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C Denef

Publications and source records attributed to C Denef.

At least 55 records · Page 3Linked to original sources
PubMed1992

Luteinizing hormone-releasing hormone and neuropeptide Y influence deoxyribonucleic acid replication in three anterior pituitary cell types. Evidence for mediation by growth factors released from gonadotrophs.

Treatment of anterior pituitary reaggregate cell cultures from 14-day-old female rats with physiological doses of the gonadotropin-releasing hormone LHRH or neuropeptide Y (NPY) for 40 h dose-dependently increased [3H]thymidine ([3H]T) incorporation into DNA of cells expressing PRL immunoreactivity (PRL-ir) and of those expressing ACTH-ir, whereas these peptides decreased the number of [3H]T-labeled cells expressing GH-ir. The effects of NPY were of the same magnitude as those of LHRH. The effects of LHRH were not seen in a gonadotroph-deprived cell population obtained by sequential velocity and buoyant density gradient sedimentation. When the latter cell population was coaggregated with purified gonadotrophs from 14-day-old rats, LHRH did enhance [3H]T labeling of lactotrophs and decreased that of somatotrophs. Gonadotroph-conditioned medium obtained by continuous perifusion of gonadotroph-rich reaggregates contained four different high molecular weight substances mimicking the effects of LHRH and NPY on [3H]T incorporation in the respective pituitary cell types. These substances were partially purified and separated from each other by concentration on a Bond-elut C18-reversed phase cartridge, ultrafiltration, and C18-reversed phase HPLC. One factor stimulated [3H]T labeling of lactotrophs, another that of corticotrophs, and two others inhibited [3H]T labeling of somatotrophs. The present data suggest that the development of PRL-, GH-, and ACTH-containing cells in the pituitary is modulated by LHRH and/or NPY and that the action of LHRH and probably also of NPY is mediated by specific paracrine growth factors released from gonadotrophs.

Adrenocorticotropic Hormone↗
PubMed1992

Evidence that folliculo-stellate cells mediate the inhibitory effect of interferon-gamma on hormone secretion in rat anterior pituitary cell cultures.

Interferon-gamma (IFN-gamma) is known to inhibit the release of ACTH, PRL, and GH by rat anterior pituitary (AP) cells, stimulated by appropriate hypothalamic releasing factors. In the present study we examined the mechanisms underlying this inhibition. Dose-response studies, revealing a maximal inhibitory effect with an IFN-gamma dose as small as 10 U/ml, suggested the existence of a limiting intermediate step. In addition, in perifusion experiments with aggregates of established hormone-secreting tumor cell lines (AtT-20 and GH3), IFN-gamma had no inhibitory effect, suggesting that an accessory cell type was involved. Studies on differentially enriched cell populations of normal rat AP, obtained by velocity and buoyant density sedimentation, indicated that the inhibitory effect of IFN-gamma on stimulated ACTH and GH release was absent in those populations that contained only few folliculo-stellate (FS) cells. The presence of a minimal proportion of FS cells was found to be necessary for the inhibition to be manifest. This was seen in monolayers, but also in cultures of AP cell aggregates, which are well known to closely mimic the behavior of the AP gland in vivo. Definitive evidence for the role of FS cells was obtained by reconstitutive coculture experiments; FS cell-poor populations, which by themselves resisted the inhibitory effect of IFN-gamma, became sensitive when cocultured with an FS cell-rich population. Basal ACTH and GH release were not influenced by preincubation with IFN-gamma in either original or fractionated AP cell populations. In contrast, basal PRL release was inhibited in both systems. In cultures of AP cell populations, separated by velocity sedimentation, this inhibition showed a pattern similar to that observed for stimulated release of ACTH and GH, i.e. more inhibition in fractions with a higher proportion of FS cells. However, in FS cell-poor cultures, inhibition of basal PRL release did occur, although to a lesser degree than in FS cell-rich cultures. Our results indicate that IFN-gamma affects AP hormone secretion through the FS cell. In addition, they suggest that IFN-gamma and the FS cell constitute a system through which the pituitary gland perceives changes in the activation state of the immune system.

Adrenocorticotropic Hormone↗
PubMed1991

Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture.

Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70% +/- 9% (mean +/- S.D.) of these so-called cup-shaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17 beta-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.

Animals↗
PubMed1991

Release of interleukin-6 from anterior pituitary cell aggregates: developmental pattern and modulation by glucocorticoids and forskolin.

The release of the immunologically active cytokine interleukin-6 (IL-6) by cultured anterior pituitary cell aggregates was found to increase with the age of the donor rats. The glucocorticoid hormone dexamethasone (DEX) dose-dependently inhibited the release of IL-6, the IC50 being 2.43 +/- 0.93 nM. The sex steroids dihydrotestosterone and estradiol were without detectable effect. The adenylate cyclase activator forskolin increased IL-6 release in aggregate cultures in the presence of DEX. It is concluded that IL-6 release by anterior pituitary cells is subject to developmental changes and under regulatory control of biologically active substances.

Adenylyl Cyclases↗
PubMed1991

Evidence for the presence of gastrin-releasing peptide immunoreactivity in rat anterior pituitary corticotrophs and lactotrophs, AtT20 cells, and GH3 cells: failure to demonstrate participation in local control of hormone release.

Using antisera raised against the N-terminal (1-16) or the C-terminal part of the bombesin (BBN)-like peptide gastrin-releasing peptide (GRP) and against the C-terminus of pro-GRP, GRP- and pro-GRP-like immunoreactivity (IR) was detected by immunostaining in freshly dispersed rat anterior pituitary (AP) cells and in reaggregate AP cells cultured in serum-free medium. Depending on the antiserum used, 6.4 +/- 0.4% to 12.4 +/- 2.7% of freshly dispersed cells were immunoreactive. Solid phase preabsorption of the antisera with the respective antigens abolished the staining. GRP-IR was detectable by double immunostaining in a subpopulation of PRL cells and ACTH-containing cells. In contrast, only very few somatotrophs, gonadotrophs, and thyrotrophs were immunoreactive, and they were much smaller than the typical mature forms of these cell types. GRP- and pro-GRP-IR were also detected in the ACTH-secreting AtT20 cell line and in the PRL- and GH-secreting GH3 cell line. GRP- and pro-GRP-IR were present in AP cell aggregates maintained in culture for 4 weeks. The finding of both GRP- and pro-GRP-IR in a subpopulation of lactotrophs and corticotrophs and their persistence in culture under serum-free conditions strongly suggest that GRP-like peptides are produced in rat AP. Although the potent GRP receptor antagonist L 686,095-001C002 effectively blocks stimulation of GH and PRL release by exogenous BBN-like peptides, it failed to affect basal GH and PRL release from perifused reaggregate AP cell cultures as well as GH and/or PRL release stimulated by epinephrine, vasoactive intestinal peptide, TRH, GH-releasing factor, and angiotensin-II or PRL release inhibited by dopamine. Thus, the latter data do not support the hypothesis often suggested that peptides endogenously present in AP cells are involved in the paracrine regulation of AP hormone secretion.

Adrenocorticotropic Hormone↗
PubMed1990

New perspectives in the function of pituitary folliculo-stellate cells.

Classical morphological studies of the folliculo-stellate (FS) cells of the anterior pituitary have suggested that these cells play roles as supporting cells, in metabolism and in macromolecular transport. Over the last 10 years the details of their activity in both trophic and catabolic processes has been clarified, and recent work has demonstrated several transport systems in these cells. Various novel peptides with growth factor or cytokine activity have been identified in FS cells and/or FS cell conditioned media. These recent functional experiments confirm and extend previous morphological and experimental studies, and in addition open new perspectives on the physiological roles of FS cells.

Animals↗
PubMed1990

Evidence for a pertussis toxin-sensitive signalling pathway in the dual action of angiotensin II on growth hormone release in pituitary cell aggregates.

In anterior pituitary cell aggregates cultured in the presence of the glucocorticoid dexamethasone (DEX) angiotensin II (AII) had a dual effect on growth hormone (GH) release. The peptide stimulated the release in aggregates from 2-week-old rats, whereas the peptide had an inhibitory effect in cultures from adult rats. Treatment of aggregates from adult rats with pertussis toxin (PT) reversed the inhibitory effect of AII on GH release in a stimulatory effect; PT treatment of aggregates from 18- to 20-day-old rats significantly enhanced the stimulation of GH release by AII. The effect of PT was seen only when DEX was added to the culture medium. The present data suggest that the glucocorticoid-dependent stimulus-effect coupling of AII on GH release involves both a stimulatory and an inhibitory component, the latter being abolished by PT, and that the stimulatory component predominates during immature life while the inhibitory one during adult life.

Age Factors↗
PubMed1990

Stimulation of growth hormone and prolactin release from rat pituitary cell aggregates by bombesin- and ranatensin-like peptides is potentiated by estradiol, 5 alpha-dihydrotestosterone, and dexamethasone.

The effect of the bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-C (NMC), and the ranatensin-like peptides, neuromedin-B (NMB), neuromedin-B30 (NMB30), and neuromedin-B32 (NMB32), on pituitary GH and PRL release was studied in perifused anterior pituitary aggregate cell cultures from 9- to 12-week-old male rats cultured in serum-free defined medium supplemented with 0.05 nM T3 and 4 nM dexamethasone (DEX). All peptides stimulated PRL and GH release. GRP and NMC stimulated hormone release in a concentration-dependent manner between 0.1-10 nM. NMB was slightly more potent than NMB30 and NMB32, but was significantly less potent than GRP and NMC. The magnitude of the PRL response to GRP and NMC inversely correlated with that of the GH response. Cultures with relatively low PRL response levels displayed high GH responses, whereas the opposite was found in cultures with high PRL response levels. The stimulatory actions of GRP, NMC, and NMB were blocked by the bombesin receptor antagonist Leu13 psi (CH2NH) Leu14-bombesin, supporting the specificity of the findings. Addition of 1 nM estradiol (E2) to the culture medium provoked an impressive (4- to 10-fold) increase in the magnitude of the GH response to NMC without changing the EC50 value (0.5 nM). In contrast, E2 significantly decreased the stimulation of GH release by rat GH-releasing factor. In the E2-treated aggregates 3 nM NMC stimulated GH release to a comparable extent as 0.1 nM GRF. 5 alpha-Dihydrotesterone (10 and 100 nM) and DEX (80 nM) also enhanced the GH response to NMC, but to a much smaller extent than E2. E2 had also a stimulatory effect on the PRL response to NMC, particularly in cultures with a low intrinsic PRL response. The PRL response to NMC was decreased by DEX and slightly augmented by 5 alpha-dihydrotestosterone. It is concluded that bombesin- and ranatensin-like peptides have a stimulatory effect on GH and PRL release at the pituitary level. Since their action on GH release is strongly potentiated by E2 and much less so by glucocorticoids, these peptides clearly distinguish their activity and specificity from that of the protagonist releasing factor GH-releasing factor, suggesting a role in sex-related differences in GH release or in the control of GH secretion during sexual maturation.

Animals↗
PubMed1990

Interferon-gamma inhibits stimulated adrenocorticotropin, prolactin, and growth hormone secretion in normal rat anterior pituitary cell cultures.

Preincubation of rat anterior pituitary (AP) cells with homologous interferon-gamma (IFN-gamma) caused a dose-dependent inhibition of ACTH secretion stimulated by CRF. The effect was seen in both monolayer and aggregate AP cell cultures and was not due to cytotoxicity. In monolayer cultures IFN-gamma also inhibited PRL and GH release stimulated by various hypothalamic releasing factors. IFN-gamma did not affect the time kinetics of the ACTH response to CRF. The dose needed for half-maximal inhibition amounted to approximately 1 (antiviral) U/ml. The effect of IFN-gamma was abrogated by an IFN-gamma-neutralizing monoclonal antibody. Furthermore, ACTH secretion by the AP cells was not affected by the anti-IFN-gamma antibody added alone, indicating that in the culture system no endogenous IFN-gamma is operational in regulating the ACTH response studied. Of the other cytokines tested [interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-alpha/beta (IFN-alpha/beta)] only TNF-alpha and IL-6 were found to inhibit CRF-stimulated ACTH release, although this inhibition was less pronounced than that caused by IFN-gamma. Lipopolysaccharide, even at high doses, did not significantly inhibit the ACTH response to CRF. These results identify IFN-gamma as one of the inflammatory cytokines that, like IL-1, TNF-alpha, and IL-6, have the potential to regulate pituitary function.

Adrenocorticotropic Hormone↗
PubMed1990

Regulation and cellular localization of the membrane-bound thyrotropin-releasing hormone-degrading enzyme in primary cultures of neuronal, glial and adenohypophyseal cells.

Using monolayer cultures from murine brain and reaggregate cell cultures of rat anterior pituitary we observed that TRH (pyroGlu-His-Pro-NH2) added to the culture medium was not taken up by these cells but hydrolyzed at the pyroGlu-His bond by an enzyme obviously located at the cell surface. This enzyme exhibited a high degree of substrate specificity and other characteristics of the membrane-bound TRH-degrading enzyme. Relatively high enzymatic activity was associated with cultured neuronal cells from embryonic rat brain while glial cells were almost devoid of this peptidase activity. Rather low, but significant activity was found on anterior pituitary cell aggregates. In agreement with previous in vivo studies we observed that the TRH-degrading ectoenzyme on adenohypophyseal cells was regulated by estradiol and stringently controlled by T3, but that the activity of the brain enzyme was not. When pituitary cells were separated according to their size and density and established in reaggregate cell culture, a close correlation was always observed between enzyme activity and the distribution of lactotrophic cells regardless of the animal models (eu- and hypothyroid adult male rats) used and the cell fractionation techniques (velocity sedimentation and sequential velocity/buoyant density sedimentation) employed. Such a close correlation was not observed with other cell types, such as the somatotrophic cells, the folliculo-stellate cells, the ACTH-producing AtT20 pituitary cells, or thyrotrophic cells. In conclusion, the high degree of substrate specificity, the tissue-specific regulation, and the very heterogeneous distribution of the TRH-degrading ectoenzyme on brain and pituitary cells strongly support the hypothesis that this enzyme serves very specialized functions in the transmission of TRH signals at specific target sites.

Aminopeptidases↗
PubMed1990

Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary.

The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.

Animals↗
PubMed1990

[Paracrine signals in the adenohypophysis].

We have demonstrated that the secretory activity of anterior pituitary cells is not only dependent on the hypothalamic releasing and inhibiting factors and peripheral hormones but that these cells are also capable to control their own function through locally produced paracrine factors. The latter substances are released by certain cell types in the gland and influence the secretory activity and proliferation of other cells in the neighbourhood. We found evidence that gonadotrophs release PRL releasing factors spontaneously as well as after stimulation with LHRH. A cholinergic system was discovered in ACTH cells. Acetylcholine released from these cells exerts a tonic inhibitory action on GH and PRL release. The folliculo-stellate cells, of which the function still remains undefined, induce an overall inhibition of excitability of most if not all hormone secreting cells. We also found new exogenous signals for secretion: a stimulatory action of angiotensin on PRL and GH secretion and of adrenaline and VIP on GH release, dual effects (stimulation and inhibition) of acetylcholine on PRL and GH release and of angiotensin on GH release. Several of the latter signals depend on intercellular communication. An important observation was that paracrine signals are under regulatory control of thyroid and glucocorticoid hormones. All these findings were obtained through the development of new technologies. A three-dimensional aggregate pituitary cell culture system with organotypic characteristics was developed. In order to study the influence of one cell type upon another, the different pituitary cell types had to be separated from each other.

Animals↗
PubMed1989

Interleukin-1 beta inhibits acetylcholine synthesis in the pituitary corticotropic cell line AtT20.

Pituitary corticotropic AtT20 cells were found to synthesize [3H]acetylcholine, when they were incubated with [3H]choline. Treatment of the cells with picomolar concentrations of human interleukin-1 beta inhibited [3H]acetylcholine production. This effect was specific, since two antisera, directed against human interleukin-1 beta neutralized the inhibitory action of interleukin-1 beta. A possible implication of the inhibitory action of interleukin-1 on acetylcholine production in neuronal transmission disorders is discussed.

Acetylcholine↗
PubMed1989

Stimulation and inhibition of prolactin release from rat pituitary lactotrophs by the cholinomimetic carbachol in vitro. Influence of hormonal environment and intercellular contacts.

The prolactin (PRL) response of perifused rat pituitary tissue to the cholinomimetic agent carbachol (CARB) was studied under various in vitro conditions. Perifusion of freshly removed hemipituitaries from 14-day-old rats with CARB did not affect basal PRL release. When established in organ culture for 3 days in a serum-free chemically defined medium, there was a significant increase of PRL release in response to CARB. This PRL releasing activity of CARB depended on the hormonal environment of the culture medium: supplementation of the culture medium with triiodothyronine (T3) and the glucocorticoid dexamethasone (DEX) completely reversed the PRL releasing activity of CARB into an inhibition of PRL release. In dispersed pituitary cells from immature rats, cultured as three-dimensional reaggregates, a similar reciprocal responsiveness to CARB existed which was also determined by T3 and DEX. This reciprocal responsiveness to CARB was preserved in adult female rats but was shifted to a more prominent inhibition in adult male rats. Tumoral PRL secreting GH3 cells, cultured as aggregates, always responded in an inhibitory way, irrespective of the hormonal environment. The expression of the reciprocal responses, in particular of the inhibitory pathway to CARB was dependent on close cellular contacts, as the inhibitory response of normal and tumoral pituitary cells, cultured as isolated cells on Cytodex beads, was completely absent. The stimulatory response of normal lactotrophs, cultured as isolated cells was, although attenuated, still preserved. The present data suggest that there exists a reciprocal responsiveness of normal lactotrophs to cholinomimetics depending on the hormonal environment and close cellular associations. In contrast, only inhibitory PRL responses occur in GH3 tumoral lactotrophs, which are not dependent on thyroid and glucocorticoid hormones.

Animals↗
PubMed1989

Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system.

Reaggregate cell cultures of mouse or rat anterior pituitary were found to produce interleukin-6 (IL-6), a cytokine known for its multiple actions in the immune system. Studies on aggregates prepared from differentially enriched pituitary cell populations revealed the presence of folliculo-stellate (FS) cells to be essential for IL-6 production. Aggregates that contained only hormone-secreting, but no FS cells, failed to produce IL-6. Furthermore, the yield of IL-6 increased with increasing proportions of FS cells present in the aggregates. It is suggested that IL-6 participates in the local regulation of the secretory function of the hypophysis and may constitute a link between events in the immune system and those in the endocrine system.

Animals↗
PubMed1989

Regulatory activity and topological distribution of folliculo-stellate cells in rat anterior pituitary cell aggregates.

An enriched population of cells immunoreactive to antiserum against S-100 protein, a marker of folliculo-stellate (FS) cells in the rat pituitary, was obtained by separation of dispersed pituitary cells from adult female rats by gradient sedimentation at unit gravity. The effect of FS cells on the stimulation and inhibition of prolactin (PRL) and growth hormone (GH) release was studied by coaggregation experiments of the FS cell-enriched population with respectively a lactotroph-enriched and a somatotroph-enriched population from adult female rats. The FS cell population not only attenuated the stimulation of PRL and GH release, but also significantly attenuated the inhibition of PRL release by 10, 30 or 300 nM dopamine (DA), and the inhibition of GH release by 0.1 nM somatostatin (SRIF). The stimulatory action of angiotensin II (AII) on PRL secretion in the presence of DA was also attenuated by the FS cells. Light microscopic evaluation of immunostained semithin sections showed a meshwork of cytoplasmic extensions of FS cells as well as follicular structures in the aggregates. There was no preferential association of FS cells with certain cell types. The permeability of the aggregates to diffusing molecules was tested at the ultrastructural level by the lanthanum hydroxide tracing technique. Lanthanum traced the intercellular gaps over the entire aggregate irrespective of whether the proportional number of FS cells was high or low, indicating that FS cells do not seal off certain areas in the aggregate by the formation of tight junctions. It is suggested that FS cells attenuate the action not only of stimulatory but also inhibitory secretagogues on hormone-secreting pituitary cells. The possible physiological relevance of the present findings is supported by the topological distribution of the FS cells in the aggregates, which closely resembles that of the intact pituitary.

Animals↗