Synthesis and renewal of proteins in duck anterior hypophysis in organ culture.

In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material.

Membrane effects of thyrotropin-releasing hormone and estrogen shown by intracellular recording from pituitary cells.

The effects of thyrotropin-releasing hormone and 17 beta-estradiol on the electrical membrane properties of a prolactin-secretin pituitary cell line (GH3/B6) were studied with intracellular microelectrode recordings. Of the cells tested, 50 percent were excitable and displayed calcium-dependent action potentials when depolarized. When injected directly on the membrane of an excitable cell, thyrotropin-releasing hormone and 17 beta-estradiol induced action potentials within 1 minute. The spiking activity was preceded by a progressive increase of the input resistance without any detectable change in the resting membrane polarization. The results reveal a rapid effect of both substances on the membrane of GH3/B6 cells. In the case of thyrotropin-releasing hormone, which has both a short-term effect on release of prolactin and a long-term effect on its synthesis, the induced electrical activity may be associated with the stimulation of prolactin production. The physiological implication of 17 beta-estradiol-induced, calcium-dependent spiking activity remains to be elucidated.

Chronic growth hormone (GH) hypersecretion induces reciprocal and reversible changes in mRNA levels from hypothalamic GH-releasing hormone and somatostatin neurons in the rat.

Effects of growth hormone (GH) hypersecretion on somatostatin-(SRIH) and GH-releasing hormone (GHRH) were studied by in situ hybridization and receptor autoradiography in rats bearing a GH-secreting tumor. 6 and 18 wk after tumor induction, animals displayed a sharp increase in body weight and GH plasma levels; pituitary GH content was reduced by 47 and 55%, while that of prolactin and thyrotropin was unchanged. At 18 wk, hypothalamic GHRH and SRIH levels had fallen by 84 and 52%, respectively. In parallel, the density of GHRH mRNA per arcuate neuron was reduced by 52 and 50% at 6 and 18 wk, while SRIH mRNA levels increased by 71 and 83% in the periventricular nucleus (with no alteration in the hilus of the dentate gyrus). The numbers of GHRH- and SRIH-synthetizing neurons in the hypothalamus were not altered in GH-hypersecreting rats. Resection of the tumor restored hypothalamic GHRH and SRIH mRNAs to control levels. GH hypersecretion did not modify 125I-SRIH binding sites on GHRH neurons. Thus, chronic GH hypersecretion affects the expression of the genes encoding for GHRH and SRIH. The effect is long lasting, not desensitizable and reversible.

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice.

The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the beta-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHbeta gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.

Properties of monoclonal antibodies to thyroliberin (TRH) induced by different immunogens: comparison with pituitary TRH receptor.

Thyroliberin E-H-P-NH2 (TRH) is a small neuropeptide pGlu-His-Pro-NH2 widely distributed in neural sites. The aim of this work was to obtain an antibody molecule with the nearest properties to that of TRH-receptor in GH3 cells. Different TRH-protein conjugates were prepared and utilized to induce monoclonal antibodies in mice. Several monoclonal antibodies were obtained using E-H-P-NH2 (TRH) coupled either to the histidyl residue (immunogen I) or to the prolyl residue (immunogen II). Antibodies generated using immunogen I and immunogen II were characterized in a radioimmunoassay system and an enzyme immunoassay system respectively. Their selectivities regarding a series of TRH related peptides were compared to those of rabbit polyclonal antibodies using three differently labelled TRH (tritiated-TRH, mono-iodinated-TRH and TRH-OH-acetyl-cholinesterase) as tracers and to prolactin secreting cells TRH receptors using 3H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

Differential pituitary gene expression profiles associated- to aging and spontaneous tumors as revealed by rat 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 the 15 genes tested. In the light of these results, differential membrane array hybridization appears suitable to identify gene expression profiles associated with pituitary aging.

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.

Effect of 17beta-estradiol on somatostatin receptor expression and inhibitory effects on growth hormone and prolactin release in rat pituitary cell cultures.

In the present study, we tested whether 17beta-estradiol (E2)-induced PRL sensitivity to somatostatin-14 (SRIF) involves selective up-regulation of discrete somatostatin receptor subtypes (ssts) in primary cultures of female rat pituitary cells. The efficacy of the endogenous peptide SRIF to inhibit GH and PRL secretion and cAMP accumulation was compared with those of octreotide (OCT), BIM-23052, BIM-23056, and BIM-23268, which have been reported to be relatively selective for rat sst2, sst3, and sst5. Experiments were performed in steroid-depleted media supplemented or not with 1 nM E2 for 96 h. SRIF, OCT, and BIM-23052 inhibited cAMP accumulation and GH release independently of E2. In contrast, all three agonists affected PRL release in E2-treated cultures only. Inhibition of cAMP accumulation by SRIF, OCT, and BIM-23052 was enhanced by exposure of cells to E2. The rank of potency of the agonists, OCT = SRIF > BIM-23052, was similar for GH and PRL inhibition. BIM-23268 was a weak agonist on GH, but not on PRL, secretion. BIM-23056 had no effect on the release of either hormone, but slightly inhibited cAMP formation in E2-treated cells. To verify whether SRIF receptor gene expression correlated with these observations, messenger RNA (mRNA) transcripts corresponding to the five ssts were measured by quantitative RT-PCR in the presence or absence of E2. Control cells expressed predominantly sst2 and sst3 transcripts; sst1 mRNA was present in moderate amounts, whereas sst4 and sst5 were only weakly expressed. E2 had a differential effect on distinct ssts; it increased mRNA concentrations corresponding to sst2 and sst3, and decreased that of sst1. These results indicate that sst2 and sst3 receptors are the major somatostatin receptors expressed in the female rat pituitary, and that both of them are positively regulated by estradiol. However, the capacity of lactotropes to respond to SRIF after exposure to E2 seems to depend more upon E2-induced up-regulation of the sst2 than of the sst3 receptor subtype.

Desensitization of type 1 angiotensin II receptor subtypes in the rat kidney.

Differences involving serine residues in the sequence of the carboxyl-terminal tail of type 1 angiotensin II (Ang II) receptor subtypes AT(1A) and AT(1B) suggest differences in desensitization ability. We examined the Ang II-induced homologous desensitization patterns of both receptor subtypes in freshly isolated renal structures: glomerulus (Glom), afferent arteriole, and cortical thick ascending limb (CTAL), whose content in each subtype mRNA is different, by measuring variations in intracellular calcium concentration. A preexposure to a maximal dose of Ang II, followed by a second application of the same concentration, induced: 1) a complete desensitization in Glom, where AT(1A) and AT(1B) mRNAs were expressed in similar proportions, and 2) no or partial desensitization in afferent arteriole and CTAL, where AT(1A) mRNA was predominant. In the absence of nephron structure containing only AT(1B) mRNA, we studied rat anterior pituitary cells that exhibit high content in this subtype and observed that desensitization was not complete. In Glom, CTAL, and pituitary cells, desensitization proceeded in a dose-dependent manner. In Glom and CTAL, desensitization occurred via a PKC-independent mechanism. These results suggest that desensitization does not depend on the nature of Ang II receptor subtype but either on the proportion of each subtype in a given cell and/or on cell specific type. This could allow adaptive biological responses to Ang II appropriate to the specific function of a given cell type.

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.

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.

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.

Multiple intracellular signallings are involved in thyrotropin-releasing hormone (TRH)-induced c-fos and jun B mRNA levels in clonal prolactin cells.

In mammosomatotropes GH3B6 cells, one of the primary responses to thyrotropin-releasing hormone (TRH) is the parallel induction of two proto-oncogenes, c-fos and jun B, which code for constituents of AP1 transcription factor. To better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signals in the induction of each proto-oncogene on the one hand, and on prolactin (PRL) release and PRL gene expression on the other hand. Northern and dot-blot analyses revealed that the activation of protein kinase C (PKC)-, Ca(2+)- or adenylyl cyclase-dependent pathways acutely increased both c-fos and jun B transcripts. However, a gene specific responsiveness was revealed using phorbol 12-myristate 13-acetate (TPA) and several combined treatments. The simultaneous activation of PKC and Ca(2+)-dependent pathways resulted in synergistic stimulations of c-fos mRNA levels only. Consistently, ionomycin plus low doses of TPA solely reproduced the potent effect of TRH on c-fos transcripts. Data collected from TRH and TPA down-regulated cells indicated that TRH probably recruits TPA-dependent PKC isoforms for stimulating c-fos but not jun B transcripts. On the contrary, the TRH-induced stimulation of either proto-oncogene likely involves Ca(2+)-dependent mechanisms because calcium agonists and the peptide exert non-additive effects. Finally, the synergistic stimulations observed in response to TRH combined with forskolin, indicate that adenylyl cyclase-dependent mechanisms are interconnected with TRH-induced proto-oncogene expression. The overall study also reveals that among the agonists tested, the dihydropyridine Bay K 8644 and forskolin only were capable to induce a long-lasting stimulation of c-fos and jun B mRNA levels, concomitant to increased levels of PRL transcripts, as does TRH. Considering that AP1 is assumed to be involved in signal transmission from the cell surface to the nucleus, it might be thus proposed that a common stimulation of c-fos and jun B gene expression is possibly involved in the activation of the PRL gene. On the other hand, the systematic coincidence between acute PRL release and proto-oncogenes expression suggest a role for c-fos and jun B in the control of genes involved in the secretory process.

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.

Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells.

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17 beta-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC50 (18-59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.

Age-associated changes in hypothalamic and pituitary neuroendocrine gene expression in the rat.

A cDNA membrane array displaying 1183 probes was used to detect hypothalamic and pituitary changes in gene expression accompanying ageing and age-associated pituitary macroadenomas. Four groups of male Sprague-Dawley rats (3-, 15-, 24-month-old and 24-month-old with prolactinoma) were compared in two independent hybridizations. cDNA array data were confirmed and completed by comparative reverse transcriptase-polymerase chain reaction on selected genes. The expression of 454 and 116 mRNAs was detected in hypothalamus and pituitary, respectively. Growth hormone (GH) mRNA alone represented 85% of total gene expression in the gland of young rats, and other pituitary hormone transcripts 2.8%, while melanin-concentrating hormone (MCH) mRNA, the most expressed neuropeptide transcript involved in neuroendocrine regulation, accounted for only 0.8% of total hypothalamic transcripts. The proportion of genes modified in the hypothalamus and pituitary was rather modest: 1.5% and 5.2%, respectively, for ageing per se, and 1.1% and 5.2% for age-associated macroprolactinomas. Among pituitary specific RNAs, GH mRNA expression was notably decreased with age. At the hypothalamic level, expression of genes directly involved in GH regulation, such as somatostatin and growth hormone-releasing hormone, was not altered, while neuropeptide transcripts involved in feeding behaviour [orexin/hypocretin, MCH, pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART)] were significantly altered. In addition, a few ubiquitous transcripts (hnRNP-K, PFKm, CCND 2, calponin and set) were differently affected in both tissues. Modifications in hypothalamic orexigenic (orexin, MCH) and anorexigenic (POMC, CART) gene expression are in keeping with an age-associated decrease in energy consumption but a higher one in the presence of macroprolactinomas.

Multihormonal regulation of the pituitary gland binding and secretory responses to hypothalamic neuropeptides in rat GH pituitary strains in culture.

The parenchyma of the anterior pituitary gland is constituted of distinct specialized glandular cell types, some of them being able to secrete two, or more, genetically related hormones. The synthesis and the release of each hormone is a discontinuous process controlled by a variety of chemical signals of hypothalamic and peripheral origin. These signals, namely neuropeptides, may cross-react at the level of one cell type and each signal may govern the activity of more than one cell type. Such integrated systems hamper the analysis of binding and secretion coupling of regulatory neuropeptides at the cellular and molecular levels. Prolactin (PRL) and growth hormone (GH) rat clonal pituitary tumor-derived cell lines (GH cells) by contrast offer a reductionist model suitable for such investigations on TRH and somatostatin hypophysiotropic neuropeptides and on their hormonal regulation, as reviewed in this paper. The tetradecapeptide somatostatin inhibits similarly PRL and GH release by GH cells. Using (125)I-Tyr-somatostatin, specific binding sites were characterized; their occupation presents dose dependence and stereospecific requirements similar to the induction of the inhibition of hormone release. In cells cultured in serum-supplemented media, TRH regulates in a biphasic manner somatostatin binding sites: TRH acutely increases and chronically decreases their number without altering their affinity. In cells cultured in serum-free media, TRH competes with (125)I-Tyr-somatostatin and displace it with a similar affinity and nearly identical efficiency as unlabelled somatostatin. These effects of TRH are totally prevented by preculturing the cells in a medium supplemented with tri-iodotyronine, which in addition increases somatostatin binding capacity. The tripeptide TRH stimulates similarly the acute release of PRL and GH by GH cells. By contrast, long-term exposure to TRH inversely regulates the production of both hormones, PRL being augmented and GH inhibited. This results from an opposite regulation of the transcription of the rPRL and rGH genes and of the half-life of their respective encoded mRNA, the stimulation of the rPRL gene being rapid and transient. These effects are mediated by the occupation of specific binding sites, extensively studied using (3)H-TRH, displaying the same dose- and time-dependence, as well as stereospecific requirements, as the regulation of hormone secretion. Factor regulating per se the PRL and GH secretion also alter TRH interaction with GH cells. TRH and tri-iodotyronine both down-regulate the number of TRH binding sites while estrogens or glucocorticoids augment them. TRH binding and secretion coupling are not always regulated in parallel. Evidences for plasma membrane located events were reported for TRH, but also for its internalization. Thus the subcellular localization of the initial signal(s) triggering acute hormone release on the one hand and alteration of specific genes transcription on the other hand is still debated.

[Neuropeptides and regulation of various proto-oncogenes in cultured anterior pituitary cells]. / Neuropeptides et régulation de certains proto-oncogènes dans les cellules antéhypophysaires en culture.

The analysis of the characteristics of a number of proto-oncogenes reveals some similarities with the site of action (plasma membrane receptor, nucleus) or the mechanisms of action (coupling protein of the "G" family, transcriptional activity) of neuropeptides and namely hypophysiotropic hypothalamic neuropeptides. The example illustrated in this review concerns the induction of the early nuclear oncogene c. fos by TRH in a prolactin secreting rat pituitary cell line. TRH regulates both, hormone release and biosynthesis, in this cell line. Kinetic and pharmacological studies suggest that the induction of the proto-oncogene mRNA accumulation is rather associated with the TRH-induced release of the stored hormone than with the increased transcriptional activity of the prolactin gene.

[Hypothalamic hypophysiotropic neuropeptide receptors]. / Les récepteurs des neuropeptides hypothalamiques hypophysiotropes.

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.