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D Gourdji

Publications and source records attributed to D Gourdji.

At least 37 records · Page 2Linked to original sources
PubMed1989

Thyroliberin and dihydropyridines modulate prolactin gene expression through interacting pathways in GH3 cells.

The stimulation of PRL gene transcription by TRH involves the two branches of the phosphatidyl inositol pathway as shown by pharmacological mobilization of intracellular Ca2+ stores and activation of protein kinase C. However, TRH receptor occupancy also results in the activation of voltage-dependent Ca2+ channels. Thus, we attempted to determine whether a specific class of voltage-dependent Ca2+ channels, the dihydropyridine (DHP)-sensitive Ca2+ channels, might also be involved in the transcriptional action of TRH. This was studied in rat pituitary tumor GH3B6 cells by runoff assay and measurement of mRNA levels, using two DHPs, BAY K8644 which increases and PN 200-110 which decreases the influx of Ca2+. We show that the PRL mRNA levels and the rate of PRL gene transcription were stimulated by BAY K8644 and inhibited by PN 200-110 in a dose-dependent manner indicating that DHP-sensitive Ca2+ channels can control the expression of the PRL gene. Furthermore, PN 200-110 abolished the BAY K8644-induced stimulations. By contrast, the stimulations of the PRL gene expression induced by TRH or by the phorbol ester TPA were not abolished by the calcium channel antagonist PN 200-110 whereas treatments combining TRH or TPA with BAY K8644 revealed the absence of any additive effect. Altogether these observations suggest that TRH, and TPA, might activate pathway(s) interacting with those triggered by the Ca2+ channel agonist for regulating PRL gene transcription but they do not support the hypothesis of a necessary implication of DHP-sensitive calcium channels in the regulation of PRL gene transcription by TRH.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

[Hypothalamic hypophysiotropic neuropeptide receptors].

The present review is dealing with the five major hypothalamic hypophysiotropic neuropeptides (H.H.N.P.) purified and synthesized so far. Four of them specifically stimulate the secretion of one or several anterior pituitary (A.P.) hormones, i.e. thyroliberin (TRH) on TSH and prolactin, gonadoliberin (GnRH) on LH and FSH, corticoliberin (CRF) on ACTH and precursor peptides and somatocrinine (GRF) on GH. The fifth one, somatostatin (SRIF), inhibits the secretion of all A.P. hormones, excepted LH and FSH. All H.H.N.P. affect, positively or negatively, in a dose- and time-dependent manner, the release of stored hormones and their neosynthesis. These responses are submitted to multihormonal modulations. They are initiated by the occupancy of high affinity specific binding sites which have been extensively characterized and morphologically localized. Informations concerning molecular characterization and cloning of receptors for any H.H.N.P. are still awaited. By contrast, the transduction mechanisms which are activated by the occupation of receptors have been extensively studied. They vary depending on H.H.N.P.: TRH and GnRH activate the catabolism of polyphosphoinositides and ensuing pathways, CRF and GRF activate and SRIF inhibits adenylate cyclase dependent pathways. In addition, Ca2+, from extracellular and intracellular sources, play a pivotal role in all cases. The intracellular mechanisms responsible for the last steps of H.H.N.P. action, i.e. exocytosis of secretory granules and transcription of target genes, are however still unknown.

Adenylyl Cyclases↗
PubMed1988

Preferential role of calcium in the regulation of prolactin gene transcription by thyrotropin-releasing hormone in GH3 pituitary cells.

TRH induces two separate events in pituitary PRL cells. It increases the release of stored PRL and enhances the rate of PRL gene transcription, which results in an increased steady state concentration of PRL messenger RNA (mRNA) and a concomitant augmentation of PRL production. The mechanisms underlying the release process involve the activation of phosphatidylinositol turnover which generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. In order to determine whether these intracellular messengers also mediate the stimulation of PRL gene expression by TRH, we have correlated the level of receptor occupancy with the rate of gene transcription and investigated the action of drugs which increase cytosolic calcium or activate protein kinase C. We have determined that sustained stimulation of transcription requires the persistent occupancy of a limited number of TRH receptor sites and that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores (A23187, ionomycin), and the calcium channel agonist BAY K 8644 enhance PRL gene transcription. However, TPA is less potent and ionomycin requires a low concentration of TPA to fully mimic TRH action, whereas BAY K 8644 alone displays the same potency as TRH. The effects of BAY K 8644 and TRH are not additive and thus suggest that the influx of calcium plays a predominant role in the regulation of PRL gene transcription by TRH.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Thyrotropin-releasing hormone increases the levels of c-fos and beta-actin mRNA in GH3/B6 pituitary tumor cells.

The effects of thyrotropin-releasing hormone (TRH) on the mRNA levels of c-fos, N-myc, beta-actin and prolactin (PRL) were studied in GH3/B6 cells, a rat pituitary cell line. TRH has previously been shown to increase biosynthesis and release of PRL, and to stimulate PRL gene transcription (13,19) in these cells. All experiments were performed on quiescent serum-deprived cells, and under these conditions, addition of TRH stimulated PRL production but did not alter cellular proliferation. Simultaneously, TRH induced time- and concentration-dependent increases in c-fos and beta-actin mRNA levels, with peak responses at 30 min and 4 h, respectively, in a dose range from 1 nM to 100 nM. The TRH effects on N-myc mRNA levels were less consistent. Addition of serum to quiescent GH3/B6 cells induced (3-5 fold) increases in c-fos, beta-actin and PRL mRNAs which differed in magnitude and kinetics when compared to TRH stimulation. Serum did not alter N-myc mRNA levels. The TRH-induced increases in c-fos and beta-actin mRNA may play a role in the secretory response.

Actins↗
PubMed1987

Is thyrotropin-releasing hormone receptor involved in thyrotrope adaptation to starvation?

The aim of the present study was to delineate the involvement of TRH receptors in the thyrotrope adaptation to starvation (i.e. plasma TSH and thyroid hormone decrease, increased sensitivity to T3) by measuring [3H]TRH binding in euthyroid, hypothyroid and T3-substituted rats (175 ng/100 g body weight). Our results show that in euthyroid rats, starvation does not significantly modify either the affinity or the number of pituitary binding sites. In hypothyroid and T3-substituted rats, starvation does not alter the negative control exerted by T3 on the number of TRH binding sites. Our data indicate that the adaptation of thyrotrope to starvation does not primarily result from alterations of TRH binding sites.

Adaptation, Physiological↗
PubMed1986

Differential implication of deoxyribonucleic acid methylation in rat prolactin and rat growth hormone gene expressions: a comparison between rat pituitary cell strains.

In order to assess the potential role of DNA methylation in the expression of rat PRL (rPRL) as compared to rat GH (rGH) gene, the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI were examined in DNA isolated from rat pituitary cell lines producing either high levels of rPRL (GH3B6) or of rGH (GC) and in a stable variant cell strain which produces minute amounts of both hormones (GH3CDL cells). The rPRL and the rGH genes were found hypomethylated in GH3B6 and GC cells, respectively, whereas in GH3CDL cells both genes were methylated, indicating a correlation between the extent of gene methylation and the level of expression. However the use of 5-azacytidine (5-azaC), which decreases DNA methylation, suggested a variable importance of gene methylation in the control of rPRL and rGH gene expression. 5-AzaC was unable to increase rPRL production to a detectable level in GC cells, whereas the cytidine analog markedly increased rPRL production and rGH production in GH3CDL cells. Further analysis using GH3CDL cells showed that the extent of the 5-azaC-induced rPRL and rGH gene demethylation was consistent with the 5-azaC-induced increase of gene expressions. However, in these cells, the stimulation of rPRL and rGH production unexpectedly increased as a function of time elapsed after drug withdrawal. The maximal stimulation, 30-fold and 7-fold, respectively, was observed 3 weeks after a 60-h exposure to 5-azaC. This pattern suggests that other events are required for the full expression of rPRL and rGH genes in addition to their own demethylation.

Animals↗
PubMed1986

[Regulation of prolactin and growth hormone gene expression in pituitary cell culture].

The rat pituitary tumor derived cell lines of the "GH" family offer a fruitful model for studying the expressions of the prolactin (rPRL) and growth hormone (rGH) genes in basic and regulated states. In order to assess the potential role of DNA methylation in the basic expressions of rPRL and rGH genes we have used different cell strains which produce either high level of rPRL (GH3B6 cells) or of rGH (GC cells) and minute amounts of both hormones (GH3CDL cells). The cleavage patterns generated by the methylation sensitive enzymes Hpa II and Msp I indicated an inverse correlation between the extent of gene methylation and the level of expression. However the use of 5-azacytidine which decreases DNA methylation suggested a variable importance of gene methylation in the respective control of rPRL and rGH genes depending on the cell lines. In an other hand we attempted to elucidate some of the mechanisms by which thyroliberin (TRH) enhances rPRL gene transcription in GH3B6 cells. Preliminary results indicated that the persistent occupancy of the TRH receptors was required to sustain at least for the first 5 hours the increased rate of rPRL gene transcription. In addition the possible relationship between the TRH-induced acute rPRL release and the stimulation of rPRL gene transcription was investigated. The results suggested that the activators of the C kinase-mediated pathway which are actually involved in the stimulation of the acute release were not sufficient alone for eliciting the maximum TRH response at the gene level.

Animals↗
PubMed1985

Immunocytochemical evidence for in vivo internalization of thyroliberin into rat pituitary target cells.

The in vivo internalization of thyrotropin-releasing hormone (TRH) was studied by using a semiquantitative immunoelectron microscopic method. Pituitary glands of normal male rats intravenously injected with 100 ng TRH and sacrificed after 5-60 min were used. Ultrathin sections were obtained by cryoultramicrotomy of fixed pituitary glands. Pituitary cellular types were identified by appropriate antiserums. An antiserum specifically directed against TRH was used. TRH-like immunoreactivity due to endogenous TRH was observed in thyrotropes and prolactin cells, but never in somatotropes, gonadotropes or corticotropes. At the subcellular level, the reaction was detected within the cytoplasmic matrix, the secretory granules, and the nucleus but only occasionally at the plasma membrane. After in vivo injection of TRH, the immunocytochemical reaction was still restricted to thyrotropes and prolactin cells, increased with time elapsed after injection up to 15-30 min and then returned to basal intensity in cytoplasm, secretory granules, and nucleus, and became very frequent at the plasma membrane. These data provide evidence for endogenous TRH within thyrotropes and prolactin cells, i.e., in physiological target cells for TRH, and support the hypothesis that normal TRH target cells can, in vivo, internalize exogenous as well as endogenous TRH into several subcellular compartments including the nucleus.

Animals↗
PubMed1983

Inverse control of prolactin and growth hormone gene expression: effect of thyroliberin on transcription and RNA stabilization.

The hypothalamic tripeptide thyroliberin (TRH) regulates prolactin (PRL) and growth hormone (GH) synthesis inversely by modulating the levels of their specific mRNA. Changes in mRNA levels could involve both transcriptional and posttranscriptional events. To examine further these possibilities, we have investigated the effect of TRH on the biosynthesis and degradation of PRL and GH RNA in a rat pituitary tumor cell line. Newly synthesized PRL and GH RNA sequences were quantified in nuclear and cytoplasmic fractions by hybridization of 3H-labelled RNA to immobilized plasmid DNA containing either PRL or GH cDNA sequences. Steady-state levels of specific RNA were estimated by RNA blot hybridization. The results indicate that TRH increases in a rapid but transient manner the transcription of the PRL gene, and suggest that it does not alter the processing and the transport to the cytoplasm. In contrast, after a lag-time, TRH seems to induce a long-lasting inhibition on GH, as well as on overall gene transcription. Furthermore, we observed an effect of TRH on mRNA stability. TRH significantly increases the half-life of PRL mRNA. Our results also support the hypothesis that TRH decreases the half-life of GH mRNA. Such post-transcriptional action of TRH amplifies and prolongs the regulations exerted at the transcriptional level.

Animals↗
PubMed1982

Effect of chloroquine on thyroliberin interaction with clonal rat prolactin cells. Cytochemical correlates.

The lysosomotropic agent, chloroquine, has been used to investigate the implication of lysosomal activity and membrane traffic in TRH binding, TRH internalization and TRH-induced stimulation of prolactin secretion in a rat prolactin cell line (GH3/B6). Chloroquine by itself does not affect cell number, cell protein and basal prolactin secretion. It does not alter TRH binding and internalization as well as both effects of TRH on the stimulation of prolactin secretion, i.e., prolactin release and prolactin production. In contrast, chloroquine partially inhibits the spontaneous dissociation of (3H)TRH from cells previously loaded with (3H)TRH and reduces prolactin release following TRH withdrawal. In addition the kinetic pattern of the TRH dissociation is modified in a manner which suggests that TRH is bound to different intracellular compartments. Chloroquine, nevertheless, does not alter the TRH-induced down regulation of (3H)TRH binding sites. Electron microscopic observations and acid phosphatases localization reveal that chloroquine elicits a disorganization of the Golgi zone and accumulation of membrane whorls within large vacuoles. This suggests that the effects of chloroquine on TRH interaction with GH3 cells may be mediated by an inhibition of membrane recycling.

Acid Phosphatase↗
PubMed1981

Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media.

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.

Animals↗
PubMed1980

Effect of 17 beta-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.

Animals↗
PubMed1980

[Prolactin secreting cell lines: a tool for the study of the mechanism of action of hypophysiotropic neuropeptides. A review (author's transl)].

This review summarizes the characteristics and advantages of cultures of clonal cell lines for studying the mechanism of action of hypothalamic hypophysiotropic factors. Results concerning the interaction of Thyroliberin (TRH) and Vasoactive Intestinal Peptide (VIP) with prolactin-secreting rat pituitary clonal cell lines are summarized here as an illustration. The TRH-prolactin cell interaction appears to be very complicated. The characteristics of 3H-TRH binding sites as well as those of the induced biological effect (PRL stimulation) appears relatively well established. In contrast, the primary event triggered by TRH, as well as the sequence of the intracellular mechanisms of the binding-secretion coupling remain to be established. VIP is shown to be another direct and potent PRL stimulating hypothalamic peptide in PRL producing cell lines. Its stimulating effect is additive to that of TRH and seems to involve a cAMP dependent process.

Animals↗