Pertussis toxin blocks the inhibitory effect of muscarinic cholinergic agonists on cyclic AMP accumulation and prolactin secretion in GH3 anterior-pituitary tumour cells.
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ABSTRACT:
SUBMITTER: Brown BL
PROVIDER: S-EPMC1144274 | biostudies-other | 1984 Oct
REPOSITORIES: biostudies-other
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Project description:Pertussis toxin (PT) caused the ADP-ribosylation of a Mr-41 000 protein in GH3-cell plasma-membrane preparations. This effect, and muscarinic inhibition of prolactin release, were reversed at similar rates by pretreatment of intact cells with PT. These results suggest that the Mr-41 000 protein is modified in intact GH3 cells, and that this protein (a component of the putative Ni unit of adenylate cyclase) is involved in the expression of muscarinic inhibition.
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Project description:BackgroundThe role of anterior pituitary hormones in systemic lupus erythematosus (SLE) remains controversial.Aims and objectivesWe determined the expression levels of human growth hormone (GH), prolactin (PRL), and their receptors in subjects presenting with SLE, and modulation of disease severity.Materials and methodsForty-seven subjects and ten healthy controls were assessed for possible association between SLE disease activity and levels of serum PRL, GH and thyrotropin-releasing hormone (TRH). In peripheral blood mononuclear cells (PBMC), specific binding and mRNA expression of receptors for GH (GHR), and PRL (PRLR) were determined by receptor-ligand binding assay (RLBA) and RT-PCR. PBMC of recruited subjects were treated with hPRL and rhGH to assess IgG production and antibodies against dsDNA.ResultsIn active SLE subjects we found elevated PRL and GH levels. Study subject PBMCs displayed augmented GHR and PRLR protein and mRNA expression. Study subjects also showed a positive correlation in serum PRL levels and specific antibodies against dsDNA, SLE disease activity index (SLEDAI), and proteinuria. However, a negative correlation was found between serum PRL levels and complement component C3. We found a positive correlation between specific binding rates of PRLR and GHR and both SLE activity and dsDNA antibody titers. Enhanced IgG and anti-dsDNA secretion was observed in cultured PBMC stimulated by PRL or GH with/without PHA, PWM, IL-2 or IL-10. In active SLE, a close association was found between augmented PRL and GH levels, expression and specific binding activities of PRLR and GHR, and changes in the specific titer of anti-dsDNA.ConclusionAnterior pituitary hormones play an important role in the pathogenesis of SLE. High levels of growth hormone (GH) and prolactin (PRL) play a role in pathogenesis of SLE, which is correlated with SLE disease activity and antibodies against dsDNA. The mechanism of GH and PRL in SLE was complicated and should be studied further.
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Project description:Prolactin (PRL) is a hormone mainly secreted by the anterior pituitary gland. In birds, PRL exerts a variety of physiological functions in target tissues expressing the PRL receptor (PRLR). In chicken, the PRLR mRNA is abundant in the anterior pituitary gland, but its regional and cellular localization are unknown. In the present study, we investigated the expression of the PRLR mRNA in cephalic and caudal lobes of the chicken anterior pituitary gland. Real-time polymerase chain reaction (PCR) revealed high levels of PRLR mRNA in both cephalic and caudal lobes. In situ hybridization revealed that the PRLR mRNA was distributed in a wide area of both lobes, and co-localized with the PRL and growth hormone (GH) mRNAs in the cephalic and caudal lobes, respectively. These results suggest that PRL exerts autocrine/paracrine effects through PRLR on PRL-producing lactotrophs and GH-producing somatotrophs in the chicken anterior pituitary gland.
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Project description:Growth hormone (GH) is an important hormone released by the pituitary gland that plays a key role in the growth and development of organisms. In our study, TargetScan analysis and the dual luciferase reporter assays were used to predict and screen for miRNAs that might act on the rat Gh1 gene, and we identified miR-543-5p. Then, the GH3 cell line and the primary rat pituitary cells were transfected with miRNA mimic, inhibitor, and siRNA. We detected the Gh1 gene expression and the GH secretion by real-time PCR and ELISAs, respectively, to verify the regulatory effect of miR-543-5p on GH secretion. The results showed that miR-543-5p can inhibit Gh1 mRNA expression and reduce GH secretion. MiR-543-5p inhibitor upregulated Gh1 mRNA expression and increased GH secretion compared with the negative control. In summary, miR-543-5p downregulates Gh1 expression, resulting in a decrease in GH synthesis and secretion, which demonstrates the important role of miRNAs in regulating GH and animal growth and development.
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Project description:The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.
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Project description:The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.
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Project description:Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.
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Project description:The PRL regulatory element-binding (PREB) protein is a transcription factor that was originally cloned from the rat anterior pituitary gland and characterized as a regulator of the PRL promoter. It is also strongly expressed in several extrapituitary tissues; however, its functional role is not well understood to date. In this study, we aimed to clone and characterize the turkey PREB gene and investigate its mRNA expression in the anterior pituitary gland and pancreas during embryogenesis. Based on the conserved sequence of chicken and mammalian PREB cDNAs, a turkey PREB cDNA fragment was obtained, and after sequencing of the fragment, the 5'-and 3'-ends of mRNA were amplified and determined. To identify the PREB gene structure, polymerase chain reaction (PCR) amplification was performed. The turkey PREB gene consists of 9 exons and 8 introns, and it encodes a 411-amino-acid protein. The expression of PREB mRNA in the anterior pituitary gland was measured during embryogenesis. Levels of PREB mRNA significantly increased at embryonic day 22, with maximum levels being detected on day 25 of ontogeny, which correlated with similar changes in levels of PRL mRNA. The highest level of PREB mRNA was detected on day 19 in the pancreas. However, the highest level of insulin mRNA was detected at embryonic day 25. These results indicate that PREB may be involved in the expression of PRL mRNA in the anterior pituitary gland, whereas insulin mRNA may be expressed independently of the expression of PREB mRNA in the pancreas during embryogenesis.
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