Corticotropin-releasing hormone and epidermal growth factor: mitogens for anterior pituitary corticotropes.

Anterior pituitary corticotropes increase in number after stimulation by adrenalectomy or corticotropin-releasing hormone (CRH). However, is this brought about by mitoses? Furthermore, as epidermal growth factor (EGF) is a potent secretagogue for corticotropes, is it also a mitogen? To address these questions, populations of corticotropes enriched to 88-97% by counterflow centrifugation were studied after growth in 0-10 nM CRH with and without 0.1-10 ng/ml EGF. Three types of assays were used to detect changes in mitotic cells or cell number. An enzyme immunoassay for bromodeoxyuridine uptake during DNA synthesis [bromodeoxyuridine (BrDU) uptake] detected a 3-fold increase in optical density readings in the presence of 0.5 nM CRH or EGF. Together the peptides increased the optical density to 4.8-fold basal levels. No further increases in BrDU uptake were seen with higher concentrations of CRH or EGF. Cytochemical detection of BrDU uptake by immunolabeled corticotropes showed BrDU in 18 +/- 2% of 3- to 5-day ACTH cells. In the presence of 0.5 nM CRH or 0.5 ng/ml EGF, this value increased to 37 +/- 3% or 34 +/- 2% of ACTH cells, respectively. Together CRH and EGF stimulated increases in mitotic activity so that 47 +/- 4% of the ACTH cells were labeled for BrDU after a 1-h exposure. Cell growth/cell death assays in 3-(4,5-dimethyltiazol-2-yl)2,5- diphenyl tetrazolium bromide were also used to detect changes in overall cell number or cell survival in the same groups of enriched corticotrope cultures. Both 0.5 nM CRH and 0.5 ng/ml EGF caused increases to 1.5- to 1.7-fold basal readings. However, higher concentrations did not stimulate increases in number, and their combined effects were not additive. These studies show that CRH and EGF can be mitogens for ACTH-bearing corticotropes, in a limited dose range. In a higher dose range, their differentiating effects may eliminate dividing cells and retard further growth of the population.

Cells that express luteinizing hormone (LH) and follicle-stimulating hormone (FSH) beta-subunit messenger ribonucleic acids during the estrous cycle: the major contributors contain LH beta, FSH beta, and/or growth hormone.

There is a 2-fold increase in the percentage of gonadotropes bearing LH beta or FSH beta mRNAs or antigens as the cells approach proestrus. The purpose of this study was to identify the source of these cells with dual labeling techniques. The first hypothesis was that they stemmed from small monohormonal gonadotropes (containing only LH or FSH) that were driven to transcribe and translate the other gonadotropin. Alternatively, they may stem from other pituitary cell types. We detected the LH beta or FSH beta mRNAs by in situ hybridization (biotinylated oligonucleotide probes were detected by peroxidase-labeled avidin). Second, immunolabeling protocols localized the pituitary hormones. The percentages of cells with LH beta antigens and FSH beta mRNA increased to 81% of the LH beta antigen-bearing cells by the time of peak expression of FSH beta mRNA. Similarly, FSH beta antigen-bearing cells increased their expression of LH beta mRNA to 40% of such cells by the morning of proestrus. During the peak period of expression (the evening of proestrus), LH antigen-bearing cells had increased their expression of LH beta mRNA to 93%. Furthermore, 81% of the same cells expressed FSH beta mRNA. Thus, at least 80% of cells with LH antigens became bihormonal as the cells approached proestrus. This partially supports the first hypothesis for the origin of the new gonadotropes. However, the dual labeling studies also showed that 47% or 60% of cells with GH antigens expressed LH beta or FSH beta mRNAs, respectively, during peak expression (14% of pituitary cells contained gonadotropin mRNAs and GH antigens). Expression by cells with other antigens was low or absent (< 5% of pituitary cells). Perhaps a subset of somatotropes expresses gonadotropin mRNAs. Alternatively, the labeling could signify the presence of GH bound to GH receptors in gonadotropes. In either case, it appears that GH cells may be somehow linked to gonadotrope function as the cells approach proestrus.

Enrichment of corticotropes by counterflow centrifugation.

Anterior pituitary corticotropes represent only 9-10% of the mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells. The separation was done by counterflow centrifugation in an elutriator containing the Sanderson chamber which was designed to separate small cells under 15 micron in diameter. The corticotropes were initially eluted at flow rates under 30 ml/min along with other cells less than 12.5 micron in diameter. They were then stimulated for 2-4 h with 0.5 nM CRH and reeluted with the use of higher flow rates to separate the enlarged corticotropes from the unstimulated cells. Reelutriation of the entire pool of small cells produced an enrichment to 60% corticotropes in five separate experiments. However, when the pool was divided into smaller cells (eluted at 20 ml/min) and medium-sized cells (eluted at 30 ml/min), and the two pools were reeluted separately, the enrichment increased to over 90% corticotropes in eight separate experiments. These corticotrope populations remained enriched for up to 14 days in culture. They also secreted in a reverse hemolytic plaque assay that recognizes ACTH-(25-39). The dual labels for ACTH and beta-endorphin showed that 60% of the corticotropes stored both peptides, whereas 30% stored only ACTH, and 10% stored only beta-endorphin. No differences in storage patterns were seen when small and medium-sized corticotropes were compared. Thus, these studies present the first report of the production of an enriched fraction of CRH-responsive corticotropes by counterflow centrifugation and the first report of heterogeneous storage of ACTH and beta-endorphin. The use of enriched fractions facilitated the analysis of these heterogeneous storage patterns in over 8000 corticotropes.

Growth and secretory responses of enriched populations of corticotropes.

The purpose of this study was to learn whether enriched populations of corticotropes could be grown without the other pituitary cell types. Corticotropes populations were enriched to 80-90% by counterflow centrifugation in an elutriator with the Sanderson chamber. After initial separation into small, medium, and large fractions, the cells were stimulated for 3 h with 0.5 nM corticotropin-releasing hormone (CRH) and then re-eluted to remove the enlarged corticotropes. More ACTH (6- to 10-fold) was released in media with 10% fetal bovine serum (FBS) than was released in media with no serum. The effects of FBS could not be mimicked by 0.3% BSA. Corticotropes grew in serum-containing media as long as they were plated at a density of at least 2500 cells per well. The corticotropes expanded in size and assumed two major morphological subtypes. Both stored ACTH and beta-endorphin. One subtype was flattened and pleomorphic. The other subtype was stellate with multiple processes. Cell counts showed a 2.5- to 3.8-fold increase in the number of labeled corticotropes during the first 21 days of culture. Then the numbers of cells declined rapidly. Basal secretion of ACTH rose 1.6-fold during the first week, plateaued after 14 days and then declined to less than 30% of first week levels. CRH stimulation produced dose-dependent increases in media ACTH. In 7 day cultures, both basal and stimulated levels of ACTH were similar to those in 7 day cultures of mixed pituitary cells (containing equivalent numbers of corticotropes). Stimulatory effects of CRH were evident for up to 42 days of culture. Arginine vasopressin enhanced CRH-mediated secretion in most cultures in the first week. Pretreatment with glucocorticoids (100 nM corticosterone) for 15 h blocked CRH-mediated secretion in all cultures. The studies showed that corticotropes do not need the other pituitary cell types for basic plating and basal and CRH-mediated secretory responses. Further tests of specific growth factors are needed to learn whether they will maintain function for longer periods.

Cytochemical studies of responses of corticotropes and thyrotropes to cold and novel environment stress.

Cold stress stimulates the release of both ACTH and TSH from the pituitary. More striking changes in ACTH content have been seen in the intermediate lobe after cold stress. Therefore, this study was designed to test responses of individual anterior lobe corticotropes to cold exposure. Male rats were exposed to either 30 min of cold (+3-5 C), 30 min of a novel, temperate environment (+24 C) or were unstressed (+24 C). Pituitaries were fixed and embedded in preparation for immunolabeling for ACTH or TSH-beta at the light (semithin sections) and electron microscopic levels. The semithin sections were used to measure areas of corticotropes and thyrotropes with Bioquant image analysis equipment. Separate groups of pituitaries were dissociated and the cells were cultured for 2 or 15 h. Then the cells were stimulated for 5-10 min with biotinylated analogs of corticotropin-releasing hormone (bio-CRH) or arginine vasopressin (bio-AVP) to detect the target cells cytochemically. A third group of dissociated cells were fixed for immunolabeling for ACTH, 16K fragment of pro-opiomelanocortin, beta-endorphin, or TSH-beta. Cold exposure resulted in a 1-4-fold increase in the levels of serum ACTH over that of unstressed rats. This was correlated with a 40% increase in the percentage of cells that contained 16K fragment and a 30-40% increase in percentages of cells storing ACTH or beta-endorphin. Cold stress also increased the percentage of cells that bound bio-CRH or bio-AVP by 45%. Analyses of semithin sections showed that areas of corticotropes increased by 21% following cold stress. The number of rows of immunolabeled (ACTH) secretion granules also increased in corticotropes from cold-stressed rats. Exposure to a novel environment for 30 min resulted in no significant increase in serum ACTH over that of unstressed rats. There was, however, a 20% increase in percentages of cells that stored 16K fragment, beta-endorphin, or target cells that bound bio-CRH. However, the corticotropes were not significantly larger. Many of the cells exhibited reduced numbers of immunolabeled secretory granules. Other corticotropes resembled those from cold-stressed rats. When TSH cells were studied, their percentages increased from 8 +/- 3% to 15.8 +/- 4% and their areas increased by 22% following exposure to cold. After exposure to a novel environment, percentages of cells that stored TSH-beta increased to 11 +/- 2%, however, no changes in areas of TSH cells were measured. These studies demonstrated that the anterior lobe corticotrope is clearly activated by exposure to both cold and novel environment.(ABSTRACT TRUNCATED AT 400 WORDS)

Heightened secretion by small and medium-sized luteinizing hormone (LH) gonadotropes late in the cycle suggests contributions to the LH surge or possible paracrine interactions.

LH secretion from pituitary cell fractions separated by centrifugal elutriation was compared to learn whether any contributed to the LH surge. After plating (1 h) and stimulation with 0-10 nM [D-Lys6]GnRH (4 h), some fractions secreted levels that were out of proportion to their enrichment. Large cells from proestrous morning rats (3-fold enriched) secreted 4.3 times more LH, and medium-sized fractions (1.5-fold enriched) secreted 2-3.4 times more LH than unseparated cells during estrus or metestrus. Normalized data (nanograms per 20,000 cells) showed that basal levels reflected the enrichment in the fractions. Data were also normalized to nanograms of LH per 1,000 LH gonadotropes to focus on LH cell activity. Basal secretion in unseparated cultures (4-6 ng/ml/1,000 LH cells) was lower than that in small or large LH cells during all stages except proestrus, when small gonadotropes became as active as those in unseparated cultures, and large gonadotropes secreted 2-3 times more LH. Basal secretion from medium-sized gonadotropes was comparable to that in unseparated cultures. [D-Lys6]GnRH-mediated secretion from unseparated, small, and large LH cells was comparable during most stages. However, during proestrus, large gonadotropes secreted 2.2 times more LH than unseparated counterparts. Stimulated medium-sized LH cells were 1.3-2.3 times more active in most stages than those in unseparated cultures and 1.75-2.8 times more active than those in large cell fractions (from proestrous PM to metestrus). Whereas this enhanced secretion late in proestrus suggests that medium-sized LH cells may support the LH surge, it also may reflect the removal of regulatory factors from cells in other fractions. Studies of autocrine or paracrine interactions with gonadotropes are needed to test this hypothesis.

Epidermal growth factor enhances ACTH secretion and expression of POMC mRNA by corticotropes in mixed and enriched cultures.

Previous studies have shown that epidermal growth factor (EGF) stimulates the release of adrenocorticotropin (ACTH) in vivo and in vitro by amplifying the effects of corticotropen-releasing hormone (CRH). The aim of the present studies was to compare responses to EGF by corticotropes in an unseparated culture with those in cultures enriched to 90-93% ACTH-beta-endorphin cells (by counterflow centrifugation). Since EGF binding sites had been identified on growth hormone (GH) or prolactin (PRL) cells (9), the enriched corticotrope cultures were studied to learn if the abundant GH or PRL cells found in unseparated cultures were required to mediate the actions of EGF. In unseparated cultures, EGF (1 or 10 ng/ml) or CRH (1-5 nM) increased the percentages of ACTH cells from 9.5 to 15% and the percentage of cells labeled for POMC mRNA from 7.5 to 12% of the population. In enriched cultures, CRH and EGF increased the percentages of cells labeled for POMC mRNA from 70% to 90-94% of the population. EGF alone stimulated ACTH secretion in both the unseparated and enriched cultures by 1.2- to 2.2-fold. EGF amplified the effects of CRH by 30-40% in both unseparated and enriched cultures. In unseparated cultures grown in serum-free media, however, 1 ng/ml EGF did not amplify the effects of CRH and 10 ng/ml EGF partly abolished the CRH stimulation. In contrast, enriched cultures grown in serum-free media continued to respond to the growth factor. the secretory responses of corticotropes in enriched cultures were similar to those of their counterparts in the unseparated cultures. This indicates that the reduction in numbers of GH and PRL cells in the enriched cultures does not interfere with EGF actions on the corticotrope population.