Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Growth hormone acts at a pretranslational level in hepatocyte cultures

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure of both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.I should like to thank Mr. P.F. Hire for his photographic assistance     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

Growth hormone-induced alteration of morphology and tubulin expression in 3T3 preadipose cells

Effects of growth hormone on morphology and cytoskeletal protein expression were examined in 3T3-F442A preadipocytes in serum-free medium. Between 2 and 5 days of culture 2 nM methionyl human growth hormone converted 3T3-F442A cells from a flat fibroblastic morphology to a rounded form with numerous membrane convolutions. Growth hormone treated cultures manifested a 30-40% reduction in cell volume. Growth hormone induced changes in morphology and volume preceded and were independent of lipogenesis. In cells treated with growth hormone, expression of alpha and beta-tubulin as determined by Western blotting was found to increase approximately 50% within 72 h as compared to untreated cells. After 7 days, tubulin levels in growth hormone treated cells were approximately 40% of control levels. This indicated that morphological changes and alteration of tubulin expression were signatures of growth hormone action on 3T3-F442A cells.     

Effect of hormones on growth rates of malignant and nonmalignant human mammary epithelia in cell culture

Summary The individual effects of seven hormones on the in vitro growth rate of different classifications of human mammary epithelium were compared. Hormones used were: 17β-estradiol, estriol, progesterone, hydrocortisone, testosterone, prolactin, and growth hormone. Cell cultures included three established breast cell lines and primary monolayer cultures established form breast fluids and excised mammary tissue from 40 women and 4 men. Specimens comprised three classifications: normal, nonmalignant atypical, and malignant. Growth was quantitated in situ and expressed as population doubling time. Principal findings were: (a) estrogens, prolactin, and growth hormone stimulated growth of normal cells more frequently than growth of malignant cells, whereas testosterone and hydrocortisone stimulated growth of malignant cells more frequently than growth of normal cells; (b) cells cultured from nonmalignant atypias generally showed hormone response profiles intermediate between those of normal and malignant cells; (c) progesterone stimulated the growth of cells from malignant specimens but not the growth of cells from normal and nonmalignant atypical samples. This research was supported by NIAID Research Training Grant 5-TO1-A1-00332-06.     

Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes

An established line of human epidermal keratinocytes was permanently transformed by transfection with plasmid vectors bearing the human growth hormone gene under the control of two different promoters. Suitable selection permitted the isolation of clones secreting abundant growth hormone in culture. When such cultures were grafted to athymic mice, human growth hormone could be detected in the blood of the mice at concentrations in the physiological range for more than 4 weeks. Retrograde transfer from epithelial cells should be studied further as a possible means of introducing exogenous gene products into the circulation of humans.     

Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. Differential effects of hydrocortisone and tissue extracts

Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 µg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1–2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10^-6 M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.     

In vitro short-term effects of SMS 201-995, bromocriptine and TRH on growth hormone cell morphology from human pituitary adenomas

This study reports, by immunocytochemistry, ultrastructure and morphometry, the in vitro effects of SMS 201-995 (10 nM), bromocriptine (1 microM) and TRH (10 microM) on the morphology of cells from two acromegalic patient adenomas containing immunoreactive growth hormone (GH). By electron microscopy, one tumor presented numerous large secretory granules (densely granulated growth hormone cell adenoma) while they were scarce and small in the other (sparsely granulated growth hormone cell adenoma); fibrous bodies could be seen in the specimen and in vitro. In the sparsely granulated growth hormone cell adenoma, TRH produced an increase in endoplasmic reticulum surface density compared to the other cultures. Bromocriptine increased the number and decreased the secretory granule diameters, while SMS 201-995 produced no significant changes in the same time. In the densely granulated growth hormone cell adenoma, the three substances increased the number of granules. TRH increased the mitochondrial volume density and endoplasmic reticulum surface density (with respect to the other cultures). SMS 201-995 decreased the mitochondrial and lysosome volume densities and endoplasmic reticulum surface density. We conclude that 1) TRH produces in cultured cells of both adenoma types an increase in cellular activity. 2) In cultured sparsely granulated growth hormone adenoma cells, bromocriptine has a stronger inhibitory effect than SMS 201-995. In cultured densely granulated growth hormone cells adenoma, bromocriptine has smaller inhibitory effect than SMS 201-995.     

Induction of human choriogonadotropin and follitropin in HeLa cell cultures by hyperosmolality.

The growth of cervical carcinoma cell (HeLa) cultures in a hyperosmolar environment stimulates increased production of the onco-developmental peptides human choriogonadotropin (hCG) and follitropin (FSH). This effect was observed in two sublines examined in this study, HeLa65 and HeLa71. hCG and FSH were measured by radioimmunoassay using antiserum against the beta-subunit of the hormone dimer, thus insuring immunochemical specificity, The amounts of hCG and FSH produced by HeLa65 and HeLa71 cells cultured in hyperosmolar medium were 2- to 50-fold higher than corresponding hormone levels in basal cultures. Synthesis of gonadotropins depended on concentration and duration of exposure to hyperosmolar medium. Levels of culture medium osmolality effective in inducing hormone production also inhibit the incorporation of 14C-thymidine into acid-insoluble macromolecules. Hyperosmolality thus stimulates the ectopic production of gonadotropic hormones while retarding cellular growth and nucleic acid synthesis.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells.

A permanent, clonal strain of rat pituitary tumor cells (GH3-cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (microng extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied. During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]eucine incorporated into rPRL as measured with immunoprecipitation and polyacryl-amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra-cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures. The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase. In spite of the marked variations in basal rPRL and rGH production the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10(-7) M) and 17beta-estradiol (10(-8)M).     

Intercellular communication between cultured granulosa cells of the marmoset (Callithrix jacchus)

Summary The influence of follicle-stimulating hormone, forskolin, insulin-like growth factor type I, epidermal growth factor, and 12-tetradecanoyl-phorbol-13-acetate on marmoset granulosa cell communication via gap junctions was investigated by morphological means and microinjection of carboxyfluorescein. Gap junctions between neighbouring granulosa cells were present in all groups. The number, but not length, of gap junctions between marmoset granulosa cells increased when the cells had been treated with follicle-stimulating hormone, insulin-like growth factor type I, and follicle-stimulating hormone plus insulin-like growth factor type I. No effect on gap junctions was seen, after exposure of the cells to the other three substances. Carboxyfluorescein and counting of the surrounding labelled cells showed that supplementation with follicle-stimulating hormone, forskolin, insulin-like growth factor type I and epidermal growth factor from the beginning of cultivation led to an increase in stained cells after 48 h. When treatment was started in 48 h cultures the substances reached their maximal activity within 30 min (forskolin and epidermal growth factor) or 3 h (follicle-stimulating hormone and insulin-like growth factor type I). Spreading of the fluorescen dye was inhibited when the medium was supplemented with 12-tetradecanoyl-phorbol-13-acetate. This effect was maximal after 30 min. Additive effects regarding the coupling of the cells were seen by combining of epidermal growth factor with follicle-stimulating hormone, but not with insulin-like growth factor type I or forskolin plus follicle-stimulating hormone.     

LHRH inactivation by reconstituted horse and fetal bovine sera: assessment by reduction of immunoreactivity and biological activity in pituitary cell cultures

Lyophilized horse and fetal bovine sera are commonly incorporated into the growth media used for primary pituitary cell cultures. LHRH degrading activity has been assumed to exist in these preparations but has not actually been demonstrated. During our studies with pituitary cultures, it became necessary to ascertain if LHRH inactivating activity could be demonstrated in these sera. Luteinizing hormone releasing hormone (LHRH) was preincubated in either serum-free medium or medium containing fetal bovine and horse serum. Whether LHRH was lost during these incubations was assessed by diminished immunoreactivity as indicated by radioimmunoassay (RIA) and by diminished biological activity as indicated by reduced release of LH from pituitary cell cultures. Both the RIA and bioassay results indicated LHRH inactivating activity; the loss of LHRH could be prevented by inclusion of bacitracin in the incubations.     

Sensitivity of preadipose 3T3 cells to growth hormone

Cultured preadipose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3-F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.     

Secretion kinetics of prolactin and growth hormone by mouse anterior pituitaries in long term organ cultures

Long-term organ cultures of mouse anterior pituitaries provides a system for generating an enriched starting material for the purification of prolactin and growth hormone. Culturing 1 anterior pituitary per ml in serum-free medium for 12 days resulted in a total yield of 40.9 μg and 6.6 μg of prolactin and growth hormone, respectively. This yield represented a 18864 net increase over the control prolactin and growth hormone content in the the unincubated pituitaries. Secondly, culturing of 1–5 anterior pituitaries per ml did not result in a negative feedback autoregulation in the culture system.     

Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling

The morphology and hormone production of pituitary adenoma cell cultures were compared in order to highlight their characteristic in vitro features. Cell suspensions were prepared from 494 surgical specimens. The 319 viable monolayer cultures were analyzed in detail by light microscopy and immunocytochemistry within two weeks of cultivation. Some cultures were further characterized by scanning, transmission and immunogold electron microscopy. The viability and detailed in vitro morphology of adenoma cells were found to be characteristic for the various types of pituitary tumors. The sparsely granulated growth hormone, the corticotroph and the acidophil stem cell adenomas provided the highest ratio of viable cultures. Occasionally, prolonged maintenance of cells resulted in long-term cultures. Furthermore, a variety of particular distributions of different hormone-containing granules were found in several cases. Both light microscopic and ultrastructural analyses proved that the primary cultures of adenoma cells retain their physiological features during in vitro cultivations. Our in vitro findings correlated with the routine histopathological examination. These results prove that monolayer cultures of pituitary adenoma cells can contribute to the correct diagnosis and are valid model systems for various oncological and neuroendocrinological studies.     

Biphasic effects of dexamethasone on growth hormone release in vitro

The effect of dexamethasone (Dex) on growth hormone (GH) release was examined in vitro in monolayer culture of normal rat pituitary cells and human somatotropinoma cells from patients with acromegaly. In either cell strain, Dex, at a concentration of 50 nM initially inhibited, but later (48 less than or equal to h) potentiated, the release of GH into the medium, with or without growth hormone releasing hormone (GHRH). The intracellular GH was significantly increased by 4-hour incubation with Dex in rat cell cultures. These results indicate a biphasic effect of glucocorticoids on GH release, irrespective of the origin of somatotrophs, and that the initial inhibitory effect is probably caused by inhibition of the release.