Maltol total alkaloid inhibits expression and secretion of prolactin by upregulating dopamine D2 receptor / 中草药

Objective: To study the role of dopamine D2 receptor (D2R) on the regulation of prolactin (PRL) secretion by malt total alkaloids. Methods: MMQ and GH3 cells of pituitary adenoma were divided into control group, bromocriptine (5 μg/mL), malt total alkaloids (4.4, 8.8, 35.2, 70.4 μg/mL), haloperidol (10, 20, 40 μg/mL), and combined administration group of total malt alkaloids and haloperidol. Cell viability was detected by CCK-8; The expressions of PRL and D2R were detected by western blotting; The level of PRL was detected by ELISA; The level of PRL and D2R mRNA were detected by qRT-PCR. Results: Compared with control group, malt alkaloids (35.2, 70.4 μg/mL) significantly reduced the expression levels of PRL protein and mRNA, and the level of PRL in the supernatant of MMQ cells (P < 0.05). Malt alkaloids (35.2, 70.4 μg/mL) significantly increased the expression levels of D2R protein and mRNA in MMQ cells. Haloperidol significantly inhibited the downregulation of malt alkaloids on the expression levels of PRL protein and mRNA, and the expression level of PRL in supernatant of MMQ cells (P < 0.05). Haloperidol significantly inhibited the upregulation of malt alkaloids on the levels of D2R protein and mRNA (P < 0.05). The level of PRL in GH3 cells had no change by malt alkaloids. Conclusion: Malt alkaloids could inhibit the expression and secretion of PRL in MMQ cell by upregulating D2R.

Troglitazone inhibits proliferation of GH3 cell in vitro / 第二军医大学学报

Objective: To study the anti-proliferation effects of thiazolidinedione compounds-troglitazone, which is a high affinity ligand of PPAR-γ, on rat pituitary adenoma GH3 cell line and explore the related mechanisms. Methods: GH3 cells were separately treated with troglitazone (10-7, 10-6 and 10-5 mol/L), dimethyl sulfoxide (DMSO) (DMSO control group) and phenol red- and serum-free F-12 medium (blank group). MTT was used to examine the cell growth in each group and FACS was used to detect the distribution of cell cycle. Semi-quantitative RT-PCR method was utilized to determine the expression of CyclinDi mRNA. ANOVA was used for statistical analysis. Results: The 72 h treatment with troglitazone inhibited GH3 cell proliferation in a dose-dependent manner. The treatment also induced cell cycle arrest in G1/S phase and significantly decreased the expression of CyclinD1 mRNA as compared to the other 2 groups (P< 0.05). Conclusion: Troglitazone can obviously inhibit the proliferation of GH3 cells; the molecular mechanism may be the decrease of CyclinD1 mRNA due to binding to PPAR-γ.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Effect of TRH on Phospholipase D Activity in GH3 Cell / 대한내분비학회지

BACKGROUND: GH3 cells are a well characterized and widely used model used for the in vitro study of growth hormone (GH) secretion. Thyrotropin releasing hormone (TRH) binds to receptors belonging to the family of G protein-coupled receptors, and secrets both GH & prolactin. Phospholipase D (PLD) is an enzyme that hydrolyses phosphatidylcholine to yield phosphatidic acid and choline, and plays important roles in cellular proliferation and hormonal secretion. To elucidate the pathway of the action of TRH in GH3 cells, we investigated the activities of PLC and PLD in GH3 cells treated with TRH or phorbor 12-myristate 13-acetate (PMA). METHODS: GH3 cells were labeled with [3H] myristate, followed by incubation of with 0.3% ethanol, prior to before the addition of the agonists. The total lipids were extracted from the harvested cells following treatment with the agonists. The PLD activity was assessed by measuring [3H] phosphatidylethanol from the [3H] phospholipid using thin layer chromatography. RESULTS: TRH (1 muM) stimulated the PLC activity by 44-fold over that of the control values. TRH (1 microM), mastoparan (5 muM), and PMA (500 muM) for 30 minutes increased PLD activity by 1.9, 1.5 and 2.2 fold, respectively, in comparison to the controls. The PLD activities after 15, 30, 60, 120 and 240 min treatments of TRH (1 microM) were 142%, 170%, 172%, 160% and 115%, respectively. CONCLUSION: These results suggest that TRH stimulates not only the PLC activity, but also the PLD activity in GH3 cells.

Effects of Epidermal growth factor (EGF) on the suppression of GH3 cell growth / 대한해부학회지

Some of the pituitary prolactinomas were reported that they don't have active dopamine receptors and do not respond to bromocriptine which is a dopamine agonist. GH3 cell line which is derived from the rat pituitary tumor cells lacks affinity of dopamine receptors and secrete prolactin as well as small amount of growth hormone. Although it has been reported that epidermal growth factor (EGF) induces functional expression of dopamine receptors on GH3 cells in vitro, there has been a contradictory result. In the present study, EGF effect on the GH3 cell response to the bromocriptine was observed in order to investigate whether EGF induces dopamine receptor expression on dopamine resistant tumors in the absence of serum. GH3 cells were cultured for 4 days in the serum-supplemented medium (SSM) followed by culture in serum-free medium (SFM) with or without EGF. Additionally, effect of tamoxifen was also observed. EGF decreased the cell number and the ratio of cell division of GH3 cells while the ratio of prolactin-immunoreac-tive cells was increased. However, EGF did not show any significant effect on the GH3 cell response to bromocriptine treatment. Although tamoxifen decreased the GH3 cell number by increasing apoptosis, it did not influence GH3 cell response to bromocriptine. Our results indicate that EGF does not increase the affinity of dopamine receptors on GH3 cells and is not useful for the treatment of the dopamine-resistant prolactinoma.

EGF Enhances the Differentiation Effect of the Extracellular Matrix Components on the GH3 Pituitary Tumor Cell / 대한해부학회지

This study was performed in order to establish the culture system optimal for the study on pituitary prolactin cells using growth factor and extra cellular matrix components as the culture substrate. The effect of epidermal growth factor (EGF) alone or along with extracellular marix components on GH3 cell growth and PRL expression was assessed using cell count, BrdU-immunocytochemistry and PRL-immunocytochemistry in in vitro cultures on plastic, laminin and Matrigel. EGF decreased the cell growth, BrdU-labeling and increased the PRL-immunoreactive cells regardless of the culture substrate by day 3 of the culture. Matrigel was the best culture substrate to decrease the cell growth and to increase the PRL expression. EGF treatment in the Matrigel culture showed about 80.5% of PRL-immunoreactive cells by day 6 of the culture. These results indicated that Matrigel is the better culture substrate than plastic or laminin to inhibit the overgrowth and to increase the prolactin expression of the GH3 cell and that EGF and Matrigel causes very effective culture environment for the long-term culture of the GH3 cell by synergistic mechanism.

Troglitazone inhibits proliferation of GH3 cell in vitro / 第二军医大学学报

Objective:To study the anti-proliferation effects of thiazolidinedione compounds-troglitazone, which is a high affinity ligand of PPAR-? , on rat pituitary adenoma GH3 cell line and explore the related mechanisms.Methods: GH3 cells were separately treated with troglitazone (10-7,10-6 and 10-5 mol/L), dimethyl sulfoxide (DMSO) (DMSO control group) and phenol red- and serum-free F-12 medium (blank group). MTT was used to examine the cell growth in each group and FACS was used to detect the distribution of cell cycle. Semi-quantitative RT-PCR method was utilized to determine the expression of CyclinD1 mRNA. ANOVA was used for statistical analysis.Results:The 72 h treatment with troglitazone inhibited GH3 cell proliferation in a dose-Dependent manner. The treatment also induced cell cycle arrest in G1/S phase and significantly decreased the expression of CyclinD1 mRNA as compared to the other 2 groups (P

Modulation of L-type Ca2+ channel currents by various protein kinase activators and inhibitors in rat clonal pituitary GH3 cell line

L-type Ca2+ channels play an important role in regulating cytosolic Ca2+ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type Ca2+ channel currents in rat pituitary GH3 cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the Ba2+ current through the L-type Ca2+ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the Ba2+ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced Ba2+ currents. The above results show that the L-type Ca2+ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in GH3 cells may be attributable, at least in part, to their effect on L-type Ca2+ channels.

Thyrotropin-releasing Hormone(TRH) Receptor Gene Expression in GH3 Cells Permanently Transfected with a Mutant Gs alpha Gene / 대한내분비학회지

BACKGROUND: Gs alpha gene mutation, that constitutively increases intracellular cAMP, is found in some acromegalic patients. It was demonstrated that increased intracellular cAMP levels suppress the expression of rat TRH receptor (TRH-R) mRNA. We previously demonstrated that transient expression of a mutant Gs alpha gene suppress the rat TRH-R gene expression in the cultured rat growth hormone-secreting tumor cell line (GH3), whereas TRH-R gene expression in adenomas with Gs alpha gene mutation (gsp oncogene) did not differ from that in tumors without the mutation. The discrepancy suggests the possibilities that the effect of permanent expression of mutant Gs alpha gene on TRH-R gene expression is different from that of transient expression of the mutant gene and hypothalamic hormones including TRH regulate the gene expression. METHODS: We investigated whether permanent expression of the mutant-type Gs alpha does not suppress the TRH receptor gene expression in GH3 cells, and whether TRH suppresses the gene expression by using reverse transcription-polymerase chain reaction (RT-PCR) and in vitro transcription. RESULTS: Permanent expression of a mutant-type Gs alpha increased basal cAMP levels up to 1.7-fold relative to the controls, whereas the wild-type cell line did not show increased cAMP levels. Permanent expression of a mutant-type Gs alpha increased TRH receptor mRNA level up to 2.8 fold compared with the controls. Treatment of the permanently transfected GH3 cells with TRH suppressed TRH-R gene expression more prominently compared to the wild type GH3 cells. CONCLUSION: These results suggest that permanent expression of mutant Gs alpha enhances the expression of TRH-R in GH-secreting pituitary tumors with gsp oncogene, but the gene expression may also be regulated by other factors including TRH.