Biomedical subjects

C Denef

Publications and source records attributed to C Denef.

At least 19 recordsLinked to original sources

PubMed1999

Detection of melanocortin-3 receptor mRNA in immature rat pituitary: functional relation to gamma3-MSH-induced changes in intracellular Ca2+ concentration?

We have previously shown that gamma3-MSH stimulates DNA replication in lactotrophs, somatotrophs and thyrotrophs of early postnatal rat pituitary in culture. Since the melanocortin-3 (MC-3) receptor is the only known receptor displaying high affinity for gamma3-MSH, the present study explored whether mRNA of the latter receptor is present in the pituitary and whether the receptor is functional. RT-PCR of RNA extracts from 14-day-old rat pituitary revealed the presence of MC-3 receptor mRNA in both the anterior and the neurointermediate lobe. The identity of the amplified products was confirmed by sequence analysis. Dispersed cells of 14-day-old female rats (24 h in culture) were exposed to gamma3-MSH, and changes in intracellular free calcium levels ([Ca2+]i) were assessed by means of fluo-3 video imaging. Gamma3-MSH evoked a rapid and maintained oscillating [Ca2+]i increase in 5%, 10% and 15% of the cells at a dose of 0.1, 1 and 10 nM, respectively. The MC-3/MC-4 receptor antagonist Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (SHU 9119) blocked the effect of gamma3-MSH in about 50% of the responsive cells. The present data suggest that the MC-3 receptor is expressed in the rat pituitary but that this receptor mediates only half of the effects of its putative ligand, gamma3-MSH, on [Ca2+]i. Part of the effect of gamma3-MSH may be mediated by a MC-3 receptor in a functional state different from the one studied previously in transfected cell lines or by a hitherto unidentified MC receptor.

Animals↗

PubMed1998

A new family of growth and differentiation factors derived from the N-terminal domain of proopiomelanocortin (N-POMC).

There is a large body of in vitro evidence that pituitary function is not only dependent on hormonal signals from the brain but also on paracrine signals produced in the tissue itself. These signals appear to be involved in the control of pituitary hormone secretion as well as in pituitary cell differentiation and development (for review see Denef C. Paracrine mechanisms in the pituitary. In: Imura H, editor. The pituitary gland, 2nd ed. Raven Press, 1994: 351-378; Denef C. Autocrine/paracrine intermediates in hormone action and modulation of cellular responses to hormones. In: Conn M, editor. Handbook of Physiology. New York: Oxford University Press, 1998 (in press)). The paracrine factors which have been identified in the pituitary belong to diverse biological molecules such as neuropeptides, acetylcholine, growth factors, cytokines and posttranslationally modified derivatives of pituitary hormone such as cleaved prolactin (PRL) and the gonadotropin alpha-subunit. Recently, we have identified several N-terminal fragments of the polypeptide proopiomelanocortin (POMC) as a novel family of growth and differentiation factors in the rat anterior pituitary.

Amino Acid Sequence↗

PubMed1998

Stimulation of Ca2+ entry in lactotrophs and somatotrophs from immature rat pituitary by N-terminal fragments of proopiomelanocortin.

We have previously shown that 10-12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC1-76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC1-26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca2+]i) in response to POMC1-76 and POMC1-26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca2+) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC1-76 or POMC1-26 induced a maintained oscillating [Ca2+]i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca2+]i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC1-76 or 5 nM POMC1-26, and 11-13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca2+]i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca2+]i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca2+ response to POMC1-76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC1-26. The rise of [Ca2+]i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca2+ stores by thapsigargin. Removal of Ca2+ from the extracellular medium or addition of cadmium completely abolished both the POMC1-76- and POMC1-26-induced [Ca2+]i increase. Nifedipine inhibited the Ca2+ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca2+ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC1-76- or POMC1-26-induced [Ca2+]i increase. The present data show that, in immature pituitary cells, POMC1-76 induces an increase in [Ca2+]i through extracellular Ca2+ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC1-76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC1-76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.

Animals↗

PubMed1998

Presence of gonadotropin-releasing hormone (GnRH) mRNA in Rathke's pouch and effect of the GnRH-antagonist ORG 30276 on lactotroph development in vitro.

Reverse transcription-polymerase chain reaction (RT-PCR) with specific GnRH cDNA primers performed on RNA from Rathke's pouches removed from pregnant rats at day 12 of gestation (e12) generated an amplified DNA fragment of the expected length (357 bp). The fragment hybridized with a labeled GnRH cDNA probe in Southern blotting. DNA sequencing demonstrated identity with the known nucleotide sequence of the corresponding segment of rat GnRH cDNA. To determine whether GnRH mRNA was located in the Rathke's pouch cells or in remnants of surrounding tissue not completely removed during preparation, the pouches were treated with collagenase. Based on light and electron microscopic examination, this treatment disconnected virtually all contaminating tissue, allowing the 'pure' Rathke's pouches to be picked-up separately. Again, RT-PCR generated a DNA fragment of the expected length, the fragment hybridized with the GnRH cDNA probe and showed the nucleotide sequence of the corresponding region of rat GnRH cDNA. In Rathke's pouches established in explant culture on e12, lactotrophs were well developed when examined 9 days later by immunostaining of prolactin in paraffin-embedded sections of the tissue. Computerized image analysis showed prolactin immunoreactivity in 8.0+/-1.1% of the section area. Addition of the potent and long-acting GnRH antagonist ORG 30276 to the crude preparation of Rathke's pouches caused a significant decrease in the relative area staining for prolactin. The latter effect was abolished by concomitant addition of GnRH. In preparations of pure Rathke's pouches (collagenase-treated), ORG 30276 failed to affect the relative area of prolactin immunoreactivity. GnRH mRNA remained expressed in explants of both crude and pure Rathke's pouches until the end of the culture period. It is concluded that the GnRH gene is expressed in Rathke's pouch as early as e12 and that GnRH may be a physiological paracrine/autocrine peptide stimulating the development of lactotrophs. Mesenchymal and/or neural factors may be essential for the latter system to function.

Animals↗

PubMed1997

Retinoids stimulate lipid synthesis and accumulation in LNCaP prostatic adenocarcinoma cells.

In a previous report we demonstrated that androgens markedly stimulate accumulation of lipid droplets in LNCaP cells. The effects were already evident at low concentrations of androgens optimal for proliferation but became much more pronounced at high concentrations optimal for differentiation. In the present report we explored whether other agonists acting by nuclear receptors and modulating LNCaP growth and differentiation also affect lipid accumulation. The agonists investigated were 1alpha,25-dihydroxycholecalciferol (VD3), all-trans-retinoic acid (atRA), and triiodothyronine (T3). Lipid accumulation was evaluated by Oil Red O staining followed by image analysis of Oil Red O-stained cells or by extraction and measurement of absorbency. Only marginal effects were noted for VD3 and T3. The atRA, on the contrary, increased lipid staining 5-12-fold. This effect required high concentrations of retinoids (10[-6] M) and was accompanied by growth stimulation. Lipid accumulation was less pronounced than that observed with maximally effective concentrations of androgens (10[-3] M R1881). Thin layer chromatography (TLC) and enzymatic determination of the various lipid fractions demonstrated that retinoids increase triacylglycerides and an unidentified lipid fraction with a slightly higher mobility. In contrast with androgens, however, they did not stimulate the accumulation of cholesterol esters. Incorporation studies with [2-14C]acetate revealed that the increased accumulation of the mentioned lipids is related both to increased synthesis and to decreased secretion. Retinoid-induced lipid accumulation is accompanied by increased steady-state levels of the mRNA encoding fatty acid synthase (FAS), a key enzyme involved in lipid synthesis, while the expression of HMG-CoA-reductase, an enzyme controlling cholesterol synthesis is only marginally affected. It is concluded that retinoids share the ability of androgens to increase lipid accumulation in LNCaP cells. The nature of the lipids affected by both agonists, however, differs at least in part suggesting that the underlying mechanisms may also be different. For the studied compounds (androgens, VD3, atRA, and T3) no simple and consistent relationship could be observed between their ability to decrease proliferation and increase differentiation on the one hand and their ability to promote lipid accumulation on the other hand.

Acetates↗

PubMed1997

Mitogenic effects of nerve growth factor on different cell types in reaggregate cell cultures of immature rat pituitary.

Treatment of reaggregate pituitary cell cultures of 14-day-old female rats with nerve growth factor (NGF) augmented the number of [3H]thymidine ([3H]T)-incorporating lactotrophs in a dose-dependent manner (0.03-3 nM). At least during short-term treatment NGF increased the total number of cells expressing prolactin (PRL) mRNA and enlarged the cytoplasmic area occupied by PRL mRNA but did not affect the number of cells and the cytoplasmic area containing PRL, suggesting that NGF recruits lactotrophs expressing PRL mRNA but not yet PRL. NGF also stimulated [3H]T incorporation in ACTH cells but not in somatotrophs, thyrotrophs and gonadotrophs. In addition, NGF augmented the total number of [3H]T-incorporating cells to a much higher extent than was expected from its effect on lactotrophs and ACTH cells, suggesting NGF also stimulates [3H]T-incorporation in non-hormone producing cells (progenitors or stem cells?). Around 40% of these [3H]T-incorporating cells in both control and NGF treated cultures showed immunoreactivity for the transcription factor Pit-1 in the nuclei, which is twice the percentage expected (18%) if these [3H]T-incorporating cells were the only known Pit-1 expressing cells in the pituitary i.e. lactotrophs, somatotrophs and thyrotrophs. The present data suggest that NGF has a mitogenic effect on several cell lineages in the pituitary: lactotrophs, corticotrophs and non-hormone-containing cells. The high proportion of mitotic non-hormone containing cells that express Pit-1 is consistent with the proposed role of Pit-1 in cell proliferation in the developing lactosomatotroph lineage.

Animals↗

PubMed1997

Paracrine communication in the anterior pituitary as studied in reaggregate cell cultures.

The classical image of the endocrine system is that secretory function of a gland is regulated from outside that gland by other organs. Focused on the pituitary gland, hormone secretion by the anterior lobe is under control of peptides and biogenic amines produced by the hypothalamus. About a decade ago, our group launched the new idea that functioning of the anterior pituitary (AP) is also regulated from within, i.e., that the constituent cell types inter-communicate to control hormone secretion. Extensive in vitro research has now provided a body of evidence that paracrine communication plays an important role, not only in regulation of hormone secretion but also in development, growth, and differentiation of the AP [reviewed in Denef (1994) The Pituitary Gland, pp. 351-378]. It further revealed that crosstalk between the cells is mediated by local, paracrine, factors. The main objective of our research is to identify those factors, their actions and the producing and target cell type(s) in order to unravel the paracrine communication network that is functional in the AP. Equally important, we set the step towards in vivo examination of the results obtained in vitro using transgenic mice. In the present article, we will review the technology used, three examples of AP cell-to-cell interactions studied, and we will discuss the value of transgenic animal models in the study of AP paracrine communication.

Acetylcholine↗

PubMed1997

Involvement of nitric oxide in the interferon-gamma-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures.

In previous work it was shown that the immune cytokine interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN-gamma. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N(G)-monomethyl-L-arginine (L-NMMA) and S-methyl-L-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN-gamma on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN-gamma these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. L-N6-(1-iminoethyl)lysine (L-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN-gamma effect on GHRH-stimulated GH secretion and partially reversed IFN-gamma inhibition of PRL release. However, L-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as L-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO2-) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of L-NMMA was confirmed by competition experiments with the natural NOS substrate L-arginine. Thus, in culture medium with lower amounts of L-arginine, L-NMMA blocked the IFN-gamma-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN-gamma was markedly reduced in L-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN-gamma on GHRH-stimulated GH and basal PRL release. Similarly to IFN-gamma, SNP did not affect basal GH release. As previously reported, inhibition by IFN-gamma occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, L-NMMA reversed the IFN-gamma effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN-gamma L-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN-gamma through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN-gamma on GHRH-stimulated GH release and partially for the IFN-gamma-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN-gamma may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.

Animals↗

PubMed1997

Expression of the low-affinity p75 nerve growth factor receptor in the developing rat pituitary gland.

We investigated, by means of in situ hybridization with a digoxigenin-labelled RNA probe, the expression of the low-affinity p75 nerve growth factor receptor (NGFR) in the developing pituitary primordium of the rat. In 13-day pc embryos, intense staining of p75 NGFR mRNA was present in the cytoplasm of all cells of Rathke's pouch. In day-17 pc embryos p75 NGFR expression was present primarily in the cells of the intermediate lobe. In the newborn rat pituitary only very weak staining was observed, predominantly in the intermediate lobe. In neural structures the staining at day 13 pc was comparable to that of day 17 pc. Since p75 expression is seen very early during pituitary development and declines during the time the expression of pituitary hormonal phenotypes are steadily increasing, we suggest that the p75 NGFR expression in Rathke's pouch may play a temporally defined role in the commitment rather than in the differentiation of the various pituitary cell types.

Animals↗

PubMed1997

Effect of POMC(1-76), its C-terminal fragment gamma3-MSH and anti-POMC(1-76) antibodies on DNA replication in lactotrophs in aggregate cell cultures of immature rat pituitary.

Treatment of aggregate cell cultures of 14-day-old rat pituitary for 40 h with purified human (h) POMC(1-76) dose-dependently augmented the number of DNA replicating lactotrophs as estimated by autoradiography of [3H]-thymidine (3H-T) incorporation in cells immunostained for prolactin (PRL). No such effect was seen on the total number of 3H-T labelled cells (the majority of which did not contain any pituitary hormone in a detectable amount) or on the total number of lactotrophs. The effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs was blocked by concomitant treatment with anti-hPOMC(1-76) monoclonal and polyclonal antibodies cross-reactive with rat POMC(1-74). The latter anti-hPOMC(1-76) antibodies also decreased the number of 3H-T incorporating lactotrophs in the absence of hPOMC(1-76). Gamma3-MSH, which is the C-terminal domain of hPOMC(1-76), mimicked the effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs but its potency was lower than that of hPOMC(1-76). Other melanocortin (MC) peptides such as alpha- and beta-MSH were also effective but were less potent than gamma3-MSH. The difference in potency was not due to partial degradation of the peptides. hPOMC(1-76) did not affect 3H-T incorporation in other pituitary cell types. In contrast gamma3-MSH also augmented the number of 3H-T labelled somatotrophs and thyrotrophs. In the embryonic kidney 293 cell line stably transfected with the MC-3 receptor, gamma3-MSH (10 nM) augmented cAMP formation up to 30 times. In contrast, hPOMC(1-76) (100 nM) was inactive in this test system, indicating this peptide is not an agonist at the MC-3 receptor. The present investigation further supports the role of rat POMC(1-74) as a paracrine growth factor in the development of lactotrophs. The active core of POMC(1-76) does not seem to be restricted to its C-terminal domain gamma3-MSH as the latter peptide displays a growth promoting effect that is different from that of POMC(1-76): it is less potent, it is not specific for lactotrophs and whereas the effect of gamma3-MSH may be mediated by the MC-3 receptor that of POMC(1-76) is not.

Animals↗

PubMed1997

Inducible nitric oxide synthase in the anterior pituitary gland: induction by interferon-gamma in a subpopulation of folliculostellate cells and in an unidentifiable population of non-hormone-secreting cells.

In the context of immune-endocrine relationships, we have previously shown that interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures. The non-hormone-secreting folliculostellate (FS) cells were found to mediate this inhibitory action. Because in the immune system IFN-gamma is a strong stimulator of nitric oxide (NO) release through the induction of NO synthase (NOS), we investigated whether the inducible form of NOS (iNOS) is present in (rat) AP cell cultures, and whether its expression is stimulated by IFN-gamma. Immunocytochemistry revealed that under basal in vitro conditions only a very few AP cells contained iNOS. Treatment with IFN-gamma caused a sixfold rise in the number of iNOS-positive cells and augmented the intensity of the staining. The increased number of iNOS-expressing cells was paralleled by elevated production of NO. Some of the iNOS-positive cells extended cytoplasmic processes between hormone-secreting cells, which is a characteristic of FS cells. Immunostaining of FS cell-poor and FS cell-enriched populations (obtained by gradient sedimentation) also suggested the presence of iNOS in a subpopulation of FS cells. By double immunofluorescence techniques we found that about 65% of iNOS-expressing cells were positive for S-100, a marker protein for FS cells. However, around 80% of the S-100-positive cells were not labeled for iNOS. On the other hand, the majority of the S-100-negative iNOS-containing cells could not be further identified by antisera against the classical AP hormones, suggesting the presence of iNOS in a still unidentified non-hormone-secreting cell type of the AP gland. This report is the first to demonstrate the expression of the inducible form of NOS in the AP gland. IFN-gamma upregulates this expression, showing that cytokines may use the same signalling mechanisms in both the immune and the endocrine system. In addition, a putative new function of a subpopulation of FS cells in the paracrine regulation of the AP gland is suggested.

Animals↗

PubMed1996

Corticotropin-releasing hormone (CRH) receptors in the mesenteric small arteries of rats resemble the (2)-subtype.

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

Adrenocorticotropic Hormone↗

PubMed1996

N-terminal 10- and 12-kDa POMC fragments stimulate differentiation of lactotrophs.

Medium conditioned by a highly enriched population of gonadotrophs, cultured as reaggregates in the presence of 0.01 nM GnRH, was concentrated, separated on a reversed-phase HPLC column, and tested for activity on lactotroph development in pituitary reaggregate cell cultures of 14-day-old rats. The number of cells expressing prolactin (PRL) mRNA was estimated by image analysis after in situ hybridization of paraffin-embedded sections. The number of these cells entering the mitotic cycle was estimated by autoradiography of [3H]thymidine ([3H]T) incorporation. One HPLC column fraction expanded the section area occupied by PRL mRNA cells without displaying an effect on [3H]T labeling of these cells, indicating that this fraction induces differentiation in the lactotroph lineage. The latter fraction was further purified on a second reversed-phase HPLC column, a gel filtration column, and a final reversed-phase HPLC column. From the last column, four substances were isolated that all selectively induced differentiation of lactotrophs. Each of them had an N-terminal amino acid sequence identical to the N-terminal domain of rat proopiomelanocortin (POMC). As determined by mass spectrometric analysis, the M(r)s were 10,091, 10,289, 12,238, and 12,247 Da, respectively. The C-terminal extension of these compounds is possibly up to Gln74 for the former two compounds and up to Gly95 for the latter two. Authentic purified human POMC(1-76) mimicked the effects of the purified 10- and 12-kDa rat POMC fragments. The present data suggest that certain isoforms of rat POMC(1-74) and human POMC(1-76) can stimulate lactotroph growth through a differentiation-inducing action on progenitor cells.

Animals↗

PubMed1996

Evidence for a trophic action of the glycoprotein hormone alpha-subunit in rat pituitary.

The effect of the alpha-subunit of luteinizing hormone (LH alpha) on lactotroph growth in 14-day-old rat pituitary was studied in vitro using a reaggregate pituitary cell culture system. LH alpha significantly expanded both the total population of cells expressing prolactin mRNA and the number of [3H]thymidine incorporating prolactin mRNA expressing cells. No such effect could be elicited by LH. Both effects were inhibited by simultaneous addition of an anti-LH alpha antiserum but not by normal rabbit serum. Anti-LH alpha antiserum added alone to the cultures caused a small decrease in the number of prolactin mRNA expressing cells and in [3H]thymidine labelling of the latter. It is concluded that LH alpha may be a trophic factor of lactotrophs not only during fetal development, as suggested by others previously, but also during the rapid expansion of this cell type during postnatal life in the rat.

Animals↗

PubMed1996

In vitro immunoneutralization of a cleaved prolactin variant: evidence for a local paracrine action of cleaved prolactin in the development of gonadotrophs and thyrotrophs in rat pituitary.

We have previously isolated a cleaved prolactin variant, secreted by rat pituitary cells in culture, that stimulated [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs when added to pituitary aggregate cell cultures of 14-day-old female rats. Using synthetic peptides homologous to the new C- and N-termini of the cleavage site, we made antisera recognizing this cleaved variant without significant cross reaction with native prolactin. Addition of these antisera to pituitary aggregate cell cultures decreased [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs but not in the other pituitary cell types. These data are further evidence that this prolactin variant, cleaved between Tyr-145 and Leu-146, may have an important role as growth regulator of the gonadotrophs and thyrotrophs in the rat pituitary.

Animals↗

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↗