| Dopaminergic neuroendocrine system and serotonergic neuroendocrine system are important parts of neuroendocrine immune system,playing a vital role in maintaining homeostasis and enhancing biological environment adaptability.In vertebrates,biogenic amines neurotransmitter are widely distributed among the central nervous system and peripheral tissue,acting as the messenger between between neurons,muscle cells and participating in various vital movements.By comparison,neuroendocrine immune system of marine invertebrates is relatively simple,but can also be actived rapidly under stress,regulating various physiological activities to maintain homeostasis.Thus,how the marine invertebrates implement environmental adaptation through the relatively simple neural endocrine-immune system is of great value to study on.In the present study,two homologue of dopamine(DA)receptor and two homologue of serotonin(5-HT)receptor were identified from Chinese mitten crab(Eriocheir sinensis)(designated as EsDAR1,EsDAR2,Es5-HTR1,and Es5-HTR7).The open reading frame(ORF)of EsDAR1 was of 1371 bp,encoding a polypeptide of 456 amino acids with a seven transmembrane region.The open reading frame(ORF)of Es DAR2 was of 1770 bp,encoding a polypeptide of 589 amino acids Es5-HTR1 was of 1788 bp,encoding a polypeptide of 595 amino acids Es5-HTR1 was of 1773 bp,encoding a polypeptide of 590 amino acids.All the recptors contains a seven transmembrane region.The cAMP contents in HEK 293 T cells increased significantly after EsDAR1 transfection and dopamine incubation,and also after Es5-HTR7 transfection and 5-HT incubation(p <0.05).While the cAMP contents in HEK 293 T cells decreased significantly after EsDAR2 transfection and dopamine incubation,and also after Es5-HTR1 transfection and 5-HT incubation(p <0.05).All the receptors appeared in the zygote,and the expression of them decreased and then increased during the juvenile development.The expression in juvenile crab is nearly the same as that in zygote.The dopaminergic system and 5-hydroxytryptamine system were all involved in the osmotic regulation process in the process of desalination.When the salinity was gradually reduced from 20‰ to 3‰,the dopamine concentration in the larva of Chinese mitten crab was increased from 53.25 ng/m L to 937.58 ng/m L.The expression EsDAR1 was up-regulated and the expression EsDAR2 was down-regulated.While,the concentration of 5-hydroxytryptamine decreased with the decrease of salinity(p < 0.05),from 623.82 ng/m L to 57.29 ng/mL.The expression of Es5-HTR1 was significantly decreased,and the expression of Es5-HTR7 was significantly increased(p > 0.05).In the process of desalination,the expression of NKA,the key enzyme regulating ion transport,also increased significantly(p < 0.05).However,after inhibiting dopamine receptors and serotonin receptors,NKA showed no significant change in the expression of salinity reduction,and the freshwater selectivity of Chinese mitten crab was also disappeared.Therefore,the dopamine receptors and serotonin receptors mediated osmotic regulation process by the regulating the activity and expression of NKA.Dopamine and serotonin bind to their receptors,affected the concentration of the secondary messenger cAMP by affecting the activity of adenylate cyclase.cAMP can activate NKA,improve its activity,and finally realize the regulation of ion transport rate.Under the condition of salinity stress for a long time,the expression of NKA will increase under the stimulation of cAMP,so as to achieve salinity adjustment.The inhibition of Cg5-HTR1 significantly reduced the expression of tumor necrosis factor(TNF)mRNA in hemocyte,down-regulated the superoxide dismutase(SOD)activity in serum,and induced the apoptosis of hemocyte(p < 0.05).These results suggested that Cg5-HTR1 was a novel member of 5-HT1 receptor family and it mediated serotonergic immunomodulation on both cellular and humoral immune responses. |
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