Regulation of hormonal secretion and DNA synthesis in the lactotrophs of the rat adenohypophysis in vitro in primary cell cultures

Changes in DNA synthesis in lactotrophs of primary monolayer cultures of the rat pituitary cells were studied, using immunoperoxidase staining in combination with autoradiography. Pituitary cell cultures were treated for 3 days with thyroliberin (TRH), bromocriptine (CB154) or somatostatin (SRIF). The proportion of lactotrophs labelled with 3H-thymidine in the total pool of labelled cells served as a criterion for the estimation of DNA synthesis in prolactin-secreting cells. Prolactin secretion by the same cultures was measured by homologous radioimmunoassay. TRH (10 ng/ml) stimulated DNA synthesis in the total population of pituitary cells, but not in lactotrophs. SRIF decreased selectively the proliferation of lactotrophs, but failed to depress or even stimulated DNA synthesis in some cell types of the rat pituitary gland in the cultures. The quantitative method of studying DNA synthesis in anterior pituitary may be used to evaluate the effects of a number of biologically active compounds on various cell systems.     

Apoptotic cell death of oestrogen activated lactotrophs induced by tamoxifen

Stimulation and inhibition of lactotroph cells cause remarkable morphological and functional changes. In keeping with these changes, the size of the lactotroph cell population undergoes striking alterations due to proliferation or cell death. Factors involved in the induction of apoptosis of pituitary cells are not well established. We demonstrated earlier that oestrogens prevent lactotroph cells of female rats to die by apoptosis induced by bromocryptine treatment, a fact that can be reversed in ovariectomised rats. In this study, we developed experimental models for in vivo and in vitro studies to gain further insight on the survival effect of oestrogens on lactotrophs. In rats pretreated with oestrogens, tamoxifen generates a massive cell death by apoptosis as validated by the TUNEL technique and DNA electrophoresis of pituitary gland. On electron microscope observations, numerous lactotrophs exhibited progressive morphological changes in the nuclei compatible with the apoptotic process. The cells remaining intact also exhibit signs of inhibition due to a significant transformation of regular lactotrophs in atypical subtypes. In pituitary cell cultures exposed to tamoxifen and oestrogen simultaneously, most of the lactotrophs displayed features of apoptosis in the nucleus. The present reports gathered new evidences on the apoptogenic potential of tamoxifen on lactotroph cells, and corroborates the contribution of oestrogens to sustain both a balanced population of lactotrophs and a competent secretory activity. The concept that opposed activities, such as inhibition and stimulation, can activate apoptosis is also strengthen by these observations.     

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-microns serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.     

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

Summary The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-m serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.     

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs. TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs. SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154. The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.     

Somatotrophs and lactotrophs: an immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult

The objective of this study was to determine the distribution of somatotrophs and lactotrophs and conduct a morphometrical analysis of immunoreactive somatotrophs and lactotrophs in the pituitary glands of White Leghorn Hens (Gallus domesticus) during the period of induced moult. We divided the periods of induced moulting into three phases viz. 7, 14 and 21 days. The labeled alkalinephsphatase method with anti-GH (growth hormone) and anti-PRL (prolactin) as a primary antibody was used to detect somatotrophs and lactotrophs, in the midsagital sections of chicken adenohypophysis. Immunohistochemistry showed that somatotrophs are not only confined to the cephalo-caudal axis but can also be found in the caudal lobe; while lactotrophs were distributed in both lobes of the anterior pituitary gland at all stages of moulting (7, 14 and 21 days). Lactotrophs were of different shapes but somatotrophs were oval to round in morphology. At the given stages of induced moulting, some hypertrophied lactotrophs were also present after 7 days of induced moult in the anterior pituitary gland. However, there were moulting-related changes: from 7 to 21 days of induced moulting the immunoreactive-PRL cell population decreased, while the mean lactotroph size was more than that of somatotrophs. Basic quantitative and morphological information relating to somatotrophs and lactotrophs during the period of induced moult in laying hens is reported here and the changes brought about by induced moulting are restricted to PRL positive cells rather than GH positive cells.Key words: Moult, pituitary gland, somatotrophs, lactotrophs, chicken.     

Different Behavior of Lactotroph Cell Subpopulations in Response to Angiotensin II and Thyrotrophin-Releasing Hormone

1. 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.     

Dependence of pituitary hormone secretion on the pattern of spontaneous voltage-gated calcium influx. Cell type-specific action potential secretion coupling.

In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.     

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 μM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT).     

Regulation of prolactin release by angiotensin

In superfused anterior pituitary cell aggregates, prolactin release is stimulated by angiotensin II (AII) in a concentration-dependent fashion between 0.1 and 10 nM. When studied in aggregates prepared from pituitary cell populations separated according to size by unit gravity sedimentation, the PRL response to AII was weak in a population enriched in lactotrophs but deprived of gonadotrophs. In other separated populations, the response increased with the proportional number of gonadotrophs. The response also increased when lactotrophs were co-aggregated with an enriched population of gonadotrophs. It is proposed that the PRL response to AII is augmented by an intercellular messenger system presumably operating between gonadotrophs and lactotrophs.     

Cytology of the hypophysis, pars anterior of the Indian five-striped palm squirrel, Funambulus pennanti (Wroughton)

By employing various staining techniques the pituitary cells of the squirrels, Funambulus pennanti, can broadly be divided into 'acidophilic', 'basophilic' and 'amphophilic' types. The acidophilic cell types include somatotrophs and lactotrophs. The basophilic cell types include thyrotrophs, folliculotrophs and interstitiotrophs while the amphophilic cells are recognized as corticotrophs. The identification of the pituitary cells and their histochemical characteristics are described at the light microscopical level. Attempt has been made to interpret the cells identified in the present study on a comparative and tinctorial basis.     

Fate of degenerating lactotrophs in rat pituitary gland after interruption of lactation: a histochemical and immunocytochemical study

Summary In the pituitary gland of pregnant and lactating rats a striking proliferation of lactotrophs occurs to meet the increased demands for prolactin. Following interruption of lactation the redundant lactotrophs undergo a massive degeneration until pre-pregnant proportions are re-established. Immunocytochemical detection of prolactin allows the recognition of degenerating lactotrophs until advanced stages of degeneration and leads to the conclusion that this process is autolytic in nature. Histochemistry of acid phosphatase reveals a remarkable accumulation of this enzyme in Golgi cisternae and lysosomes. At later stages of degeneration the acid phosphatase spreads throughout the entire cell. The presence of increased numbers of necrotic cells appears to activate phagocytosis of stellate cells and, to a lesser extent, of follicular cells. Stellate cells responsible for the secondary processing of cell residues are isolated cells characterized by a prominent oval nucleus and an electron-lucent cytoplasm with scarce organelles and extensive cytoplasmic processes. They appear as scavenger cells engulfing cell remnants and debris. Immunocytochemistry of S-100 protein discloses differential staining of two types of cell, one forming clusters of 2–4 cells with faint immunoreactivity, while the other type consists of isolated cells with a stellate profile and stronger labelling to S-100 protein.     

Subcellular localisation of VEGF in different pituitary cells. Changes of its expression in oestrogen induced prolactinomas

Summary Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.     

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.     

The cytoarchitectonics of the adenohypophysis in light of the concepts of cellular flows]

Analysis of published data allows to suggest a possible model of adenohypophyseal cytoarchitectonics based on the concepts of tissue self-renewal and streaming in this gland. The model provides a framework for understanding: a) the existence of ambiguous cellular elements; b) change of relationship between different types of hormone secreting cells with sex-dependent prevalence of somato- or lactotrophs; c) relatively high proliferative activity of lactotrophs in mature pituitary gland; d) specific spatial arrangement of various types of hormone secreting cells; e) multiple effects of some bioregulators on the secretion of two or more pituitary hormones; f) the existence of polyfunctional pituitary adenomata containing and secreting several adenohypophyseal hormones simultaneously. Possible approaches to thorough evaluation and testing of the model in experiments using organ or cell cultures of adenohypophysis are discussed.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Differential expression of ionic channels in rat anterior pituitary cells.

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.     

Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique

Summary The morphological characteristics of bovine pituitary cells separated by a rapid enrichment procedure are described. Single-cell suspensions were prepared from pituitary glands of steers by use of a collagenase technique and separated by discontinuous gradient centrifugation. The separation of prolactin and growth hormone-containing cells was assessed by radioimmunoassay of hormone content and immunocytochemistry, and the distribution of fibroblasts assessed after establishing cell cultures. Morphometric analysis of the fine structure of two fractions respectively enriched and depleted in the proportion of immunocytochemically-identified lactotrophs was performed after labelling with anti-prolactin antiserum coupled to immunogold complex. Cells recovered from the higher-density fraction were more highly granulated, suggesting that this was a major characteristic determining separation. Cells labelled for prolactin could not be distinguished from unlabelled cells on the basis of their granule size range, but unlabelled cells had a significantly greater coefficient of variation. These data suggest that granule density and distribution, but not granule size per se, are useful characteristics for the identification of bovine lactotrophs.     

Secretory activity of lactotrophs and its regulation by hypothalamic hormones in primary cultures of pituitary cells of rats of different ages]

Basal prolactin (PRL) secretion and the responses of lactotrophs to thyroliberin, dopamine and somatostatin were studied in the experiments employing primary monolayer cultures of pituitary cells obtained from developing rats of different ages. High responsiveness of PRL-secreting cells to the action of hypothalamic hormones was observed in the group of neonatal rats, although basal PRL release was about two orders lower in pituitary cultures of neonatal rats as compared to the cultures of immature, pubertal and adult animals. The investigation performed could reveal quantitative, but not qualitative differences in the reactions of lactotrophs of various age groups. It is concluded that postnatal development in the rat is coupled with significant changes of basal PRL release and to a lesser extent, with changes of lactotroph responsiveness to hypothalamic hormones.