Biomedical subjects

C Denef

Publications and source records attributed to C Denef.

At least 19 recordsLinked to original sources

PubMed1995

Gonadotropin-releasing hormone influences the release of prolactin and growth hormone from intact rat pituitary in vitro during a limited period in neonatal life.

Freshly isolated pituitaries from female rats between the day of birth and 14 days were challenged with GnRH in a perifusion system. At the day of birth GnRH significantly stimulated GH release but had no effect on PRL release, whereas PRL secretion was strongly stimulated by TRH. GnRH-stimulated PRL release was seen from day 3 up to about day 9 after birth, with a maximum in the magnitude of the response on day 5. GH release showed also a maximal response to GnRH on day 5. However, although stimulated PRL release declined rapidly at the end of the GnRH pulse, GH release did not and even increased over a period of about 20 min. The latter secretion dynamics are consistent with previous observations in reaggregate cell cultures that the GnRH effect on GH release is dual (stimulation and inhibition). The poststimulus rise of GH release was most pronounced with pituitaries of 7- and 9-day-old rats. Finally, in pituitaries of 14-day-old rats the effect on GH release was largely diminished. The present data show that GnRH affects the secretion of lactotrophs and somatotrophs in intact rat pituitary glands only during a restricted period of early postnatal life in a cell-type specific manner and shed new light in establishing the role of GnRH in modulating PRL and GH release.

Animals↗

PubMed1995

Interaction of alpha T3-1 cells with lactotropes and somatotropes of normal pituitary in vitro.

A pituitary cell population of 14-day-old female rats, containing lactotropes and somatotropes but deprived of gonadotropes, was prepared by unit gravity sedimentation through a serum albumin gradient and allowed to reaggregate either as such or after mixing this population with cells of the gonadotropic alpha T3-1 cell line. In a perifusion system gonadotropin-releasing hormone (GnRH) had no effect on prolactin (PRL) and growth hormone (GH) release in the former aggregates but stimulated PRL release in the latter. In the alpha T3-1 cell-containing aggregates GnRH showed a biphasic effect on GH release: inhibition during exposure to GnRH followed by a rebound secretion upon removal of the peptide. The aggregation capacity of alpha T3-1 cells with the normal pituitary cells was demonstrated by using an alpha T3-1 cell clone stably transfected with the reporter gene beta-galactosidase. Perifusion of the gonadotrope-deprived aggregates with medium conditioned by alpha T3-1 cell provoked a rapid stimulation of PRL release and a biphasic effect on GH release. Medium conditioned by the corticotropic cell line AtT20 also stimulated PRL release but had no concomitant effect on GH release. Medium conditioned by alpha T3-1 cells, when added for 40 h to aggregates of 14-day-old rat pituitary, provoked an increase in the number of 3H-thymidine (3H-T)-labelled lactotropes and a decreased in the number of 3H-T-labelled somatotropes. The conditioned medium was concentrated on Sep-Pak C18 and ultrafiltrated through an Amicon membrane with 3-kD molecular weight cut-off and the retained molecules separated by reversed-phase HPLC. The material stimulating 3H-T labelling of lactotropes eluted from the column with a different retention time than material inhibiting 3H-T labelling of somatotropes, suggesting that the effect on lactotropes is mediated by (a) molecule(s) different from that affecting somatotropes. The effects of alpha T3-1 cells and their secretion products on lactotropes and somatotropes were comparable to those we previously observed using enriched populations of normal gonadotropes. The HPLC elution profiles of the substances affecting 3H-T incorporation as well as the specificity of these effects were also similar to that of the substances isolated previously from gonadotrope-conditioned medium. The present data, therefore, support previous conclusions on the paracrine control of the lactotrope/somatotrope lineage by the gonadotropes. Further purification and chemical characterization of the growth factors with selective action on lactotropes and somatotropes may lead to a better understanding of the development of the latter lineage.

Analysis of Variance↗

PubMed1995

Production of transferrin-like immunoreactivity by rat anterior pituitary and intermediate lobe.

Using a specific anti-rat transferrin (Tf) antiserum, Tf-like immunoreactivity (Tf-lir) was detected by immunostaining in intact rat pituitaries and in reaggregated pituitary cells cultured in serum-free medium. Tf-lir cells were present in the anterior pituitary (AP), and in the intermediate (IL) and neural lobes (NL). In the AP, Tf-lir cells were oval or polygonal. An unusual topographical distribution was found. Tf-lir cells mainly occurred as dense clusters in the lateral wings. In the central part of the AP, Tf-lir was found in flattened perisinusoidal cells. Double immunostaining for Tf and the different pituitary hormones showed that Tf-lir co-localized with some gonadotrophs and somatotrophs (7% and 3% of Tf-lir cells, respectively, in typical sections). No co-localization was seen with PRL, ACTH, TSH, or alpha-MSH. The distribution of Tf-lir cells and their cell shape completely differed from that of S-100-positive cells in the AP. In the IL, clusters of large stellate Tf-lir cells were found. Again, their distribution completely differed from S-100-positive cells. In the NL, diffuse staining was found. Double immunostaining of paraffin-embedded sections of reaggregate cell cultures of the AP did not reveal any co-localization of Tf-lir with ACTH, alpha-MSH, LH, FSH, TSH, GH, or PRL. In aggregates consisting of NL + IL cells, Tf-lir was located in clusters: no co-localization with ACTH or alpha-MSH could be demonstrated. Reaggregate cell cultures of AP and NL + IL secreted Tf-lir as measured by radioimmunoassay, at least during 21 days of culture. After metabolic labeling with [35S]-methionine and immunoprecipitation of [35S]-methionine-labeled material present in the culture medium of both AP and NL + IL aggregates with anti-Tf antiserum, a 35S-labeled substance was found, which on SDS-PAGE showed an apparent M(r) of approximately 78 KD, corresponding to the M(r) of rat Tf. The present data show that a specific population of cells of rat anterior pituitary is capable of synthesizing, storing, and secreting transferrin or a substance closely related to it. Cells different from melanotrophs and S-100 cells in the IL, as well as pituicytes in the NL, also appear to produce this material. We suggest that transferrin or a transferrin-like substance may have a local role in the transport of iron or other metals or may play a role as growth factor in the three lobes of the pituitary gland.

Animals↗

PubMed1995

Interaction of LHRH with growth hormone-releasing factor-dependent and -independent postnatal development of somatotrophs in rat pituitary cell aggregates.

Addition of LHRH for 40 h to aggregate cell cultures of 14-day-old rat pituitary significantly decreased the number of [3H]thymidine ([3H]T)-incorporating cells which immunostained for GH protein as well as the number of [3H]T-labelled cells expressing GH mRNA detectable by in situ hybridization with a digoxigenin-labelled riboprobe. The effect at the level of GH protein was seen at a dose of 1 nM LHRH. However, the effect at the GH mRNA level required a higher dose of LHRH (10 nM) or a longer incubation time (64 h). Treatment of the cultures for 40 h with 0.1 nM GH-releasing factor (GRF) provoked a 54% increase in the number of [3H]T-labelled cells containing GH mRNA and a 30% increase in the number of cells immunostained for GH protein. The latter effects of GRF were completely blocked by simultaneous addition of LHRH (1 nM) to the cultures. In the absence of GRF, LHRH (1 nM) also had an inhibitory effect on the total number of cells containing GH mRNA and a comparable effect on the total number of cells stained for GH protein. The present data show that LHRH is capable of inhibiting the GRF-independent as well as the GRF-dependent development of somatotrophs in postnatal rat pituitary in culture.

Animals↗

PubMed1994

Modulation of epidermal growth factor receptor binding and action by N-acetyl-TGF alpha (34-43) methyl ester.

Controversial data have been reported concerning the ability of N-acetyl-TGF alpha (34-43) methyl ester (TGF alpha A) to antagonize the mitogenic effect or the receptor binding of TGF alpha and EGF. We reinvestigated the effect of TGF alpha A on various EGF-sensitive cell lines and in reaggregate and monolayer cell cultures of rat anterior pituitary. In reaggregate pituitary cell cultures, EGF dose-dependently increased [3H]thymidine ([3H]T) incorporation in lactotrophs and decreased [3H]T incorporation in somatotrophs. TGF alpha A (5 microM) completely blocked the effect of 0.1 nM EGF on both lactotrophs and somatotrophs. It had no intrinsic effect on its own. However, depending on the batch of [125I]EGF used, a dose of 5-10 microM TGF alpha A did not or only partially inhibited the binding of 3 x 10(-10) M [125I]EGF to pituitary monolayer cell cultures. In the EGF-sensitive cell lines A-431, BS-C-1, NRK-52E, and pituitary GH3 cells, none of the EGF effects were antagonized by TGF alpha A. On the contrary, the EGF effect was slightly augmented in A-431 cells. In GH3 cells TGF alpha A was clearly mitogenic on its own. It is concluded that, although TGF alpha A negatively interacts with EGF action and binding in normal pituitary, this is most likely not due to a direct competition with the binding of EGF.

Animals↗

PubMed1994

Autoradiographic demonstration of 125I-Tyr4-bombesin binding sites on rat anterior pituitary cells.

Because several direct effects of bombesin related peptides on pituitary hormone release have been demonstrated, we chose to study the presence of bombesin binding sites in the adult male rat anterior pituitary in aggregate cell culture by autoradiographic localization of 125I-tyrosine4-bombesin (125I-Tyr4-BBN) binding and immunocytochemical localization of the anterior pituitary hormones. When aggregates were cultured in medium without hormonal supplements, the number of cells with detectable 125I-Tyr4-BBN binding was below 1%. In cell aggregates cultured in the presence of 1 nM estradiol (E2) 125I-Tyr4-BBN binding was detected on 5.4 +/- 0.8% of the cells after redispersion and on 5.8 +/- 1.1% of the cells in sections of paraffin embedded aggregates. The binding cell types were mainly lactotrophs and somatotrophs. The binding of 125I-Tyr4-BBN (3 or 5 nM) was specific because it was inhibited by the addition of an excess of unlabelled Tyr4-BBN or the bombesin receptor antagonist L686,095. In aggregates cultured in the presence of 1 nM E2 and 4 nM dex, the percentage of cells with detectable 125I-Tyr4-BBN binding was significantly lower than that in aggregates cultured in the presence of 1 nM E2 alone. Binding on somatotrophes almost completely disappeared. The present data show that specific 125I-Tyr4-BBN binding sites are present on anterior pituitary cells, are detectable mainly on a small subpopulation of lactotrophs and somatotrophs, and are affected by hormonal conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Isolation of two peptides from rat gonadotroph-conditioned medium displaying an amino acid sequence identical to fragments of secretogranin II.

Pituitary cells from 14-day-old rats were separated by unit gravity velocity sedimentation, and a highly enriched population of gonadotrophs was established in reaggregate cell culture in serum-free and serum albumin-free defined culture media. The medium conditioned by these aggregates was concentrated, ultrafiltrated, and the concentrated substances consecutively separated by two reversed-phase HPLC steps, a gel filtration step and an additional reversed-phase purification step. Two peptides could be isolated that displayed an N-terminal amino acid sequence identical to fragments of rat presecretogranin II: one was cleaved at the dibasic amino acid residues K182-R183 and the other at the dibasic amino acid residues K569-R570. The latter peptide was completely purified and separated into three variants with the same N-terminal amino acid sequence. From an analysis by electrospray ionization mass spectrometry, it was deduced that the three forms extended up to amino acid residue 612 (A), which is, in presecretogranin II, also flanked by two basic amino acids (K613R614). Two variant forms had a molecular mass that was 17 Da higher. The present data support the hypothesis that secretogranin II is a precursor of putative regulatory peptides.

Amino Acid Sequence↗

PubMed1994

Bioactive peptides in anterior pituitary cells.

The anterior pituitary (AP) has been shown to contain a wide variety of bioactive peptides: brain-gut peptides, growth factors, hypothalamic releasing factors, posterior lobe peptides, opioids, and various other peptides. The localization of most of these peptides was first established by immunocytochemical methods and some of the peptides were localized in identified cell types. Although intracellular localization of a peptide may be the consequence of internalization from the plasma compartment, there is evidence for local synthesis of most of these peptides in the AP based on the identification of their messenger-RNA (mRNA). In several cases the release of the peptide from the AP cell has been shown and regulation of synthesis, storage and release have also been described. Because the amount of most of the AP peptides is very low (except for POMC peptides and galanin), endocrine functions are not expected. There is more evidence for paracrine, autocrine, or intracrine roles in growth, differentiation, and regeneration, or in the control of hormone release. To demonstrate such functions, in vitro AP experiments have been designed to avoid the interference of hypothalamic or peripheral hormones. The strategy is first to show a direct effect of the peptide after adding it to the in vitro system and, secondly, to explore if the endogenous AP peptide has a similar action by using blockers of peptide receptors or antisera immunoneutralizing the peptide.

Animals↗

PubMed1992

Isolation of cleaved prolactin variants that stimulate DNA synthesis in specific cell types in rat pituitary cell aggregates in culture.

Using an affinity column of monoclonal antibodies against rat prolactin (PRL), PRL immunoreactive material secreted by rat pituitary aggregate cell cultures was purified in milligram amounts from the culture medium. Further purification of the PRL immunoreactive molecules was achieved by anion-exchange and reversed-phase h.p.l.c. At least four variants could be distinguished which showed PRL-like bioactivity in the Nb2 lymphoma bioassay. On SDS/PAGE two variants migrated as single bands under both reducing and non-reducing conditions. The two other variants migrated as single bands under non-reducing conditions, but yielded under reducing conditions two fragments, the larger of which had molecular masses of approx. 16 and 17 kDa respectively. These variants were therefore considered to be cleaved PRL (clPRL) variants, and were designated clPRL-1 and clPRL-2 respectively. These variants were not artefactually produced at low pH during the h.p.l.c. purification step. Each of the clPRLs represented about 0.6-1.0% of total PRL. The cleavage site of clPRL-1, determined by N-terminal and C-terminal amino acid sequence analysis, was in the large disulphide loop between Tyr-145 and Leu-146. Purified clPRL-1 added to pituitary aggregate cell cultures from 14-day-old female rats in the presence of the dopamine agonist bromocriptine increased [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs, but not in other pituitary cell types. Under the same experimental conditions the main forms of PRL and clPRL-2 were inactive. These data demonstrate that PRL is cleaved within the pituitary gland between specific amino acid residues, and that PRL cleaved between Tyr-145 and Leu-146 may have a specific biological role as a paracrine growth regulator in this tissue.

Amino Acid Sequence↗

PubMed1992

Negative regulation by dexamethasone of the potentiation of neuromedin C-induced growth hormone and prolactin release by estradiol in anterior pituitary cell aggregates.

The bombesin-like peptide neuromedin C (NMC) stimulated the release of growth hormone (GH) and prolactin (PRL) from adult male rat anterior pituitary cell aggregates cultured in serum-free medium. This effect, particularly on GH release, was significantly increased when the cells were cultured in the presence of estradiol (E2). The GH response to NMC was not affected by 4 nM dexamethasone (Dex) but was enhanced in the presence of 80 nM Dex. However, the strong stimulatory action of E2 on the GH response to NMC was completely abolished by 4 nM Dex. In contrast, the PRL response to NMC was reduced by 4 nM Dex and this to a proportionally similar extent in the presence or absence of E2. The present data show that glucocorticoids can be both positive and negative regulators of the same response system.

Animals↗

PubMed1992

Stimulation of prolactin secretion from rat pituitary by luteinizing hormone-releasing hormone: evidence against mediation by angiotensin II acting through a (Sar1-Ala8)-angiotensin II-sensitive receptor.

In aggregate cell cultures of 15- to 20-day-old rat pituitary maintained in serum-free medium, luteinizing hormone-releasing hormone (LHRH) (10 nM) stimulated prolactin (PRL) release, confirming our previous results and those of others with serum-supplemented medium. Since angiotensin II (AII) stimulates PRL release and a renin-angiotensin system is expressed in gonadotrophs, LHRH stimulation of PRL release might be mediated by AII. To evaluate this hypothesis, the influence of (Sar1,Ala8)AII and (Sar1,Ile8)AII two peptide AII receptor antagonists, of DUP753, a nonpeptide and stable AII receptor antagonist, of a converting enzyme inhibitor, and of angiotensinogen on LHRH-induced PRL release was tested in various in vitro conditions of 15- to 20-day-old female rat pituitary. In aggregates maintained in serum-free medium with or without dexamethasone (DEX) and triiodothyronine (T3), or maintained in serum-supplemented medium, the effect of LHRH on PRL release was not affected by (Sar1Ala8)AII (0.1 microM), (Sar1,Ile8)AII (10 microM) or DUP753 (10 microM). Only a high dose (10 microM) of (Sar1,Ala8)AII attenuated the LHRH-induced PRL release. The latter attenuation was seen only with aggregates cultured in the DEX/T3 medium and not with aggregates cultured in the presence of serum. A dose of 1 or 10 nM (Sar1,Ala8)AII also failed to block the effect of LHRH used at 1 nM. In contrast, (Sar1,Ala8)AII dose dependently as well as DUP753 (10 microM) abolished the AII-induced PRL release. (Sar1,Ala8)AII also failed to affect the LHRH-induced PRL release in pituitary cell aggregates from 6-week-old male rats. However, in aggregates from both immature and 6-week-old rats, (Sar1,Ala8)AII provoked a small and statistically significant attenuation of the LHRH-induced PRL release when a 100 nM dose of LHRH was used. In freshly isolated hemipituitaries from 5-day-old rats, (Sar1,Ala8)AII (1 or 10 microM) did not affect the LHRH- (10 nM) induced PRL release. In single cells obtained by redispersion of aggregates and mounted in a Biogel P2 column, LHRH still stimulated PRL release. Again this effect could not be blocked by DUP753. Treatment of aggregate cell cultures with the angiotensin-converting enzyme inhibitor captopril or with angiotensinogen did not alter the LHRH-induced PRL release. It is concluded that AII is not the paracrine factor mediating the effect of LHRH at low nanomolar doses on PRL release, at least not through the classical AII receptor. The involvement of AII acting on a non-(Sar1,Ala8)AII-sensitive receptor cannot be excluded and warrants further investigation.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1992

Angiotensin II is retained in gonadotrophs of pituitary cell aggregates cultured in serum-free medium but does not mimic the effects of exogenous angiotensins and luteinizing-hormone-releasing hormone on growth hormone release.

Angiotensin II (AII)-like immunoreactivity (LIR) was detected by immunostaining in 7.5 +/- 1.1% of cells obtained by redispersion of pituitary cell aggregates from 15- to 20-day-old female rats, cultured for 5-7 days in serum-free medium supplemented with thyroid hormone and dexamethasone. Also, renin-LIR was retained in these cultures. As shown by double immunostaining of paraffin-embedded sections of the aggregates, this AII-LIR was localized only in gonadotrophs. AII-LIR was detected at least up to 5 weeks in culture. On reversed-phase, high-performance liquid chromatography (HPLC), this AII-LIR co-migrated with authentic AII. In perifused aggregate cell cultures of 15- to 20-day-old female rat pituitary maintained in serum-free medium supplemented with dexamethasone (DEX) and triiodothyronine (T3), AII stimulated GH release. AI and AIII had a similar effect. To evaluate the possible involvement of endogenous AII in the local regulation of GH release, gonadotrophs were stimulated with luteinizing hormone-releasing hormone (LHRH). LHRH displayed a transient inhibitory effect on GH release, which was followed by a rebound of GH release after withdrawal of the peptide. Treatment of aggregates with pertussis toxin reversed this inhibitory effect into a significant stimulation of GH release. In aggregates cultured in serum-supplemented medium, LHRH provoked a significant stimulation of GH release which was still followed by a post-stimulus rebound release. In hemipituitaries from 5-day-old rats, a significant stimulatory effect of LHRH on GH release was found without rebound secretion. To evaluate the possible involvement of endogenous AII in the effects of LHRH on GH release, the influence of (Sar1,Ala8)AII, a peptide AII receptor antagonist, and of DUP753, a non-peptide AII receptor blocker was tested in various in vitro conditions. The effect of LHRH on GH release in aggregates cultured either in serum-free medium supplemented with DEX and T3 or in serum-supplemented medium was not affected by (Sar1,Ala8)AII, not even after enhancing the LHRH-induced GH release by treatment of the aggregates with pertussis toxin. A hundred times lower concentration of (Sar1,Ala8)AII, however, abolished the AII-induced changes in GH release. Also DUP753 (10 microM) failed to block LHRH-induced GH release in aggregates. (Sar1,Ala8)AII also failed to block the effect of LHRH on GH release from hemipituitaries. It is concluded that LHRH has inhibitory and stimulatory effects on GH release in cultured pituitary cell aggregates.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin II↗

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↗