Distribution, cloning and sequencing of GnRH, its receptor, and effects of gastric acid secretion of GnRH analogue in gastric parietal cells of rats.
Our objective was to study the distribution of gonadotropin-releasing hormone (GnRH) and its receptor, cloning and sequencing of GnRH and its receptor gene in cultured gastric parietal cells of rats. The distribution of GnRH and its receptor mRNA were investigated through immunocytochemical ABC methods and in situ hybridization methods in cultured gastric parietal cells of rats. After isolation of the total RNA from the parietal cells, RT-PCR was conducted to obtain GnRH and its receptor cDNA. Then, the products of PCR was purified, digested by the restriction enzyme of Hind III and EcoR I, and DNA fragments of interests were cloned into pUC19 vector. The products of PCR were analyzed by sequencing with Sanger's method after identified by PCR and digestion of restriction enzyme. Gastric parietal cells showed GnRH and its receptor immunoreactivity; positive material was located in cytoplasm other than in nuclei. GnRH and its receptor mRNA hybridized signals were also detected in cytoplasm with negative nuclei. The specific amplified band of GnRH and its receptor sequences were detected through Agarose gel electrophoresis, and GnRH gene sequence is identical to that of GnRH which has been reported in rat hypothalamus and GnRH receptor sequence is identical to that of the pituitary of rat. GnRH analogue (Alarelin) could inhibit the gastric acid secretion both by direct actions on parietal cells and by inhibiting vagous function. Our data suggest that GnRH could be produced by gastric parietal cells of rats and may modulate physiological function of gastric parietal cells of rats through autocrinal and paracrinal way.
Chen
Lei
L
Department of Histology and Embryology, The Fourth Military Medical University, Xi' an, 710032, People's Republic of China. chenleis@mail.tsinghua.edu.cn
Sun
Xu-De
XD
Zhao
Jing
J
Yang
An-Gang
AG
Huang
Wei-Quan
WQ
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005744:Gastric Acid / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007275:Injections, Intravenous; D008297:Male; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011966:Receptors, LHRH / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020539:Sequence Analysis, Protein
D012333:RNA, Messenger; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone