PACAP is transiently expressed in anterior pituitary gland of rats: in situ hybridization and cell immunoblot assay studies

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.     

Dual effect of osmotic cell swelling on prolactin secretion by acutely dispersed adenohypophyseal cells

Cell swelling induced by acute exposure to the permeant molecule urea or by medium hyposmolarity evoked a prompt PRL secretory burst from dispersed rat anterior pituitary cells. However, during continuous exposure greater than or equal to 10 min to these conditions inhibition of basal and TRH-induced PRL secretion occurred and there was an "off" burst of PRL secretion following return to basal conditions. Compared with continuous TRH stimulation which causes biphasic PRL secretion with a rapid high amplitude first phase secretory burst followed by a sustained low level second phase of secretion, cell swelling induced only "first phase" secretion. Removing Ca2+ from the medium or adding 50 microM verapamil markedly depressed the "off" secretory burst following return to basal conditions but had no effect on the initial high amplitude burst. Our data suggest that the effect of cell swelling on PRL secretion is complex and that there are at least two mechanisms for PRL secretion in normal anterior pituitary cells; these are differently affected by cell swelling and Ca2+ influx.     

Artesunate inhibits cell proliferation and decreases growth hormone synthesis and secretion in GH3 cells

To determine the effect of artesunate (ART) on the rat pituitary adenoma GH3 cell line to evaluate its potential as a novel agent in growth hormone (GH) adenoma and to investigate its underlying mechanisms of action. The MTT assay was used to assess cell proliferation. DAPI staining was used to visualise apoptotic changes in the nucleus. We also analyzed cell apoptosis and cell cycle stage by flow cytometry, semi-quantitative RT-PCR analysis for the expression of GH mRNA and apoptosis-induced factor (AIF) mRNA, analysis of GH protein by western blot, ELISA detection of secreted GH, and the caspase inhibition assay. We found that ART inhibited the proliferation of GH3 cells in a dose- and time-dependent manner, with an IC50 of 9.53 ± 4.12 μM. The IC50s of ART against of two normal cell lines (mouse embryonic fibroblasts, and rat bone mesenchymal cells) were much higher than the IC50 recorded for the GH3 cells. ART induced apoptosis and blocked GH3 at G2/M arrest. The pan caspase inhibitor V-ZAD-FMK partly attenuated the inhibitory effect of ART. ART increased the expression of AIF mRNA and reduced GH mRNA levels, GH synthesis and the secretion of GH level in GH3 cells. ART can inhibit proliferation and induce apoptosis in GH3 cells by caspase-dependent pathways. Additionally, ART can inhibit GH synthesis and secretion. Thus, we propose ART as a probably anti-tumour candidate drug in the treatment of GH adenoma.     

Steroid 5α-reductase type 1 immunolocalized in the anterior pituitary of intact and castrated male rats

The localization of 5α-reductase was immunohistochemically studied in the anterior pituitary of male rats, using a polyclonal antibody against 5α-reductase rat type 1. The immunoreactive cells were concentrated in the central region and on the border of the intermediate lobe in the anterior pituitary, but not in the intermediate or posterior lobe. The immunoreaction was located mostly in the cytoplasm and occasionally in the cell nuclei. The immunoreactive cells showed alterations in size and number and in the intensity of the immunoreaction after gonadectomy. One week after castration, the cells became larger and the immunoreactivity increased. Two weeks after castration, the number of immunoreactive cells increased. Double immunostaining using antiluteinizing hormone β-subunit or anti-follicle stimulating hormone β-subunit antibody revealed that most of the cells containing 5α-reductase were gonadotrophs. Electron microscopically, the immunoreactive cells showed lamelliform rough endoplasmic reticulum and a depletion of secretory granules 1 week after castration. One week later, the rough endoplasmic reticulum was developed and dilated and the number of secretory granules increased. These results suggest that 5α-reductase is located in the gonadotrophs of rat anterior pituitary and that it is involved in the feedback regulation of gonadotropin secretion by androgens.     

A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion

Background  

Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells.     

Regulation of hormonal secretion and DNA synthesis in the lactotrophs of the rat adenohypophysis in vitro in primary cell cultures

Changes in DNA synthesis in lactotrophs of primary monolayer cultures of the rat pituitary cells were studied, using immunoperoxidase staining in combination with autoradiography. Pituitary cell cultures were treated for 3 days with thyroliberin (TRH), bromocriptine (CB154) or somatostatin (SRIF). The proportion of lactotrophs labelled with 3H-thymidine in the total pool of labelled cells served as a criterion for the estimation of DNA synthesis in prolactin-secreting cells. Prolactin secretion by the same cultures was measured by homologous radioimmunoassay. TRH (10 ng/ml) stimulated DNA synthesis in the total population of pituitary cells, but not in lactotrophs. SRIF decreased selectively the proliferation of lactotrophs, but failed to depress or even stimulated DNA synthesis in some cell types of the rat pituitary gland in the cultures. The quantitative method of studying DNA synthesis in anterior pituitary may be used to evaluate the effects of a number of biologically active compounds on various cell systems.     

Ether as an anesthetic for decapitation in the rat: gonadotropin secretion by subsequently established anterior pituitary cell cultures

Historically, for the establishment of dispersed anterior pituitary cell cultures, rodents have been killed by decapitation without anesthesia. Because decapitation fails to induce immediate unconsciousness, the American Veterinary Medical Association Panel on Euthanasia has recently recommended that rodents should not be decapitated without previous anesthesia or sedation. Investigators are therefore confronted with the dilemma of wishing to euthanize rodents humanely yet not wishing to potentially compromise experimental data through the use of anesthetics. We present our observations on the effects of diethyl ether anesthesia administered prior to decapitation on the gonadotropin secretory characteristics exhibited in vitro by cultured rat anterior pituitary cells. Neither light nor complete (surgical level) ether anesthesia had any statistically significant effect on either luteinizing hormone or follicle-stimulating hormone responsiveness or cell content of luteinizing hormone and follicle-stimulating hormone or DNA content. The data indicate that ether anesthesia would appear to be acceptable for those studies involving subsequent in vitro luteinizing hormone and follicle-stimulating hormone secretion.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.     

Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

Short term kinetics of luteinizing hormone secretion studied in dissociated pituitary cells attached to manipulable substrates

With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.     

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

Measurement of peptide secretion and gene expression in the same cell

A combined reverse hemolytic plaque-in situ hybridization assay was developed to allow analysis of the relationship between peptide secretion and gene expression within individual cells. We used the pituitary lactotroph as a model system, but this strategy should be widely applicable. It can be used to test hypotheses regarding if and when peptide secretion and gene expression are coupled in any system in which antibodies to the secreted peptide and probes complementary to the mRNA are available. Using the mRNA hybridization signal to identify certain cell types, this method may also be useful in further studies on the biochemical mechanism of peptide secretion. In addition, questions regarding whether a cell known to secrete a given peptide contains other specific mRNAs and the relationship between these mRNAs and the secretion of the peptide can be studied using this strategy. We found striking heterogeneity among lactotrophs in both gene expression and PRL secretion and a lack of correlation of these parameters within individual lactotrophs under every treatment examined. We also present the first direct visualization and quantitation of the percentage of nonsecreting PRL mRNA-containing cells after estradiol treatment and in the presence or absence of the PRL secretagogue, TRH. Finally, we found that in ovariectomized rats, nonsecreting lactotrophs exhibited significantly higher levels of PRL mRNA than lactotrophs that were actively secreting PRL during the assay.     

The role of flow cytometry in the study of cell growth in the rat anterior pituitary gland

Flow cytometry is a suitable technique for studying in vivo and in vitro the cell cycle kinetics of different animal and human tissues, both in normal and tumoral conditions. The rat anterior pituitary gland is a model to investigate cell growth and replication of differentiated, neuroendocrine cells, and we report current evidence on its cell cycle kinetics as well as on the role played by flow cytometry in this type of study. The proliferation potential of normal anterior pituitary cells is related to a number of different conditions, including heterogeneity of cell types, age and sex of donors, and circadian influences. In addition, the trend of cell proliferation in both in vivo and in vitro studies is similar, suggesting that cultured anterior pituitary elements may, at least in part, retain growth features analogous to those of the intact gland. Sorting of selective cell types and analysis of the relation between proliferating anterior pituitary cells and the light-dark cycle have shown that flow cytometry may be useful to investigate the replication process of the gland. By using a combination of flow cytometry, light microscopic immunocytochemistry and morphometry, we have reported a peculiar trend of proliferation in primary monolayer cultures of rat anterior pituitary gland, characterized by a non-linear reduction in their proliferation rate with advancing age, primarily dependent on a reduced transition of cells from the G0/G1- to the early S-phase pool. These studies indicate that flow cytometry offers insights into cell cycle check points of anterior pituitary cells, and suggest that it might be applied to the study of growth of selective pituitary elements, both in normal and tumoral conditions.     

Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular,luteal or ovariectomized state

Objectives were to determine if neuropeptide Y (NPY) had direct effects GnRH induced secretion of LH from the anterior pituitary gland, and if endogenous steroids modulated the effect of NPY. To accomplish these objectives, 15 Hereford heifers were assigned to one of three ovarian status groups: follicular, luteal, or ovariectomized. One animal from each of the three ovarian status groups was slaughtered on each of 5 days and anterior pituitary gland harvested. Anterior pituitary gland cells within ovarian status were equally distributed and randomly assigned to one of three cell culture treatments: no NPY or GnRH (control), 10 nM GnRH, or 100 nM NPY+10 nM GnRH. Anterior pituitary cell cultures were incubated with or without NPY for 4 h and further incubated for an additional 2 h with or without GnRH and supernatant collected for quantification of LH. Treatment of anterior pituitary cell cultures with GnRH or GnRH+NPY did not affect LH release in cultures obtained from follicular (S.E.=5%; P=0.58) or ovariectomized (S.E.=7%; P=0.22) heifers. Both GnRH and GnRH+NPY increased LH release from anterior pituitary cell cultures from heifers in the luteal phase (S.E.=14%; P < or = 0.05) compared to control cultures. Cultures from luteal phase heifers treated with GnRH did not differ from those treated with GnRH+NPY (P=0.34). These data provide evidence to suggest that effects of NPY on LH release may occur primarily at the level of the hypothalamus.     

Accumulation of secretory granules in pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary 2A8 clonal cells derived from the epithelium of Rathke's pouch of the fetal rat are essentially agranular when grown in vitro, in spite of their active secretion of hormones, i.e., ACTH, prolactin and growth hormone. These cells do not produce detectable amounts of thyrotrophic or gonadotrophic hormones in vitro. When the growth medium (Ham's F10) was supplemented with rat median eminence extract (MEE), l-thyroxin and fresh serum from hypophysectomized rats, some of the 2A8 cells accumulated and stored secretory granules which were characteristic of the cells of the intact pituitary gland. Typical thyrotrophic and gonadotrophic cells also became recognizable and all six anterior pituitary hormones were released into the culture medium. Growth of 2A8 cells in this modified culture medium resulted in an increased production of all hormones with increasing time in culture.These results indicate that the processes that lead to the accumulation of typical mature secretory granules which characterize the individual pituitary cell types are initiated or promoted by some unidentified factor or factors which are present in fresh rat serum. It is also apparent that fresh rat serum can promote the differentiation of gonadotrophs and thyrotrophs in vitro.Supported by USPHS Grant Am 12583 and Institutional Research Grant of The University of Texas Health Science Center at San Antonio, TexasThe authors wish to thank Mrs. Pauline Polette for her skillful technical assistance     

Galanin Inhibits ACTH Releasein Vitroand Can Be Demonstrated Immunocytochemically in Dispersed Corticotrophs

Double labeling of dispersed anterior pituitary cells revealed the coexistence of galanin immunoreactivity with adrenocorticotropic hormone (ACTH) as well as prolactin. However, only laser confocal microscopy showed three different areas of immunoreactivity within the corticotroph cytoplasm, two of them for ACTH and galanin separately and the third containing both immunoreactivities. To determine a possible relation between ACTH cells and galanin, 4-day cultures of anterior pituitary cells from female rats were examined by cell blot assay, and they showed ACTH release inhibition by 10⁻⁶Mgalanin. Furthermore, after 17 β-estradiol treatment to maximize lactotroph galanin releasein vitro,the cell blotting-assessed secretory level of corticotroph cells was very similar to that of cells in the presence of 10⁻⁶Mgalanin. In fact, immunoneutralization with galanin antiserum quenched the inhibitory effect of galanin on ACTH secretion. Our study suggests that locally produced galanin can modulate corticotropin release.     

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone.

While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10^?6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.