Ana Lucía De Paul

Different behavior of lactotroph cell subpopulations in response to angiotensin II and thyrotrophin-releasing hormone.

Abstract1. In the present investigation we have extended the study of lactotroph subpopulations in primary pituitary cell cultures. Male rats with or without previous estrogenization followed by A-II or TRH treatments were selected as experimental models.2. The TRH increased up to 50% the PRL released in both whole and ORQX + EB rats (P < 0.05). In contrast, A-II treatment introduced no changes in PRL secretion from cell cultures derived from whole male rats but attained a significant augmentation (about 75%) of PRL secreted by ORQX + EB pituitary cells.3. The addition of TRH and A-II to cultures of ORQX + EB-derived lactotrophs induced cytological changes compatible with a high secretory activity. In estrogen-treated rats the prevailing lactotroph subpopulation is type I. In cell cultures from control and A-II treated whole male pituitaries, the majority of lactotrophs consists of atypical subpopulations of II and III cells, with smaller secretory granules (between 150 and 300 nm in diameter).4. Morphometry of immunostained lactotrophs performed on light microscopic preparations revealed that about 30–36% of the total cell count were lactotrophs. This percentage was fixed and did not change significantly after TRH and A-II treatments.5. The present results confirm the presence of morphological and functional subtypes of lactotroph cells in rat pituitary. Typical PRL cell population shows the highest responsiveness to angiotensin II and TRH action. This functional heterogeneity of lactotroph subtypes may reflect an important and scarcely explored factor in the regulatory process of prolactin secretion.

Heterogeneity of pituitary lactotrophs: immunocytochemical identification of functional subtypes

The existence of functional lactotroph subpopulations was confirmed in primary pituitary cell cultures of female rats submitted to estrogen treatment and stimulation with thyrotrophin releasing hormone (TRH) and angiotensin II (A-II). In cell cultures of pituitary tissue, prolactin (PRL) producing cells represent about 50% of the total cell count, most of which (90%) correspond to a typical lactotroph subpopulation characterized by large secretory granules, 500-900 nm in diameter, and well developed rough endoplasmic reticulum (RER) and Golgi complex. Few atypical lactotrophs were detected with a quiescent appearance and containing smaller secretory granules, often indistinguishable from granular content of other pituitary cells. Depletion of endogenous estrogen caused by ovariectomy (OVX) decreased the pituitary lactotroph population about 34%, with a relative increase of atypical forms (56%). Replacement therapy with benzoate estradiol (EB) to OVX rats did not reverse the proportion of typical and atypical lactotrophs gauged in control pituitary glands. The predominant lactotroph population of OVX rat was an atypical PRL producing cell which displayed a quiescent appearance compatible with a reduced secretory activity. By contrast, estrogen administration to OVX rats caused a striking development of the RER, a hypertrophy of the Golgi complex and an increased storage of mature and immature secretory granules in the majority of lactotrophs. These features are compatible with a reactivated protein synthesis. Estrogen also enhanced significantly (p < 0.05) the responsiveness of lactotrophs to A-II and the PRL secretion in both intact and OVX + EB treated rats increased by 40% and 30% respectively. By contrast, A-II did not produce any statistically significant response of lactotrophs from OVX female rats. At variance to this observation, in all models tested TRH increased significantly the PRL secretion (p < 0.05). The correlation of PRL secretion and morphology of different lactotroph subtypes authenticates the existence of a lactotroph subpopulation unresponsive to A-II in pituitary cell cultures from rats depleted of estrogen.

Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.

Activation of PKC epsilon induces lactotroph proliferation through ERK1/2 in response to phorbol ester.

The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.

Behaviour of a somatotroph population under a growth hormone releasing peptide treatment

In this investigation, we studied the effects of Momany peptide (GHRP-5), on somatotroph secretory activity. Acute and chronic administration of GHRP-5 provokes a significant release of growth hormone that can be closely correlated with ultrastructural changes in somatotroph populations. After 3, 5 and 7 days of GHRP-5 treatment, two somatotroph cell subpopulations coexist. One of them has an enhanced secretory activity and the other presents a quiescent appearance. Therefore, pituitary growth hormone content was not affected in the first seven days of GHRP-5 treatment. After 14 days, there was a significant depletion of growth hormone pituitary content coincident with the highest levels of serum growth hormone. These results concur with the surge of a new hyperactive somatotroph subtype characterised by numerous immature secretory granules that are discharged bypassing the maturation step. Acute and chronic treatments caused no changes in somatotroph cell density, the area immunostained for growth hormone and the levels of total mRNA for transcription factor pit-1.The results of pituitary cell cultures incubated with specific blockers for different signalling pathways demonstrated an involvement of the phospholipase C–inositol phosphate system in GHRP-5 stimulated somatotroph secretion.GHRP-5 treatment enhanced significantly the release of growth hormone, thereby eliciting ultrastructural modifications in somatotrophs that can be correlated with an increased secretory activity devoid of cell density changes.

Bromocriptine induces parapoptosis as the main type of cell death responsible for experimental pituitary tumor shrinkage.

Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.

17β-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.

Thyroid hormone receptor α1–β1 expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα1–β1 isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα1–β1 isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α1 and β1 isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes

Electron Microscopy is an indispensable tool to investigate the intricate structures of the cell and organelles, and also to study the cellular biological processes implicated in the responses to changes in the microenvironment. However, several cellular events may be missed if conventional ultrastructural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins which can become rearranged as part of their own dynamic processes. Thus, immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology, and ultrastructural studies, by placing macromolecular functions within a cellular context.

17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells.

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.