Expression And Significance Of Neuropeptide Y And Its Receptor In Pituitary Adenoma

Neuropeptide Y (NPY) is a widely distributed peptide in the brain of several species where the highest concentration has been reported in some hypothalamic nuclei. NPY which acts as a neuroendocrine modulator participates in regulating many physiological functions. Thus, it is very easy to speculate that NPY also plays a certain role in pituitary tumorigenesis as well as other biological behavior. Our study was conducted to determine the expression and its significance of NPY as well as its receptors (Y1R and Y2R) in pituitary adenomas. Therefore, we developed rat pituitary tumor models by estradiol benzoate aiming to evaluate NPY expression in pituitary and hypothalamus. In addition, it was also carried out to define the expression of NPY as well as its receptors in human pituitary adenomas, to investigate the relationship between NPY as well as its receptors expression and the subtypes of pituitary ademona and to explore their clinical significance. So experiments were performed as follows.Part I : Animal ExperimentObjective The aim of this study was to explore the expression of neuropeptide Y as well as its receptors (Y1R and Y2R) in estrogen-induced pituitary tumors and hypothalamic nuclei of Wistar rats, to investigate their roles in pituitary tumorigenesis. Methods 40 female 4-week-old Wistar rats were divided randomly into experimental and control groups. All animals were surgically ovariectomizedbefore experiment. The rats were induced with estradiol benzoate by peritoneal injection in experimental group. Each pituitary gland and hypothalamus were weighed and collected for histopathological and other examination. Blood serum prolactin and plasma neuropeptide Y concentrations were measured with radioimmunoassay (RIA) kit. The gene expression of NPY as well as its receptors in pituitary glands and hypothalamic tissues was also detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Some pituitary tissues were also examined using immuno-electron microscopy in order to study the distribution of NPY at the subcellar level. The data were analyzed by the SPSS 11.5 for windows. Results (1) In experimental group, significant increases in pituitary weight were noted three months after peritoneal injection of estrogen (52.35 ± 4.97) mg. The serum prolactin concentration also increased sharply (198.23 ± 93.07) ng/ml. They were approximately 4 fold compared to pititutary glands (12.02 mg ± 0.77 mg) and serum prolactin concentration (50.12 ng/ml ± 14.24 ng/ml) of control group. But plasma NPY concentration in experimental group (6865.52 pg/ml ± 2898.31 pg/ml) was significantly lower than that in control group (9889.71 pg/ml ± 3779.19 pg/ml). The pituitary tissues which showed proliferation and atypia with formation of blood-filled spaces were diagnosed as adenomas by light microscopically. The PRL expression level in pituitary tumors was very higher than that in control pituitary (P <0.001). (2) The expression level of NPY in pituitary and hypothalamus of experimental groups were significantly lower compared to that of control group (P <0.001). (3) RT-PCR detection indicated that the gene expression of Yl and Y2 recepors were decreased in the pituitary tumors induced by estrogen (P <0.001). The YI receptor gene expression of hypothalamus in experimental groups was significantly lower than that in control groups (P <0.001), but no significant differences were observed with regard to Y2 receptor gene expression ofhapothalamus between experiment and control groups. (4) Immuno-electron microscopy examination demonstrated that positive NPY staining with a high density of gold particles located mainly in the secretory granulas. Conclusion These data suggest that chronic treatment with estrogen can result in pituitary adenoma in female Wistar rats. Down regulation of NPY and its Yl receptor expression in hypothalamus may at least in part be responsible for estrogen-induced pituitary tumorigenesis in rats. NPY may modulate pituitary tumorigenesis via direct interactions with Yl receptor located in hypothalamus nulei. Eexpression of NPY as well as its receptors (Y1R and Y2R) was decreased by estrogen in the pituitary glands, with possible involvement in estrogen induced pituitary tumorigenesis in rats.Partll: Clinical ResearchObjective The aim of this research was to study the expression of neuropeptide Y (NPY) as well as its receptors (Y1R and Y2R) in human pituitary adenoma and to investigate its effect on tumorigenesis and other oncobiological behavior. Methods The study included specimens from patients with pituitary adenomas who underwent surgery because of their clinical diagnosis. The clinical data about patients were collected. Using a highly specific anti-NPY polyclonal antibody, immunohistochemical analysis was performed on the surgically removed pituitary adenomas in order to study the distribution of NPY at the pituitary adenoma and the relationship between NPY expression level and different subtypes of pituitary adenoma. To further find the histological evidence of NPY expression in pituitary tumor cells, an immuno-electron microscopic method was also used with the aim of localizing NPY at the cellular and subcellular levels. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was alse carried out to estimate the expression of NPY as well as its receptors (Y1R and Y2R) in fresh pituitary adenoma tissues. In addition, the gene expression of Yl and Y2 receptor attumor cells level was investigated using in situ hybridization (ISH). Statistical calculations were performed by SPSS 11.5 for windows. Results (1) NPY as well as its receptors (Y1R and Y2R) was detectable in pituitary adenoma. The expression level of NPY and Y2 receptor was significantly different among pituitary adenoma (P<0.05). The expression of NPY and Y2 receptor in gonadotrophin adenomas was in the highest level, but in prolactin adenomas was in the lowest level. A positive correlation was demonstrated between NPY and Y2 receptor expression among pituitary adenomas. A positive correlation was also found between NPY expression and serum growth hormone concentration in growth hormone adenomas (PO.05). (2) Immuno-electron microscopy discovered that positive NPY staining with a high density of gold particles located mainly in the secretory granulas. In addition, gold particles were sparsely detected in the rough endoplasmic reticulum and cell matrix. (3) No significant differences were observed with regard to NPY as well as its receptors (Y1R and Y2R) expression between invasive and noninvasive pituitary adenomas (P >0.05). (4) Based on in situ hybridization, it was observed that Yl and Y2 receptors existed in both tumor cells and vascular endothelial cells. The gene expression of Y2 receptor has more advantages than Yl receptor in vascular endothalia cells in pituitary adenoma. (5) NPY and Y2 receptor expression in pituitary apoplexy were significantly higher compared to that in no-pituitary apoplexy (P<0.05). Moreover, difference of Y2 receptor expression existed mainly in vascular endothelial cells. Conclusion (1) NPY as well as its receptors (Y1R and Y2R) is expressed in various pituitary adenomas, and their epression level was correlated with hormonal status and biological characteristic. (2) NPY as well as its receptors participated in regulating on endocrinal behavior of pituitary adenoma. (3) NPY may play a certain role in modulating the tumor's hemodynamic change. Y2 receptor may possiblly involve in this process. (4) NPY as well as its receptors (Y1Rand Y2R) may not involve in modulating the pituitary tumors invasive behavior.Taken together, all of these studies support the potential role of NPY as well as its receptors (YIR and Y2R) in pituitary tumorigenesis and tumor's other biological behavior, but their regulatory mechanism has to be elucidated further.