Size heterogeneity of rat pituitary prolactin.
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ABSTRACT:
SUBMITTER: Wallis M
PROVIDER: S-EPMC1162041 | biostudies-other | 1980 Sep
REPOSITORIES: biostudies-other
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Project description:The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.
| S-EPMC8764672 | biostudies-literature
Project description:Gene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP). Prolactin promoter activity in transgenic pituitaries varied with time across different regions of the gland. Although amplitude of transcriptional responses differed, all regions of the gland displayed similar overall patterns of reporter gene expression over a 50-hour period, implying overall co-ordination of cellular behaviour. By contrast, enzymatically dispersed pituitary cell cultures showed unsynchronised fluctuations of promoter activity amongst different cells, suggesting that transcriptional patterns were constrained by tissue architecture. Short-term, high resolution, single cell analyses in prolactin-d2EGFP transgenic pituitary slice preparations showed varying transcriptional patterns with little correlation between adjacent cells. Together, these data suggest that pituitary tissue comprises a series of cell ensembles, which individually display a variety of patterns of short-term stochastic behaviour, but together yield long-range and long-term coordinated behaviour.
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Project description:Clonal strains of rat pituitary tumour (GH4C1) cells are known to possess specific intracellular binding sites for calcitriol (1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin D3). GH4C1 cells respond to calcitriol by a selective increase in prolactin(PRL)-gene expression. The interaction between calcitriol and glucocorticoids was studied by using this cultured-cell model. It was found that cortisol potently antagonized the induction of PRL mRNA and PRL production by calcitriol. The effects were concentration-dependent and were evident at glucocorticoid concentrations that did not alter basal PRL production. Inhibition was half-maximal at 3.2 nM-cortisol and 0.4 nM-dexamethasone. Calcitriol-induced PRL mRNA fell by more than 50% at 25 h and reached the control level 50 h after treatment with cortisol. The inhibition by cortisol of calcitriol induction of PRL production was selective when compared with effects on other inducers of PRL-gene expression [thyroliberin, epidermal growth factor and phorbol myristate acetate ('12-omicron-tetradecanoylphorbol 13-acetate')]. Potent antagonism by glucocorticoids of vitamin D action on specific gene expression has been demonstrated. Further studies with this cultured-cell model may help to explain the mechanism of this hormonal interaction, which assumes particular importance at major sites of vitamin D action such as the intestine.
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Project description:Heavy metals, such as methylmercury, are key environmental pollutants that easily reach human beings by bioaccumulation through the food chain. Several reports have demonstrated that endocrine organs, and especially the pituitary gland, are potential targets for mercury accumulation; however, the effects on the regulation of hormonal release are unclear. It has been suggested that serum prolactin could represent a biomarker of heavy metal exposure. The aim of this study was to evaluate the effect of methylmercury on prolactin release and the role of the nitrergic system using prolactin secretory cells (the mammosomatotroph cell line, GH3B6). Exposure to methylmercury (0-100 μM) was cytotoxic in a time- and concentration-dependent manner, with an LC50 higher than described for cells of neuronal origin, suggesting GH3B6 cells have a relative resistance. Methylmercury (at exposures as low as 1 μM for 2 h) also decreased prolactin release. Interestingly, inhibition of nitric oxide synthase by N-nitro-L-arginine completely prevented the decrease in prolactin release without acute neurotoxic effects of methylmercury. These data indicate that the decrease in prolactin production occurs via activation of the nitrergic system and is an early effect of methylmercury in cells of pituitary origin.
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Project description:The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1-1mum-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1mum-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1mum) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1mum) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3mug/ml for 4h) increased pituitary cyclic AMP concentrations 4-5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.
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Project description:BackgroundPituitary carcinomas (PCs), defined as distant metastases of pituitary neoplasms, are very rare malignancies. Because the clinical presentation of PCs is variable, early diagnosis and management remain challenging. PCs are always refractory to comprehensive treatments, and patients with PCs have extremely poor prognoses.Case presentationWe describe one case of a prolactin-secreting pituitary adenoma (PA) refractory to conventional therapy that evolved into a PC with intraspinal metastasis. A 34-year-old female was diagnosed with an invasive prolactin-secreting PA in 2009 and was unresponsive to medical treatment with bromocriptine. The tumor was gross totally removed via transsphenoidal surgery (TSS). However, the patient experienced multiple tumor recurrences or regrowth despite comprehensive treatments, including medical therapy, two gamma knife radiosurgeries (GKSs), and four frontal craniotomies. In 2016, she was found to have an intradural extramedullary mass at the level of the fourth lumbar vertebra. The intraspinal lesion was completely resected and was confirmed as a metastatic PC based on histomorphology and immunohistochemical staining. The literature on the diagnosis, molecular pathogenesis, treatment, and prognosis of patients with prolactin-secreting PCs was reviewed.ConclusionPCs are very rare neoplasms with variable clinical features and poor prognosis. Most PCs usually arise from aggressive PAs refractory to conventional therapy. There is no reliable marker to identify aggressive PAs with a risk for progression to PCs; thus, it is difficult to diagnose these PCs early until the presence of metastatic lesions. It is still very challenging to manage patients with PCs due to a lack of standardized protocols for diagnosis and treatment. Establishing molecular biomarkers and the pathobiology of PCs could help in the early identification of aggressive PAs most likely to evolve into PCs.
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Project description:Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.
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Project description:Sex dependency in pain perception is well documented and is thought to be attributable to the effect of reproductive hormones on nociceptive processing. In the present study, we evaluated whether estradiol alters gene transcription in the trigeminal ganglia (TG) of ovariectomized rats (OVX). These experiments demonstrated a dramatic (40-fold) upregulation of prolactin (PRL) expression in TG by 17-beta-estradiol (E2). PRL expression was restricted to TG neurons and was highly overlapped with transient potential receptor vanilloid type 1 (TRPV1) (approximately 90%) in TG. Additionally, PRL is released from neurons during stimulation. Both forms of PRL receptors (PRLRs), short and long, were also present in TG neurons. Moreover, expression of the long PRLRs was under control of estradiol. We next evaluated the novel hypothesis that PRL acts as a neuromodulator of sensory neurons. PRL pretreatment significantly enhanced capsaicin-evoked inward currents, calcium influx, and immunoreactive calcitonin gene-related peptide release from cultured TG neurons. This PRL modulation of capsaicin responses was abolished by withdrawal of E2 from TG cultures. Biochemical analysis demonstrated that PRL increased (>50%) phosphorylation levels of TRPV1 in TG. In a behavioral test, PRL pretreatment significantly potentiated capsaicin-evoked nocifensive behavior in female rats at proestrous and in OVX rats after E2 treatment. The in vivo potentiating effect of PRL on capsaicin responses was also dependent on E2. Collectively, these data demonstrate that PRL is a novel modulator of sensory neurons tightly regulated by E2. These findings are consistent with the hypothesis that PRL could contribute to the development of certain pain disorders, possibly including those modulated by estrogen.
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Project description:1. Newly synthesized prolactin was obtained from cultures of rat pituitary tumour cells (GH4C1 cells) after incubation with [35S]methionine. 2. Radioactive synthesized and secreted prolactin was quantified by an immunoprecipitation method by using disc-gel electrophoresis of the dissolved immunoprecipitate in the presence of sodium dodecyl sulphate. By using a microanalytical modification, hormone synthesis and secretion could also be studied in single-cell cultures. This technique was combined with a cytoimmunofluorescence method in which rhodamine-conjugated antibodies were used for studying intracellular prolactin. 3. The presence of radioactive synthesized and secreted prolactin was demonstrated in nine out of 13 single-cell cultures. Cell cultures containing 10 cells or more and clonal populations originating from one cell always secreted radioactive prolactin. 4. Thyroliberin treatment (2 muM) for 24h increased the extracellular accumulation of radioactive prolactin in five out of seven single-cell cultures and always in populations of cells. 5. The number of cells showing prolactin specific fluorescence increased from 20 to 50% and the intensity of this fluorescence became greater after thyroliberin treatment. 6. Studies of [35S]prolactin secretion from single cells and immunochemical detection of intracellular prolactin showed that some cells in an unsynchronized population did not secret radioactive prolactin or show prolactin specific fluorescence. 7. The quantitative effect of thyroliberin as studied in single-cell cultures suggested that the main if not the only effect was to increase prolactin synthesis in cells already producing hormone.
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Project description:Aggressive prolactin pituitary tumors, which exhibit aggressive behaviors and resistance to conventional treatments, are a huge challenge for neurosurgeons. Many studies have investigated the roles of microRNAs (miRNAs) in pituitary tumorigenesis, invasion and metastasis, but few have explored aggressiveness‑associated miRNAs in aggressive pituitary tumors. Differentially expressed miRNAs (DEMs) between aggressive and nonaggressive prolactin pituitary tumors were screened using the GSE46294 miRNA expression profile downloaded from the GEO database. The potential target genes of the top three most highly upregulated and downregulated DEMs were predicted by miRTarBase, and potential functional annotation and pathway enrichment analysis were performed using the DAVID database. Protein‑protein interaction (PPI) and miRNA‑hub gene interaction networks were constructed by Cytoscape software. A total of 43 DEMs were identified, including 19 upregulated and 24 downregulated miRNAs, between aggressive and nonaggressive prolactin pituitary tumors. One hundred and seventy and 680 target genes were predicted for the top three most highly upregulated and downregulated miRNAs, respectively, and these genes were involved in functional enrichment pathways, such as regulation of transcription from RNA polymerase II promoter, DNA‑templated transcription, Wnt signaling pathway, protein binding, and transcription factor activity (sequence‑specific DNA binding). In the PPI network, the top 10 genes with the highest degree of connectivity of the upregulated and downregulated DEMs were selected as hub genes. By constructing an miRNA‑hub gene network, it was found that most hub genes were potentially modulated by hsa‑miR‑489 and hsa‑miR‑520b. Targeting hsa‑miR‑489 and hsa‑miR‑520b may provide new clues for the diagnosis and treatment of aggressive prolactin pituitary tumors.
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