Studies On The Cloning And Tissue Expression Of Neuropeptide S And Its Receptor And Effect Of Heat Stress On Their Expression In Rabbit

Neuropeptide S is a20-amino acid peptide identified in2002. NPS selectively binds and activates an orphan G protein-coupled receptor named NPS receptor (NPSR). Reinscheid RK termed this molecule neuropeptide S (NPS) because the amino-terminal residue of NPS in all species is always serine(S). NPS and NPSR mRNA are mainly expressed in central nervous system and lowly in peripheral tissues. NPS/NPSR system has been shown to modulate a variety of physiological functions, including wakefulness, anxiety, food intake, macrophage immune responses and oxidative damage. At present the studies on NPS and its receptor have been focused on rat and mouse, but the cloning and expression of NPS and NPSR in rabbit were not reported. Therefore we studied in detail the cloning and expression of NPS and NPSR in rabbit in order to provide basic information for further studies on NPS physiological functions. In addition, we studied the expression of rabbit NPS and NPSR mRNA in the hypothalamus-pituitary-adrenal axis in response to heat stress. The main results are as follows:1. The first complete coding sequence (CDS) of NPS and partial CDS of NPSR were obtained from rabbit brain using the approach of gene cloning. The length of NPS and NPSR were270bp and847bp respectively. The sequences of the product were confirmed to be that of NPS and NPSR in rabbit and enrolled in GenBank(accession numbers: EU978456and FJ713102). Amino sequece of rabbit NPS precursor had high homology compared with chimpanzee, Dasypus novemcinctus, human and horse (76.7%、75.6%、75.6%,75.6%amino acid homology,respectively). Amino sequece of rabbit NPS precursor had low homology compared with rat, cattle, mouse, dog, opossum,chicken, guinea pig, European shrew, macaque and galago (between52.2%and71.1%amino acid homology). NPS is essentially conservative with some differences between the different species in the composition of the mature peptide portion of the first half. Composition to14species showed NPS precursor relatively conservative during evolution, but evolution in different species exist local variations between branches of interspecies. 2The expression of NPS and NPSR mRNA in rabbit were studied by RT-PCR. The results revealed that the expression of NPS and NPSRA mRNA was extensive mainly in the central nervous system, and low expression in peripheral tissues and organs, including the pons, hypothalamus, pituitary, brain stem, spinal cord, stomach, gall bladder,duodenum, jejunum, ileum, colon, cecum, heart, liver, spleen, lung, kidney, testicle, uterus,ovary, aorta, pancreas, salivary gland, thyroid gland, thymus and mammary gland. The results in this study were similar to that in rat, which indicated the conservative of NPS/NPSR in phylogeny.3The influence of heat stress on expression of NPS/NPSR mRNA in rabbit hypothalamus-pituitary-adrenal axis was studied by relative quantity RT-PCR(42℃, control,0.5h,1h,2h,n=6). The results were as follows:(1) Expression of NPS and NPSR mRNA was found in the hypothalamus, pituitary and adrenal gland of rabbit at all phases of the heat stress, but showed different expression patterns.(2)After the heat stress NPS mRNA expression level of the hypothalamus increased gradually, reached the highest at1h compared with the control showing significant difference (P<0.01), and followed by decline at2h. After heat stress NPS mRNA expressed the highest level at0.5h in the adrenal gland compared with the control showing significant difference (P<0.01), and followed by a downward trend afterwards. After the heat stress expression of NPS mRNA in the pituitary had different patterns from that in the hypothalamus and adrenal gland (P<0.01).(3)NPSR mRNA expression patterns in the hypothalamus-pituitary-adrenal was similar to that of NPS during heat stress.The results in this study showed that NPS and its receptor gene in rabbit was conservative and that rabbit was suffered from heat stress. The NPS and its receptor gene expression in the HPA axis changed, suggesting that in the heat stress NPS/NPSR system may play a physiological role. These results provided the experimental data for studying physiological functions of NPS in rabbit.