Study On Regulation Mechanism Of GHS-R For The Growth Hormone Secretion And Cell Proliferation Of Pituitary Somutotrophinomas

Objective To study the mechanism of GHRP-6-activated signal transduction by binding to growth hormone secretagogues receptor (GHS-R). Explore the protein kinase C (PKC) isoforms which play the critical role in the signaling pathways.Methods Firstly, measure the effect of GHRP-6 on growth hormone (GH) release and cAMP-responsive element-binding protein (CREB) phosphorylation. Secondly, detect the PKC isoforms which mediated these responses of GHRP-6:GH3 cells were treated with PKC activator (phorbol ester, PMA) or inhibitors (G66983 and rottlerin), and a dominant negative mutant of PKCσwas transfected into the cells by lipo2000. Genomic sequence of this isoform gene was retrieved from Genebank. The siRNA was synthesized by Company. Measure the expression of this isoform to detect the transfection efficiency. Measure the effect of GHRP-6 on GH release and CREB phosphorylation after transfection. Examine this isoform activity after treatment of GHRP-6 to verify the result.Results GHRP-6 stimulated GH secretion in both time-and dose-dependent manner (P<0.05) and enhanced the effect of GHRH on GH secretion (P< 0.05).This study provided the first evidence that GHRP-6 could stimulate CREB phosphorylation. These responses of GHRP-6 were reduced by the PKC inhibitors and knockdown of PKCσ. Moreover, PKCσcould be activated by GHRP-6.Conclusion PKC, especially PKCσ, mediated CREB phosphorylation and GH secretion induced by GHRP-6 in GH3 cells. Objective To study the mechanism of GHRP-6-activated signal transduction by binding to growth hormone secretagogues receptor (GHS-R). Explore the downstream mediatory molecules of PKC in the signaling pathways.Methods Firstly, GH3 cells were treated with adenylate cyclase (AC) inhibitor, MDL-12,330A, in different dose for 2 h. Then, cells were treated with GHRP-6 (100 nM). Measure the cell cAMP level after AC inhibition by using cAMP-enzyme-linked immunosorbent assay (ELISA) kit. Examine the effect of GHRP-6 on GH secretion after AC inhibition by using GH-ELISA kit. Western blot was used to detect the effect of GHRP-6 on CREB phosphorylation.Results MDL-12,330A reduced basal and GHRP-6-induced cAMP production (P< 0.01). Inhibition on AC decreased CREB phosphorylation and GH secretion induced by GHRP-6 (P< 0.01).Conclusion GHRP-6-induced CREB phosphorylation and growth hormone secretion depends on AC activation in GH3 cells. Objective Measure the effects of ghrelin and Nitric Oxide (NO) on growth hormone (GH) secretion and cell proliferation in rat GH3 cells and explore the possible mechanism of these responses.Methods GH3 cells were incubated by ghrelin on different concentrations or for different times to measure the effect of ghrelin on GH secretion; then, to determine the effect of ghrelin on GH secretion and cell proliferation after SNAP and NAME treatment; GH levels in the cells medium were determined by enzyme linked immunosorbent assay (ELISA) kit. The cell proliferation rate was measured by MTT and the expression of cells proteins were examined by Western blotting.Results Ghrelin induced GH secretion in both time-and dose-dependent manner (P< 0.01) and ghrelin induced cell proliferation (P< 0.05); the stimulatory effects of ghrelin were reduced by SNAP (P< 0.05) but NAME. SNAP could also inhibit the basal GH secretion and cell proliferation (P< 0.05); ghrelin activated extracellular signal-regulated kinase (ERK) signaling pathway and SNAP blocked this pathway.Conclusion Nitric oxide blocks growth hormone secretion and cell proliferation induced by ghrelin in GH3 cells via blocking ERK signaling pathway.