Inhibition Of Prolactin Synthesis Research Articles

Effects of resveratrol on cell growth and prolactin synthesis in GH3 cells

Resveratrol (RE), a phytoestrogen, has antiestrogenic properties. Estrogen plays a key role in the development and progression of pituitary prolactinoma. Moreover, RE is a potent cancer chemopreventive agent that inhibits the initiation, promotion and progression of carcinogenesis. The present study investigated the antitumor effects of RE on GH3 pituitary tumor cells. A concentration- and treatment duration-dependent biphasic effect of RE on the proliferation of the GH3 cells was demonstrated. After three days of treatment, RE stimulated proliferation at low concentrations and inhibited proliferation at high concentrations. However, when the treatment duration was reduced to 6 h, RE inhibited proliferation in a concentration-dependent manner. In addition, RE induced apoptosis with the activation of caspase-3 and -8, and decreased the percentage of prolactin (PRL)-immunopositive GH3 cells. Furthermore, RE suppressed expression of the PRL gene and inhibited the cell proliferation and PRL synthesis induced by 17β-estradiol (E2). In GH3 cells, the proliferation response exhibited higher sensitivity to E2 compared with the PRL response; by contrast, the PRL response was more sensitive to RE than the proliferation response was. These results indicate that RE, an antiestrogenic compound, exerts its antitumor effect on GH3 cells through the suppression of GH3 cell growth and through the inhibition of PRL synthesis. The RE-induced cell apoptosis was shown to be caspase-dependent. Therefore, the present study provides support for the use of RE in the chemoprevention and chemotherapy of pituitary prolactinoma.

Suppression of Prolactin Expression by Cabergoline Requires Prolactin Regulatory Element-binding Protein (PREB) in GH3 Cells

The prolactin regulatory element-binding protein (PREB) is a transcriptional factor that regulates prolactin (PRL) promoter activity in the anterior pituitary. Prolactinomas are the most common pituitary tumors. Administration of cabergoline, a selective dopamine D2-receptor agonist, has become the initial therapy of choice for most patients with prolactinomas. Although activation of the D2 receptor results in the inhibition of PRL synthesis, the details of the underlying mechanisms remain unknown. Samples of ten prolactinomas and ten nonfunctioning pituitary adenomas were analyzed by immunohistochemistry to detect the expression of PREB. The effect of cabergoline on PREB expression was assessed by western blotting and real-time polymerase chain reaction (PCR) analysis. Reporter gene analysis of PRL was employed to examine the role of PREB on cabergoline-induced suppression of PRL transcription. Immunohistochemical analysis revealed strong positive PREB expression in the prolactinoma tissue, but extremely weak or undetected expression in the nonfunctioning pituitary tumor tissue. Western blots probed with a PREB-specific antiserum revealed that the relative abundance of the PREB protein in the GH3 cells decreased in a dose-dependent manner in response to cabergoline treatment, as did the relative abundance of PREB mRNA. Although cabergoline inhibited the activity of the PRL promoter, mutation of PREB-binding site within the promoter abrogated the ability of cabergoline to inhibit the PRL promoter activity. We have demonstrated that PREB is expressed in prolactinomas and that the suppression of PRL expression by cabergoline requires the transcriptional factor PREB.

Effects of prolactin inhibition during late gestation on the immunity of gilts and foetuses

The goal of the present study was to determine the effects of inhibiting prolactin (PRL) synthesis for three consecutive 20-d periods during the second half of gestation, on the immune status of gilts and their foetuses. Crossbred gilts were randomly assigned as controls (n = 12) or received 10 mg of bromocriptine orally three times daily from days 50 to 69 (BR50, n = 12), days 70 to 89 (BR70, n = 12), or days 90 to 109 (BR90, n = 12) of gestation. All gilts were injected subcutaneously with ovalbumin (OVA) on days 53 and 72 of gestation. Blood samples were collected on days 50, 60, 70, 90 and 109 of gestation to evaluate the antibody response. Cellular immunity, as measured by lymphocyte proliferative response and production of interferon-γ, was characterized on controls and BR50 gilts until the end of gestation. Six foetuses from five litters per treatment were selected and their thymus and spleen were excised and weighed. Splenocytes and thymocytes were assayed to characterize lymphocyte sub-populations and to evaluate splenocyte responses to mitogenic stimulation. Average spleen weight in foetuses from BR90 gilts was significantly lower (P < 0.05) than that of foetuses from control or BR70 gilts. In all gilts treated with bromocriptine,percentages of CD4+ and CD8+ lymphocyte populations in foetus spleens were numerically reduced compared with those obtained in foetus from control gilts but the differences were not significant. In BR50 gilts, inhibition of PRL synthesis did not affect the proliferative response of lymphocytes to mitogenic stimulations compared with control group, but tended (P = 0.13) to increase the production of interferon-γ. A time effect showed that production of interferon-γ by leukocytes was reduced (P < 0.02) on days 80 and 100 compared with days 50 and 70, regardless of bromocriptine treatment. The antibody response of gilts to OVA was not affected by bromocriptine treatments. In conclusion, inhibition of PRL synthesis by bromocriptine did not significantly affect immune response of pregnant gilts. In foetuses, although the differences between treatments were not significant, data suggest that the foetal development of immune tissues seems to be impaired by bromocriptine treatment. Key words: Bromocriptine, prolactin, immunity, pregnancy, sow

Antisense strategy unravels a dopamine receptor distinct from the D2 subtype, uncoupled with adenylyl cyclase, inhibiting prolactin release from rat pituitary cells.

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [3H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.

An Early Pineal-Induced Suppression of Pituitary Prolactin mRNA Levels in Light-Deprived Male Hamsters

Light deprivation by blinding or exposure to short photoperiod results in depressed prolactin (PRL) cell activity and gonadal regression in the male Syrian hamster after at least 42-56 days of treatment, and both of these events can be prevented if pineal removal accompanies light deprivation. In the present study, we investigated changes in PRL cell activity after 11, 21, or 42 days of blinding by measuring changes in: (1) pituitary PRL mRNA levels; (2) newly synthesized pituitary PRL, and (3) pituitary-RIA-PRL. The purpose of this study was to characterize the early (i.e. prior to gonadal regression and loss of reproductive function) effects of light deprivation on PRL production in the pituitary gland of the male hamster, and to determine whether a reduction in PRL mRNA levels precedes the inhibition of PRL synthesis and storage. After 11, 21, and 42 days of blinding, PRL mRNA levels in the male hamster pituitary (relative PRL mRNA/pituitary) were significantly depressed compared to intact controls by 33, 41, and 65%, respectively. Pituitary PRL synthesis (dpm of 3H-PRL/pituitary) and RIA-PRL (micrograms PRL/pituitary) were significantly depressed (by 49 and 53%, respectively) only after 42 days of blinding. All of these statistically significant depressions were prevented when the surgical blinding procedure was accompanied by pineal removal. There were no differences in testicle, pituitary, or body weight as a result of treatment. These results support the hypothesis that light deprivation by blinding causes a pineal-dependent decline in PRL mRNA levels, which leads to a decrease in PRL synthesis and pituitary RIA-PRL.

Cytoplasmic protein phosphorylation related to multihormonal regulation of prolactin in pituitary cells

The specialized functions of mammotropic cells from the anterior pituitary and of the pituitary tumor derived GH cell lines can be regulated by a large number of neurohormones and other substances. Although the biological responses to many of these regulatory signals are well documented, much less is known about the intracellular mechanisms involved in their action. Various "second messengers" are generated, which in turn regulate other intracellular activities like protein phosphorylation reactions. Thyrotropin-releasing hormone stimulates both prolactin synthesis and release by mammotrophs of the anterior pituitary, or by GH(4)C(1) cells in culture. Thyrotropin-releasing hormone simultaneously enhances the phosphorylation of a small number of cytoplasmic proteins in GH(4)C(1) cells, as revealed by two-dimensional polyacrylamide gel electrophoresis after (32)PO(4)(3?) labelling. On a pharmacological basis, two distinct subsets were identified among the seven phosphoproteins affected by thyrotropin-releasing hormone: set I, proteins 1-6, whose phosphorylation could be related to the regulation of prolactin synthesis; set II, proteins 7 and 8, whose phosphorylation was related to the modulation of prolactin release (Sobel A. and Tashjian A. H. Jr. (1983) J. Biol. Chem.258, 10312-10324). Phosphorylation of all these proteins but protein 1 was alkali-resistant, indicating that it might take place on tyrosine residues. Protein 1, which could be visualized by silver-staining and whose phosphorylation resulted in a shift of its position on two-dimensional gels, was present also in normal rat pituitary cells in primary culture. Moreover, thyrotropin-releasing hormone induced its phosphorylation as in GH(4)C(1) cells, thus indicating that this reaction is a normal regulatory process, not related to the tumoral origin of GH(4)C(1) cells. This observation allowed us to study some physiologically important regulatory mechanisms which were lost in GH cells as, for example, the inhibition of prolactin synthesis and release by dopamine: in normal anterior pituitary cells in culture, dopamine also prevented the thyrotropin-releasing hormone induced phosphorylation of protein 1. This result is in good agreement with those obtained with GH(4)C(1) cells, and extends them. The same technique allowed us to detect a protein with two-dimensional gel migration properties identical to those of protein 1 in certain tissues, like the adrenal medulla, but not in others, like the adrenal cortex. This tissue specificity might be related to the specific regulatory function of protein 1 in the cell.

The inhibition of prolactin synthesis in GH 3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E 2 (1 nM) stimulated the synthesis of PRL in GH 3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E 2. [ 3H]E 2 and [ 3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH 3 cells cultured in media containing [ 3H]E 2 or [ 3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8–9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [ 3H]E 2 was inhibited but the binding of [ 3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E 2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.

Evidence for a coupling of prolactin secretion and synthesis by rat pituitary cells in culture

The relationship between the release and the synthesis of prolactin by rat pituitary cells in culture was studied using a microtubule-disrupting drug, vinblastine. (1) Prolactin secretion was inhibited by vinblastine in short-term incubations. Vinblastine did not act via the dopamine pathway, since a potent anti-dopaminergic drug, fluphenazine, was unable to reverse the inhibiting action of the antimicrotubular agent. (2) Continuous treatment by vinblastine induced a progressive decrease of the rate of incorporation of [ 3H]leucine in prolactin. The half-inhibition time was about 2 days. This inhibition of prolactin synthesis was selective, since total protein synthesis remained unaffected. (3) Measurements of radioimmunoassayable prolactin showed that the inhibition of hormone release by vinblastine led to a transient increase of the intracellular content of prolactin. The phase of over-accumulation was followed by a progressive reduction of the total (cell + medium) prolactin. This result is in agreement with the observed inhibition of de novo synthesis of prolactin and indicates that a degradation process takes place in pituitary cells in culture. In conclusion, the use of vinblastine allows us to demonstrate that the rate of prolactin synthesis is dependent upon the secretory status of the cell.

Increased responsiveness of the dopamine-mediated inhibition of prolactin synthesis after destruction of the medial basal hypothalamus.

Long term destruction of the tuberoinfundibular dopaminergic neurons results in increased responsiveness of the anterior pituitary to the suppression of PRL release by dopamine. In the present study we tested whether long term destruction of these neurons by medial basal hypothalamic lesions would also increase the potency of dopamine in suppressing the release and apparent synthesis of PRL. Anterior pituitaries from ovariectomized control rats and ovariectomized rats lesioned for 2 weeks were incubated for 3 h in medium 199 containing [3H]leucine in the presence or absence of various concentrations of dopamine. Labeled PRL was separated by polyacrylamide gel electrophoresis and quantitated by liquid scintillation spectroscopy. Concentrations of dopamine ranging from 10(-8)-10(-6) M caused a dose-dependent suppression of labeled newly synthesized PRL released into the medium from anterior pituitaries of medial basal hypothalamus-lesioned animals. With pituitaries from the nonlesioned rats, newly synthesized PRL release was progressively inhibited by 10(-7) and 10(-6) M dopamine, while 10(-8) M dopamine actually significantly stimulated PRL release. Total labeled PRL (that released and that remaining in the gland) was equally suppressed by all three concentrations of dopamine in pituitaries from lesioned animals, but only a minor effect was observed at the highest concentration of dopamine with the control pituitaries. Therefore, the potency of dopamine to suppress the release and apparent synthesis (total labeled PRL) of newly synthesized PRL from pituitaries of long term lesioned animals was increased.

In vivo evidence for catecholaminergic inhibition of prolactin secretion in the teleostPoecilia latipinna

Injections of L-dopa in freshwater (FW) fish reduced the size of the prolactin (PRL) cell nuclei, suggesting inhibition of PRL synthesis. A single injection of 6-hydroxydopamine (6-OHDA) in one-third seawater (1/3SW) fish reduced pituitary PRL content and increased PRL cell nuclear size at 6 h and 12 h, indicating stimulation of both synthesis and release of PRL. Two daily injections of 6-OHDA in 1/3SW fish led to PRL cell nuclear enlargement and elevated pituitary PRL content at 48 h after the second injection, indicating strong stimulation of PRL synthesis. Consideration of other parameters (plasma and body sodium levels, plasma osmotic pressure) suggests that the PRL cell responses to 6-OHDA were not mediated by internal osmotic changes. Pretreatment with 6-OHDA also appeared to accelerate the PRL cell activation induced by the transfer of fish from 1/3SW to FW.