Immunocytochemical localization of annexin 5, a calcium-dependent phospholipid-binding protein, in rat endocrine organs

Annexin 5, a unique calcium- and phospholipid-binding protein, has been investigated for its specific distribution in rat endocrine organs by immunocytochemistry with a specific antiserum to recombinant rat annexin 5. Follicular epithelial cells and parafollicular cells of the thyroid gland, adrenocortical cells of the zona fasciculata and zona reticularis, luteal cells, testicular interstitial cells, and Sertoli cells were shown to contain annexin 5. To examine whether the synthesis of annexin 5 would be affected by a change in humoral signal, the distribution of annexin 5 in the anterior pituitary was examined three weeks after ovariectomy. The withdrawal of ovarian hormones induced huge castration cells in the anterior pituitary gland, which contained abundant annexin 5. Annexin 5 was not detected in the pineal gland, the parathyroid gland, the islet of Langerhans, the adrenal medulla, zona glomerulosa cells, and granulosa cells. Since annexin 5 was shown to exist in many of the endocrine tissues examined, to be localized in specific cell types, and to be abundant in castration cells, it is suggested that annexin 5 contributes to secretory cell functions, which may be common to endocrine cells secreting chemically different hormones.     

Gene expression and immunohistochemical localization of megalin in the anterior pituitary gland of helmeted guinea fowl (<Emphasis Type="Italic">Numida meleagris</Emphasis>)

Megalin/the low density lipoprotein receptor-related protein-2 (LRP-2) is expressed in a variety of epithelia and mediates endocytosis of numerous substances. Megalin is also shown to bind clusterin with high affinity. In the pituitary gland, clusterin is localized in endocrine cells, folliculostellate (FS) cells and colloids. The present study examines the expression pattern of megalin within the gland and assesses its cellular localization to that of clusterin so as to deduce their functional implications in colloidal accumulation as relevant in vivo. Quantity of megalin mRNA expression in pituitary and other endocrine tissues was quantified by real-time PCR using SYBR-green I detective system. High levels were detected in kidneys and pituitary. In situ hybridization showed megalin mRNA in FS cells. Megalin protein detected by immunohistochemistry was also observed in FS cells. Immunoelectron microscopy clearly showed the localization of megalin in peripheral region of colloid-containing follicles and on vesicular structures in FS cells. Immunolabeling was also found to be associated with membranes of vacuoles in apoptotic endocrine cells and cell remnants engulfed by FS cells. Double immunofluorescence labeling was performed to determine whether megalin and clusterin in the anterior pituitary were present within the same cell. Simultaneous localization was detected in almost all FS cells surrounding colloids and in several foci of FS cells surrounding endocrine cells. These findings suggest that megalin may drive ingestion of clusterin complexes with products of digested apoptotic endocrine cells in FS cells, and thereby providing a potential mechanism for a receptor mediated uptake of degenerating endocrine cells and secretion of colloid.     

Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells?

Folliculo-stellate cells (FS-cells) in the anterior pituitary gland are star-shaped cells and form tiny follicles. FS-cells are positive for S-100 protein and produce many cytokines or growth factors, such as interleukin-6 (IL-6), leukemia inhibitory factor (LIF), basic fibroblastic growth factor (bFGF) and vascular endothelial cell growth factor (VEGF). Therefore, it is generally accepted that FS-cells regulate endocrine cells through these growth factors. FS-cells also exhibit a phagocytotic activity and are known to work as scavenger cells. In addition to these functions, FS-cells are considered to have some unknown functions. In order to reveal the biological significance of FS-cells in the anterior pituitary gland, we performed a morphological study and obtained some new findings. First, we were interested in the colloid formation in the senescent porcine pituitary gland. We analyzed the colloids and found that clusterin is a major protein in them. We also found that the accumulation of clusterin in the colloids is related to the phagocytotic activity of FS-cells. In our next study, we found that FS-cells have the potential to differentiate into striated muscle cells. From FS-cells show multi-potent cell character and other cytological evidence, we propose that FS-cells are candidate of organ-specific stem cells in the anterior pituitary gland.     

Electrical activity in endocrine pituitary cells in situ: a support for a multiple-function coding

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ. Three patterns of voltage fluctuations were distinguishable by their durations (1-100 s). These patterns could be ordered on top of each other, namely in response to secretagogues. Thus, pituitary endocrine cells express in situ a cell code in which small-amplitude voltage fluctuations lead to a multimodal arrangement of spike firing, which may finely tune calcium-dependent functions.     

Changes in thyrotroph and somatotroph cell populations induced by stimulation and inhibition of their secretory activity

Summary The populations of cells which produce immunoreactive growth hormone (GH) and thyroid stimulating hormone (TSH) in the rat pituitary gland do not occur in fixed percentages but vary greatly under different physiological and experimental conditions. These variations can be directly correlated to the levels of stimulation and/or inhibition of the specific secretory activity. In both types of cell, sustained stimulation with trophic hormones or blockage of the feedback mechanisms induces remarkable growth in the specific cell population. Conversely, the interruption or inhibition of the stimulus thwarted the hormonal secretion and caused a massive degeneration of redundant cells. The stimulation of both GH and TSH cells is accompanied by an enhanced secretory activity as judged by their higher concentrations in serum and hypertrophy of the cytoplasmic organelles involved in synthesis and intracellular processing of the hormones. By contrast, interruption of the stimulus is followed by a variable degree of disruption of the cytoplasmic organization, including a sizable degeneration of cells. In stimulated rats, the concentrations of both GH and TSH decreased significantly in pituitary tissue due to mobilization of the hormonal stores contained in secretory granules. On the other hand, the withdrawal of stimuli blocked the hormonal release; this is reflected by the accumulation of both hormones and secretory granules in pituitary tissue. The strict correlation between the size of the GH and TSH populations with stimulation and inhibition of hormonal secretory activity reported in this investigation further supports the critical role played by the cell renewal process in endocrine secretion.     

Immunohistochemical localization of huntingtin-associated protein 1 in endocrine system of the rat.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.     

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.     

Immunohistochemical characterization of chicken pituitary cells containing the vasotocin VT2 receptor

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.     

Non-hormonal cell types in the pituitary candidating for stem cell

Hormone balances in the body are primarily governed by the hypothalamus-pituitary system. For its pivotal role, the pituitary gland relies on an assortment of different hormone-producing cell types, the proportions of which dynamically change in response to fluctuating endocrine demands. Mechanisms of pituitary cellular plasticity are at present far from understood, and may include proliferation and transdifferentiation of hormonal cells. Whether new cells also originate by recruitment from stem cells is unsettled, although this idea has frequently been proposed. Here, I will review these data by focusing on the non-hormonal cell types that have been advanced as candidates for the pituitary stem cell position.     

Distribution of an enzyme which acetylates α-melanocyte stimulating hormone in rat brain and pituitary gland and effects of arcuate nucleus lesions

This study describes the distribution of an α-melanocyte stimulating hormone (α-MSH) acetyltransferase (MAT) in rat brain and pituitary gland. Highest activities of MAT were found in the neurointermediate lobe of the pituitary gland with the anterior lobe containing slightly less. Within the brain, lowest MAT activities were measured in the hypothalamus, the region which contained the highest concentrations of α-MSH. Relatively high enzyme activities of MAT were measured in the hippocampus, cortex and cerebellum—regions with very low α-MSH concentrations. The fact that MAT activity levels did not parallel α-MSH concentrations indicates that MAT was not solely localized to α-MSH synthesizing neurons or endocrine cells. Furthermore, arcuate nucleus lesions which depleted brain α-MSH failed to deplete MAT activity. Although MAT was not solely localized to α-MSH synthesizing cells, it may have functional significance for α-MSH acetylation due to compartmentalization with α-MSH in α-MSH synthesizing endocrine cells and neurons. Alternatively a second regionally specific MAT may exist.     

Presence of chromogranin A, B and C in bovine endocrine and nervous tissues: a comparative immunohistochemical study

Summary Antisera against chromogranin A, B and C were used to study the distribution of these acidic proteins in bovine endocrine and nervous tissues. The three chromogranins occur together in several endocrine organs (adrenal medulla, anterior pituitary, endocrine pancreas) and in sympathetic ganglion cells. In the posterior pituitary, only chromogranin C and in the intermediate lobe only A and C are found. The parathyroid gland contains only A, and enterochromaffin cells are immunoreactive for A and B. Cells of the thyroid gland and some cells of the anterior pituitary apparently do not contain any chromogranins. It is concluded that the three chromogranins are not always stored together and that they are not present in all endocrine cells. This distinct localization of the chromogranins indicates some special, although still undiscovered, function for these proteins.     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Phenylethanolamine N-methyltransferase-(PNMT)-like immunoreactivity in the rat parathyroid gland

A phenylethanolamine N-methyltransferase-(PNMT)-immunoreactivity, present without the other catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), has been previously detected in the central nervous system and in endocrine cells of the islets of Langerhans and the pituitary intermediate lobe of the rat. In the present study a similar PNMT-like immunoreactivity is demonstrated in the rat parathyroid gland. The immunoreactivity was distinctly localized to the cell periphery, and present in all glandular cells. The thyroid gland was negative. In the parathyroid TH- and DBH-immunoreactivity was seen only in vascular nerve fibers; no glandular cells were stained. The functional significance of the PNMT-like immunoreactivity is not known. The absence of TH- and DBH-immunoreactivity and the low level of adrenaline detected in the parathyroid, and the peripheral localization of the immunoreactivity may indicate an alternative enzyme function or the detection of an immunologically related protein common to pancreatic, pituitary and parathyroid endocrine cells.     

The antisecretory factor: synthesis and intracellular localisation in porcine tissues

The antisecretory factor, AF, is a 41-kDa protein, cloned and sequenced from a human pituitary library. AF is a potent inhibitor of experimental intestinal hypersecretion in rats and pigs. An antiserum against the C-terminal of the truncated, recombinantly produced AF protein was raised in rabbits. The affinity-purified antiserum was used to study the expression of AF in mucosal membranes and in the pituitary gland of the pig; distinctly stained cells were found in lymphoid cells in the connective tissue of all parts of the gastrointestinal, respiratory and urinary tracts. Cytoplasmic AF was demonstrated in endocrine and epithelial cells in the pituitary gland. In situ hybridisation with a digoxigenin-labelled mRNA probe also demonstrated specific cytoplasmic staining in epithelial and lymphoid cells in all of these tissues. The cells stained by either method were similarly distributed topographically within the tissues. The results suggest that a specific defined cell population in these various tissues possesses the capability of both synthesising and storing the AF protein within the cellular cytoplasmic compartment.     

Quinacrine affinity of endocrine cell systems containing dense core vesicles as visualized by fluorescence microscopy

Summary Intravenous injections of low amounts of the fluorescent antimalarial acridine derivative quinacrine into rats and mice lead to selective high affinity binding of the drug, which can be visualized by fluorescence microscopy, to several hormone-producing cell systems. Injection of 1 mg/kg causes strong drug accumulation in the granules of renin-producing juxtaglomerular cells of the kidney and in several types of cells of the pancreatic islets, and a moderate binding to parafollicular cells in the thyroid gland, chromaffin cells of the adrenal medulla, several gastrointestinal cell systems including APUD cells, cells of the anterior pituitary gland, neurosecretory neurons and mast cells. These different cell systems all have large dense-core storage granules.The present results, together with our earlier finding of a population of gastrointestinal nerve fibers which demonstrate a similar selective high affinity binding of quinacrine might all be explained by a binding of quinacrine to large dense-core storage granules, since such granules are known to be present also in certain gastrointestinal nerve fibers. This is further supported by the finding that endocrine cell systems lacking such granules, such as steroid producing cells in the adrenal cortex and testis, do not accumulate quinacrine. Peptide storage, possibly mediated by or accompanied by purines such as ATP within the granules or an acidic intragranular pH constitute possible binding mechanisms.