Danielle Gourdji

Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cAMP production in a rat pituitary cell line (GH3/B6). Additive effects of VIP and TRH on PRL release

The recent localization of VIP [l] (a peptide initially discovered in the gastrointestinal tract) within the hypothalamus, the hypothalamo-hypophyseal portal blood and the adenohypophysis [2-61, strongly suggested its possible regulatory role on the pituitary gland. Kato et al. [7] and Vijayan et al. [8] have shown that VIP was actually able to stimulate the release of several adeno-hypophyseal hormones including prolactin (PRL) in vivo, but not in vitro. They concluded, therefore, that VIP was acting on the secretion of pituitary hormones by means of indirect mechanisms [7,8]. Ruberg et al. nevertheless, demonstrated a direct stimulating effect of VIP on PRL release by incubated rat hem&pituitaries [9]. We have investigated the responsiveness to VIP of a rat prolactin-secreting celI line, the GH3/B6. The hormonal secretion of these cells, or of related strains, has already been shown to be sensitive to various physiological modulators of PRL secretion in vivo, including thyroliberin (TRH) [lo] (for recent reviews see refs. [ 11,121). Such an homogeneous population of target cells provides, therefore, a suitable model for investigating a hypothetical effect of VIP on prolactin cells at the cellular and subcellular levels. Here, we show that the GH3/B6 cells are highly responsive to VIP which stimulates in a concomitant manner both their PRL release and 3’,5’cyclic adenosine monophosphate (CAMP) production. These effects of VIP were compared to those of TRH, a well-established hypothalamic PRLstim-

Binding of a tritiated thyrotropin-releasing factor to a prolactin secreting clonal cell line (GH3)

Abstract The GH3 pituitary clonal cell line, which secretes prolactin and is responsive to thyrotropin-releasing factor, TRF, has been used to study the binding of a strongly labelled 3H-TRF (60 Ci/mM). This binding is time-dependent. It increases linearly in the first 15 min of incubation, thereafter reaching a plateau up to 60 min, independent of the dose (from 0.27 to 27 nM). This kinetic is similar to that of the increase of prolactin release induced by TRF. The specificity has been tested by comparing the affinity of GH3 pituitary cells with those of 3T3 fibroblasts and C6 glial cells and by competition experiment with unlabelled TRF and other peptides. After a constant period of incubation (30 min), the amount of bound molecules is dose-dependent. The two different rates of augmentation with increasing doses for low (1.35 to 155 nM) and high (155 to 1 080 nM) concentrations of 3H-TRF, suggest the involvement of at least two components in the3H-TRF binding to GH3 cells. Moreover, the presence of radioactive material within the cells is revealed by an autoradiographic study performed after a 30 min incubation with 3H-TRF.

A cell culture approach to the study of anterior pituitary cells.

Publisher Summary This chapter discusses a cell culture approach to study the anterior pituitary cells. Pituitary cells maintained in vitro for months or years differ from pituitary organ cultures in which tissue organization is maintained, and from tissue culture starting from small fragments of tissue which grow and can display selection in some cell populations. Among cell cultures one must distinguish (1) primary cultures that start from normal adult fully differentiated anterior pituitary cells, and (2) continuous cell lines that consist of homogeneous populations of pituitary glandular cells which continuously grow in vitro. The chapter discusses the functional and morphological features of pituitary cells grown in these two situations. The effects on these models of factors that regulate the secretion of anterior pituitary hormones are analyzed and discussed. Anterior pituitary continuous cell lines were found to be a useful model for the analysis of mechanisms of action of agents that quantitatively regulate their hormonal secretion. In several cases they appear to be very close to the in vivo situation, e.g., control of prolactin (PRL) secretion by TSH-releasing hormone (TRH) and estrogens, control of growth hormone secretion by thyroid hormone, and control of adrenocorticotropic hormone (ACTH) secretion by glucocorticoids. Because of their cellular homogeneity, they have the advantage in regard to primary culture of allowing investigations at the molecular level, which are undergoing rapid progress.

The rat prolactin gene: a target for tissue-specific and hormone-dependent transcription factors

The expression of the prolactin (rPRL) gene is essentially restricted to glandular cells of the pituitary: the la.ctotropes and the mammosomatotropes, which also express the growth hormone gene. This developmentally acquired specificity is complemented by a complex network of neuroendocrine regulations ensuring a rate of rPRL synthesis adapted to its multiple functions, from reproduction and behaviour to immune responses (see Hoshino, 1988). In the last years a number of studies have underlined the key role of the 5’-flanking regions of the rPRL gene in this dual control: most, if not all, the nucleotide sequences involved in tissue-specific and hormone-regulated expression reside within a two kilobases stretch, itself organised into two domains (see review in Rosenfeld et al., 1987; Davis et al., 1988). Our knowledge in the functional properties of these domains has benefited of rapid progress in molecular biology approaches and the availability of clonal mammosomatotropic cell lines as model systems (Gourdji et al., 1982). This made it possible to delineate DNA &-active sequences mediating cell-specific and hormone-dependent regulations and to identify important tissue-specific transcription factors. Interestingly, concomitant progress in our knowledge in constitutive and regulated rPRL gene expression revealed that both should result from the combined involvement of cell-specific and ubiquitous transcription factors. Finally, several reports indicate that both types of control probably involved changes in DNA structure. The aim of this paper is to update major advances in these three different mechanisms that govern the rPRL gene transcription, except that

Effect of thyreotrope-releasing hormone (TRH) on prolactin turnover in culture

A kinetic study of the influence of thyreotrope-releasing hormone (TRH) on prolactin turnover and synthesis by a new rat pituitary prolactin cell line (SD1) has been performed by means of pulse-chase experiments. After a 10-min [3H]leucine pulse, the chase was carried out in the presence or absence of TRH (54 nM), cycloheximide (3.6 X 10(-5)M) and/or [14C]-proline. The prolactin content of the cells in the medium was estimated using a radioimmunoassay technique. The specific radioactivity of prolactin in the medium was estimated after its isolation by disc gel electrophoresis. This kinetic study demonstrated, firstly, a rapid intracellular transit of newly synthesized prolactin (15 + 10 min or less); secondly, the existence of at least two intracellular prolactin pools; thirdly, a rapid effect of TRH on release of stored prolactin, which is independent of de novo protein synthesis, and fourthly, a delayed stimulating effect of TRH on prolactin synthesis.

Preparation of highly labelled3H‐thyreotropin releasing hormone (PGA‐HIS‐PRO(NH2)) by catalytic hydrogenolysis

The synthetic tripeptide pyroglutamyl-histidylprolineamide (TRH) has been shown to possess the biological potencies of the natural thyreotropin releasing factor (TRF) [ 1,2] . This achievement encouraged a large variety of physiological investigations and efforts towards the preparation of labelled hormone. The first attempts reported concerned tritium labelling of extractive TRF [3]. The specific radioactivity obtained, of the order of 8 mCi/mg was not sufficient for precise cellular localisation under physiological conditions. The simplicity of TRH structure suggested a labelling of the molecule by total synthesis, using labelled amino acids. This aim has been reached by Flouret [4] and by Monahan and Young [5] using, respectively, 14C-histidine or 3H-proline. The specific radioactivities obtained were those of the precursor amino acids, 250 mCi/mmole and 50 Ci/mmole, respectively. Previous work had shown that a variety of hormonal peptides could be tritium labelled by catalytic replacement of iodine atoms previously bound on tyrosyl residues(s) [6,7] . The specific radioactivities

CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines

In the present report, we have investigated the role of DNA methylation on the binding and trans-acting properties of transcription factors involved in the regulation of the rat prolactin (rPRL) gene, specifically Pit-1. To this aim we took advantage of a model system composed of three GH3 rat pituitary tumor cell lines that greatly differed in the extent of rPRL gene methylation and in the level of rPRL gene expression. Northern blot analyses indicated that identical species of Pit-1 mRNA were present to similar extent in the three GH3 cell lines. Electrophoretic mobility shift assays further demonstrated that Pit-1 was present in nuclear extracts and displayed equal affinities to bind the 1P responsive element encompassing the -65 to -38 region of the rPRL promoter, whatever the GH3 cell line tested. These data suggested that differential expression of the rPRL gene among cell lines did not result from variable amounts of Pit-1. By combining in vitro methylation and transient transfection experiments with a rPRL promoter-driven CAT construct, we showed that extensive methylation at CpG sites abolished the expression of the reporter gene. Furthermore, in vivo competition assays demonstrated that CpG methylation inhibited gene expression by preventing the binding of transcription factors We propose that related mechanisms linked to DNA methylation might alter the activity of the endogenous PRL gene in the low expressing cell line.

Differential Pituitary Gene Expression Profiles Associated- To Aging and Spontaneous Tumors as Revealed by cDNA Expression Array

Aging of the rat pituitary is often accompanied by the occurrence of adenomas. We asked whether complementary DNA hybridization array was adapted to identify gene expression patterns linked to aging and associated spontaneous adenomas. Thus, [32P]dATP-labeled cDNAs were prepared from pituitaries of three-month-old rats (Y) and tumor-bearing 20-28-month-old rats (OT). The cDNAs were hybridized to identical membrane arrays allowing to study simultaneously 588 known genes (Clontech 7738-1). Among the 79 genes detected, the GH gene was predominantly expressed in both groups. Twenty-eight genes in the OT group and 15 in the Y group were found to be expressed at a higher level. The largest differences were of about 17 fold and were observed for the galanin and glutathione S transferase genes in the Y and OT groups, respectively. Relative RT-PCR was applied to validate the OT versus Y expression pattern obtained via cDNA array hybridization. The results were consistent for 14 out of the 15 genes tested. In the l...

Effect of 17β-Estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.

Characterization of Thyroliberin (TRH) Binding Sites and Coupling with Prolactin and Growth Hormone Secretion in Rat Pituitary Cell Lines

The secretory response induced by the synthetic hypothalamic hormone thyroliberin (TRH) (L-pyroglutamyl-L-histidyl-L-prolineamide) (Burgus et al., 1969) on anterior pituitary involves, as in any hormone-target cell interaction, the recognition of receptors. The characterization of binding sites relevant to hormone release or synthesis or both constitutes an approach to identification of these receptors. Cell culture of homogeneous populations of target cells is a useful model system for such investigations, as demonstrated by the large body of information that has been obtained in the past few years. Indeed, since the initial discovery by Tashjian et al.(1971) of the TRH prolactin-promoting activity in GH3 and GH1 cells, the use of these or of similar rat prolactin-secreting clonal cell lines became preeminent in studying the TRH mechanism of action at the cellular level(see the reviews in Tixier-Vidal et al., 1975b, 1979a). Although they are continuously dividing, and despite some tumoral aspects of their behavior, these cell lines retained, even after several years of growing in culture, their specific differentiation: (1) They synthesize and release prolactin (PRL) and growth hormone (GH), biologically and immunologically indistinguishable from rat hormones (Tashjian et al., 1968, 1970; Gourdji et al., 1973a). (2) They respond in the same manner as normal rat pituitary cells to several drugs or hormones that are known to regulate PRL in vivo, such as estrogens (Tashjian and Hoyt, 1972; Brunet et al., 1977), CB 154 (Gourdji et al., 1973b), dopamine (Tixier-Vidal et al., 1979b), and TRH, since it has now been shown that TRH is actually a PRL-stimulating factor in a wide range of species including rat and man (cf. the review in Vale et al, 1977). Its effect on PRL is biphasic, as demonstrated in SD1 cells (Morin et al., 1975) and in GH3 cells (Dannies et al., 1976), as well as in normal rat pituitary primary culture (Vale et al., 1973): a short-term effect on PRL release (150-350% of the control) and a secondary stimulation of PRL synthesis (130-500% of the control) that involved an increase of the messenger RNA (mRNA) coding for PRL in GH3 (Evans et al., 1978). TRH also elicits an acute release of GH in GH3 B6 (Faivre-Bauman et al., 1976; Ostlund et al., 1978), GH1 (Morin and Labrie, 1975), and SDX (Gourdji et al., 1975), but in contrast to PRL, this stimulating effect is transitory and turns into a long-term inhibiting action on GH synthesis (Tashjian et al., 1971; Tashjian and Hoyt, 1972).