ganglion and hemocytes at both mRNA and protein level. Unexpectedly, CgGAD was more abundant in hemocytes than that in ganglion, and was mostly located in the granulocytes without phagocytic capacity. Moreover, CgGAD could also dynamically respond to LPS stimulation, its expression level decreased at first and increased at later stage, which was conformed to the change of GABA after LPS stimulation.LPS/GABA stimulation could influence both the humoral and cellular immune response of oysters. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the NOS activity increased significantly, but these increased trends were remarkably inhibited by GABA stimulation. Similarly, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. Furthermore, compared with LPS+PBS group, LPS+GABA stimulation could also inhibit the increase of Ca2+ concentration and ROS production in the primary cultured hemocytes. However, the inhibitory role of GABA became invalid when the antagonist of GABA A or B receptors was added. Compared with GABA, Glu could increase the Ca2+ concentration and the ROS production, and then induce apoptosis of primary cultured hemocytes, however, these effects could be inhibited by three different type receptors (NMDA, AMPA and metabotropic) antagonists of Glu.All results collectively suggested that, CgGAD, as GABA synthase, could modulate the dynamic balance between Glu and GABA during the immune response of oysters, and participate in the regulation of neuro-endocrine-immune network. Besides, as the molecular marker of GABAergic system, CgGAD was mainly distributed in the immune tissues of oyster, which also provided a novel insight to the co-evolution between nervous system and immune system. In addition, the antagonistic fluctuation of Glu and GABA in hemolymph during immune response w- as witnessed, together with their excitory or inhibitory effect on immune response, which benefits for oysters to maintain the immune homeostasis. |
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