Studies Of GnRH And Its Receptor In Rat Pancreas

Gonadotropin-releasing hormone (GnRH) is a deca-peptide synthesized by hypothalamus and released from nerve terminals in a pulsatile manner into the hypophysial-portal circulation, stimulating gonadotropin target cells in anterior pituitary. The gonadotropin response to GnRH stimulation is manifested by releasing the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH) that play a central role in control of production. Recently, GnRH and its receptor have been found exist in many extra-hypothalamus-pituitary tissues, such as gonad (ovary, oviducU prostate,testis,placenta > et al. ) and immune system ( T and B lymphocytes ). GnRH and its receptor are not only expressed in normal tissues, but also widely exist in various carcinoma that derived from reproductive ( ovary cancer , prostate cancer ) or non-reproductive tissues (breast cancer lung cancers pancreas cancer and hepatocellular carcinoma, et al.). These indicate that they may have certain function in these neoplastic tissues.Our previous studies found that GnRH and its receptor co-existed in digestive system (such as gastric-intesinal), andmeanwhile, we found their mRNA also present in the same organs mentioned above. We have demonstrated that cDNA sequence of GnRHR in submaxillary was identical to the relative cDNA sequence of GnRHR in pituitary. The suppressive effect of GnRH analogue on the secretion of never growth factor (NGF) in submaxillary showed that these organs are the targets of GnRH. Till now, there still have been no report concerning the expression and function of GnRH and its receptor in pancreas. Based on our previous studies, we conducted the following experiments to further clarify the following uncertainties:Using immunohistochemistry and in situ hybridization, we detected the expression of GnRH and its receptor in rat pancreas. Meanwhile, we carried out RT-PCR and gene clone, further investigating GnRH and its receptor transcription units and proving their gene sequencing.In order to study the regulatory role of GnRH in rat pancreas, we established the functional experiments in vitro and in vivo. Further more, we used ELISA and radioimmunoassay to investigate the changes of trypsin, glucagon and insulin in cultural medium, serum and duodenum secretion.Annexin-V apoptosis kit and flow cytometric analysis were performed to detect the effect of GnRH on pancreatic cell apoptosis.At last, ELISA, radioimmunoassay , laser confocal scanning microscope and immunohistochemistry were used to study the mechanism of GnRH analogue impact on intracellular PKC activity,cAMP concerntratiom MAPK level , iNOS concerntration and [Ca2+]j mobilization in rat pancreatic cells in order to reveal the mechanism of GnRHR signal transaction pathways.The main results were as follows:Using immunohistochemistry, we detected that GnRH is distribute in the exocrine portion of rat pancreas, while its receptor located in the endocrine portion with the same sites as A cells. We used in situ hybridization to detect GnRHR mRNA and found that it was located in both exocrine and endocrine portion of rat pancreas.After having detected both GnRH and its receptor molecule and their mRNA in rat pancreas, we carried out RT-PCR, further proving that GnRH and its receptor transcription units do exist in the pancreas and possess the same sequence as the hypothalamus GnRH mRNA and pituitary GnRHR mRNA, respectivly.By the experiment of pancreatic tissue culture in vitro, we administered different concentration of GnRH analogue into the medium and incubated for Ih. It was found that both trypsin and glucagon concentration in the medium increased in a dose-dependent manner, while insulin decreased in a dose-dependent manner.By the animal experiment in vivo, we administered GnRH analogue through the caudal vein and duodenum, recpsctively. It was found that administering through caudal vein resulted in the same results as them in vitro, while administering through duodenum resulted that try...