Rabbit Neuromedin U And Its Receptors:cDNA Cloning,Expression And Effects Of Heat Stress On THEM

Neuromedin U(NMU)is a structurally highly conserved neuropeptide,initially isolated from porcine spinal cord and there are two peptides of NMU(NMU-25 and NMU-8)with similar biological activities.NMU has been found in both peripheral organs such as gastrointestinal(GI)tract and the central nervous system(CNS).NMU interacts with two G protein-coupled receptors,NMU-R1 and NMU-R2.NMU-R1 is expressed mainly in various peripheral tissues while NMU-R2 is expressed in the central nervous system.NMU has now been found to be expressed in several different tissues,which suggests that NMUis involved in numerous functions,such as appetite regulation and energy balance control,muscle contraction,stress and tumor progression.However,the present studies of NMU mainly focused on the human,rat,mouse and pigs and so on,studies of NMU and its receptor of the rabbit have not been reported in the literature.Therefore,we studied the cDNA cloning and distribution of NMU and its receptor,and investigated the effect of heat stress on NMU,NMU-R1 and NMU-R2 mRNA expression in the rabbit hypothalamus,pituitary gland,adrenal gland and testis by RT-PCR method.The results are as follows:(1)cDNA cloning of rabbit NMU,NUM-R1 and NMU-R2 genes.In this study,wecloned rabbit NMU,NUM-R1 and NMU-R2 cDNA fragments using RT-PCR method.The clonedof cDNA fragments size of NMU,NMU-R1 and NMU-R2 was 443,366 and 203 bp,respectively,and the cDNA sequences were submitted to the GenBank database(GenBank ID:kp276160.1,KM-236787.1 and KM-236788.1)respectively.(2)Bioinformatics analysis of rabbit NMU,NUM-R1 and NMU-R2 cDNA.The homology of the cloned nucleotide sequence of rabbit NMU,NUM-R1 andNMU-R2 genes was((83%,80%,78%,83%,80%,78%),(87%,84%,81%,80%,86%,80%)and(85%,89%,80%,83%,89%,82%))respectively identical to those of the correspondinghuman,pig,mouse,cattle,sheep and rat.The homology of the predicted amino acids ofrabbit NMU,NUM-R1 and NMU-R2 was((84%,75%,73%,73%,74%,72%),(84%,78%,75%84%,83%,75%)and(87%,90%,75%,87%,88%,76%))respectively identical to those of the corresponding human,pig,mouse,cattle,sheep and rat.The rabbit NMU-R1 and NMU-R2 amino acid sequence were found to have a typical transmembrane structure of G protein coupled receptors.NCBI database map views program analysis showed that the NMU,NMU-R1 and NMU-R2 gene was located on chromosome 2,3 and 1 respectively.(3)Expression of NMU,NUM-R1 and NMU-R2 genes in30 rabbit tissues.The results of RT-PCR analysis showed that the expression of NMU,NMU-R1 and NMU-R2 mRNAs gene was found in the central nervous system and the peripheral tissues,and NMU mRNAs were more abundant in the peripheral tissues and a high level of NMU mRAN gene expression was found in the ileum,testis,liver,rectum and hypothalamus.Moderate NMU mRNA expression was found in the duodenum,hippocampus,colon,parotid gland,adrenal gland,while other tissues showed a low level of NMU mRNA expression.While a high level of the NMU-R1 mRNA was mostly detected in the gastrointestinal tract,such as liver,colon,Ileum,cecum and sacculus rotundus.Moderate NMU-R1 mRNA expression was found in the pons,hypothalamus,and spinal cord.While a low level of NMU-R1 mRNA expression was found in the cerebellum.A high level of NMU-R2 mRAN expression was detected in the hypothalamus,spinal cord,cerebellum,hippocampus,spleen,kidney,colon,ileum,mandibular gland,parotid gland and adrenal gland.Moderate NMU-R2 mRNA expression was found in the in jejunum,sacculus rotundus,stomach and uterus.While a low level of NMU-R2 mRNA expression was found in other tissues.(4)Effect of heat stress on NMU,NMU-R1 and NMU-R2 mRNA expressionThe effect of heat stress on NMU,NMU-R1 and NMU-R2 mRNA expression in the hypothalamus,pituitary gland,adrenal gland and testis in rabbit was studied using relative real-time PCR.The results showed that NMU,NMU-R1 and NMU-R2 were expressed in different patterns during the heat stress.The expression of NMU,NMU-R1 and NMU-R2 mRNA in rabbit hypothalamus was up regulated earlier than that in the pituitary gland,adrenal gland and testis in order to respond to heat stress and then regulate function of the HPA axis.The study provides the experimental data for further research in the physiological function of NMU and its mechanisms of action in the rabbit.