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Aminopeptidase B (Ap-B), a ubiquitous enzyme, catalyses the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. The physiological function of Ap-B still remains an open question, even though its activity suggests that it could be involved in inflammatory processes and proliferation of tumor cells. This study was conducted to determine the expression of Ap-B in the developing and adult retina as a path to envisage physiological roles of Ap-B. RT-PCR and in situ hybridization were used to detect expression of Ap-B mRNA and activity tests, Western blotting and immunofluorescence microscopy were performed to identify and localize the enzyme in the rat retina. These biochemical and morphological methods show that Ap-B is expressed in the retina from embryo to adult. Expression level is restricted to specific layers (pigmented epithelium, outer and inner plexiform layers and ganglion cell layer) and is developmentally regulated. Moreover, a preliminary analysis indicates that Ap-B, the glucose transporter GLUT3 and choline acetyltransferase (ChAT) share a similar expression pattern in retina. Altogether, Ap-B appears predominantly expressed in neuronal cells lying in retinal layers containing neuritic extensions and synaptic junctions. Such expression is up-regulated during ontogenesis allowing to hypothesized that Ap-B participates in processes accompanying retinal neuronal cell differentiation.

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.

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.

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.

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 = SRIFBIM-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.

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

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.

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)-, Ca2+- 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 Ca2+-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 Ca2+-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.

The hypothalamic neuropeptide TRH stimulates PRL release and PRL gene transcription in GH3B6 rat pituitary cells. In this model, TRH also raises c-fos proto-oncogene mRNA levels. c-fos is assumed to be involved in the transduction of external signals to the nucleus as a component of AP1 transcription factor, a complex that contains a member of the jun proto-oncogene family. Hence, the main aim of this study was to look for the expression of c-jun, jun B, and jun D proto-encogenes in TRH-stimulated GH3B6 cells, compared to c-fos mRNA levels and PRL secretion. This was performed using serum-starved cells. Northern blotting and dot blotting revealed that jun B mRNA were expressed at noticeable levels in control cells and were strongly stimulated by TRH. This stimulation was dose-dependent and exhibited an early peak and a long-lasting plateau phase stabilized at 4 h, similar to TRH-induced c-fos mRNA induction. c-jun mRNA was expressed at minute levels in control cells and was transiently stimulated by TRH. The jun D message was not detected. TRH-induced jun B and c-fos responses were characteristic of immediate early genes as shown by the superinduction observed under cycloheximide treatment and the total inhibition elicited by actinomycin D. Finally, the responses to serum at 30 min and 4 h revealed differences among the proto-oncogenes studied and indicate that the action of TRH is specific. Altogether, the common biphasic pattern of jun B and c-fos mRNA regulation by the neuropeptide suggests that the heterodimer Jun B/c-Fos could participate in TRH-induced PRL secretion and PRL gene transcription in GH3B6 cells.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteinsor = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Thyroliberin E-H-P-NH4 (TRH) is a small neuropeptide p Glu-His-Pro-NH 2 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-NH 2 (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 3 H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

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, 17β-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.

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

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.

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.

The hypothalamic neuropeptide TRH, which stimulates prolactin (PRL) release and PRL gene transcription, also raises c-fos proto-oncogene mRNA levels in GH3B6 rat pituitary cells. C-fos is assumed to be involved in the transduction of external signals to the nucleus as a component of AP1 transcription factor, a protein complex that contains a member of the jun proto-oncogene family. We have thus looked for the member(s) of the jun family that could be the partner of c-fos in TRH-stimulated GH3B6 cells. The common biphasic pattern of jun B and c-fos mRNA regulation under TRH exposure, i.e., an early peak and a long-lasting plateau phase, suggested that jun B was the best candidate. Then, 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 signalings in the induction of each proto-oncogene. This was done taking as a model that the effects of TRH on PRL release and PRL gene transcription has been previously ascribed to the coupling of the TRH receptor to the activation of both protein kinase C- and calcium-dependent mechanisms. An extensive pharmacological analyses revealed that PKC-, Ca2+ but also protein kinase A-dependent mechanisms are involved in TRH-induced c-fos and jun B mRNA early responses in GH3B6 cells. The overall study also revealed specific features in the control by TRH of each proto-oncogene by some intracellular messengers. Finally, considering the fact that second long lasting phase of proto-oncogene expression was found associated with increased PRL mRNA accumulation whatever the stimulus, it might be proposed that AP1 [c-Fos/Jun B] factor could be involved in the regulation of PRL gene expression. Such hypothesis was furthermore supported by preliminary gel-shift experiments. Nevertheless, in view of the systematic coincidence between acute PRL release and early proto-oncogene induction, a role for c-fos and jun B in the control of genes involved in the secretory process might also be suggested.

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

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.

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.

In this study we show that the electrophysiological properties of a clonal line of prolactin secreting (PRL) rat pituitary cells (GH3/B6) are altered by local application of γ-aminobutyric acid (GABA). The effects of GABA on these cells are: 1) decrease in membrane conductance and 2) hyperpolarization of 5 to 10 mV. When the cells were spontaneously active, GABA reduced and usually arrested action potential firing. This effect was completely reversible. No desensitization of GABA effects was observed even after several applications. The reversal potential of the GABA induced responses was found to be near — 40 mV. Pharmacological studies were performed in order to assess the specificity of the response to GABA and to attempt to characterize the ionic mechanism involved. GABAergic receptor antagonists such as picrotoxin and bicuculline prevented the effect of GABA, whereas dopaminergic receptor antagonists, such as haloperidol or chlorpromazine had no effect. Furosemide, known as a blocker of chloride ion transport in a number of systems, competed with GABA. This substance induced a response similar to that observed with GABA and reduced the effect of GABA when administered before GABA. This study demonstrates a direct and specific effect of GABA on the electrical activity of a tumoral line of rat pituitary cells. Since this electrical activity has previously been shown to be calcium dependent and involved in the secretion of PRL by these cells, the data presented here suggest GABA as an inhibitory regulator of PRL secretion directly at the pituitary level.

The in vivo internalization of thyrotropin-releasing hormone (TRH) was studied by using a semiquantitative immunoelectron microscopic method. Pituitary glands of normal male rats or of 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 (1) was still restricted to thyrotropes and prolactin cells, (2) increased with time elapsed after injection up to 15–30 min and then returned to basal intensity in cytoplasm, secretory granules, and nucleus, and (3) became very frequent at the plasma membrane. These data (1) provide evidence for endogenous TRH within thyrotropes and prolactin cells, i.e., in physiological target cells for TRH, and (2) 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.

GH3 cells which had been exposed for 30 min to [3H]-TRH and had performed their maximum biological response, i.e. increase of prolactin (PRL) release, retained intracellular radioactivity which consisted of chemically unmodified TRH (>93%). Such preloaded GH3 cells were found able to spontaneously release into the culture medium a radioactive material [3H]-RM) which was analyzed. The kinetics of binding of [3H]-RM to intact GH3 cells and competition with unlabeled TRH were indistinguishable from [3H]-TRH. [3H]-RM was able to stimulate PRL release from GH3 cells. In addition thin layer electrophoresis (TLE) revealed that 90% of [3H]-RM migrated like TRH reference. All these features strongly suggest that [3H]-RM is identical to [3H]-TRH.

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.

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.

A direct fluorination of the imidazole side chain of thyroliberin using a photochemical method is described. Preparation of 5-fluoro-imidazole-thyroliberin and 2-fluoro-imidazole-thyroliberin was achieved by successive diazocoupling, amination and fluorination. Specifically substituted derivatives were purified and identified at each step. 5-fluoro-imidazole-thyroliberin was found as active as TRF on short-term prolactin release in GH3 cell lines and more active (4.5 times) than the parental peptide on long-term experiments. 2-fluoro-imidazole-thyroliberin biological activity test shall be delayed since its chemical stability is not known precisely.

[3H]-Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and n

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17β-estradiol (E 2 ) 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 × 10 −8 M E 2 . The binding of 3 H-TRH (30 min at 37°C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E 3 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 ED 50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E 2 on the increase in number of binding sites but respected its potentiating effect on prolactin release

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.

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 125I-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 125I-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 3H-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.

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.

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.

Three different aspects of the control of prolactin secretion in birds are discussed. 1. (1) The problem of autonomy of prolactin secretion in birds. Four bird species have been examined in this respect. The analysis of results obtained from long-term culture experiments and from cytological studies on cultured or autografted adenohypophysis leads to the conclusion that there is autonomous prolactin secretion in the Pekin duck, but not in the pigeon, and probably not in the tricolored blackbird and the quail. 2. (2) The in vitro evidence and modalities of action of the prolactin-stimulating factor(PSF). The existence of a prolactin-stimulating factor in bird hypothalamic extracts appears as a general fact consistently observed in the 6 species examined. By long-term experiments it was shown that PSF stimulates both synthesis and release of prolactin and that increased KCl concentration does not exert the same degree of stimulation. In short-term incubation experiments, it appears that the effects of the PSF depend on the initial prolactin content of the pituitary. If the latter is high, the PSF acts more on release, but if it is low, the most conspicuous effect observed was on synthesis. The PSF content of the hypothalamus does not vary to the same extent as the pituitary prolactin level in the duck, quail, and turkey, but the contrary was observed in the pigeon. 3. (3) The evidence for additional hormonal control of prolactin cells. In the Pekin duck and the quail, testosterone (0.5 μg/ml) inhibits prolactin release and induces appearance of lysosomes which could participate in the intracellular degradation of secretory product.

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.

It was previously shown that after a 30 min. incubation with [3H]-TRH intact GH3 cells have already performed their optimal precocious biological response to TRH (i.e increase of prolactin release) and that chemically unmodified ( greater than or equal to 93%) [3H]-TRH entered the cells and remained associated to them. The present work demonstrates that such cells spontaneously release a radioactive material ([3H]RM) when post-incubated at 37 degrees C. The binding to GH3 cells (time course and competition with unlabelled TRH) as well as the biological effect on prolactin release and the electrophoretic mobility of the [3H]-RM released in one hour were compared to those of [3H]-TRH. According to those criteria [3H]-RM is undistinguishable from [3H]-TRH at 90%. Our data, therefore, support the hypothesis of a continuous flow of TRH molecules on both sides of the plasma membrane during TRH-GH3 cells interaction.

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).

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.

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.

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 effects of sex steroids on the central nervous system are commonly explained according to the general scheme of steroid hormone action involving the nucleus following binding to a cytoplasmic receptor and translocation to the nucleus. However, since estrogen acts on brain and pituitary with latencies ranging from seconds and minutes to hours and days, it is unlikely that a single mechanism of action is involved. At the hypothalamic level, the short latency effect of sex steroid has been reported on the electrical activity of hypothalamic neurons (Kelly et al., 1976; Dufy et al., 1976), which is hardly compatible with the latencies required by genomic activation. Moreover, action potentials are not only a property of neurons, but also are detected in endocrine cells including normal (Taraskevich and Douglas, 1977) and clonal pituitary cells (Kidokoro, 1975), where they have been related to the secretory process.