Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions

Summary Immunofluorescent ACTH cells are present in the developing chick pituitary gland from the 9th day of incubation.Rathke pouch grafts from 4–5 day or 5.5 and 6.5 day-old chicks, grafted into chick chorioallantoic membrane and grown for 12 days, gave rise to tinctorially normal pituitary glands in both cases.The early grafts were of pouch epithelium alone, separated from mesenchyme by trypsinization. The later grafts were surrounded by their attached mesenchyme, from which they are virtually inseparable.In 17 out of 18 of the 4.5 day grafts no immunofluorescent ACTH cells developed. (In the 18th case a few feebly stained single cells). In 16 out of 30 of the 5.5 and 6.5 day grafts ACTH cells were present in normal numbers.Of the 3 hypotheses put forward to explain these findings only one appears valid. This is that the ACTH cells are contributed directly by the mesectoderm (neural crest) surrounding the 5.5 and 6.5 day pituitary primordia.     

Protein kinase C mediates the effect of vasopressin in pituitary corticotrophs

The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 nM TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKC-depleted cells, cAMP responses to stimulation with 100 nM CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC- depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 +/- 0.47 and 1.93 +/- 0.11 pmol 32P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [3H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP- and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [125I]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

Adrenalectomy increases beta-lipotropin secretion over beta-endorphin secretion from anterior pituitary corticotrophs

Previous studies have indicated that acute stress in vivo or ovine corticotropin releasing hormone (oCRH) in vitro, releases both beta-lipotropin (beta-LPH) and beta-endorphin (beta-END) from the anterior lobe, with beta-END predominating over beta-LPH by 2:1. However, repeated stress shifts this ratio to proportionately more beta-LPH released with re-stress or oCRH in vitro. Alternative hypotheses were that the glucocorticoids released during stress altered the processing of proopiomelanocortin (POMC) or that the increased biosynthetic drive resulted in an inability of the processing enzymes to keep pace with biosynthesis. To distinguish between these alternatives, adrenalectomy studies were performed. Following removal of glucocorticoid negative feedback there is greatly increased secretion of beta-END-IR from anterior lobe corticotrophs with a subsequent increase in biosynthetic drive. Under these conditions of increased biosynthetic drive in the absence of steroids, the corticotroph secretes primarily beta-LPH, suggesting that increased biosynthetic drive alters the posttranslational processing rate of POMC.     

AgNOR numbers in rat pituitary corticotrophs following adrenalectomy or corticotrophin releasing factor administration

Using a combined silver staining/immunoalkaline phosphatase technique, nucleolar organiser regions (AgNORs) were visualised and quantified in rat anterior and intermediate lobe pituitary corticotrophs following bilateral adrenalectomy or sham surgery. Compared to sham operated animals, the mean number of AgNORs was increased in anterior lobe corticotrophs in adrenalectomized rats and there was a shift to the right in the distribution. At 2 weeks after adrenalectomy, AgNOR numbers were greater than at 6 weeks. AgNOR numbers were also quantified in anterior lobe corticotrophs of intact rats receiving daily intraperitoneal injections of ovine CRF-41 at 50 micrograms/kg, which has been shown to stimulate ACTH release and to produce morphological evidence of increased corticotroph stimulation. CRF-41 did not produce an increase in AgNOR numbers, compared to saline injected controls.     

Lysosomes in anterior pituitary corticotrophs of the rat. A combined immunocytochemical and enzyme cytochemical study

E600 resistant non-specific esterase activity or acid phosphatase activity were localized in corticotrophic cells identified by postembedding immunocytochemistry (PAP of protein A-immunogold techniques). The lysosomal system of this cell type consists of dense bodies, of a population of small lysosomes mostly situated at the cell periphery in the vicinity of secretory granules as well as of tubular structures. These latter were located either in the central part of the cytoplasm and probably belonged to the Golgi apparatus or at the cell periphery, partly in the extensions. Small lysosomes occurred to be in continuity with enzyme-containing tubules. In a few structures lysosomal enzyme activity and ACTH immunoreactivity overlapped. Some autophagic vacuoles seemed to contain secretory granule matrix. It is suggested that the concept of crinophagy can be extended to the corticotrophs, though the lysosomal system may be involved in the specific function of this cell type by other mechanisms as well.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Cytochemical identification of human and murine pituitary corticotrophs and somatotrophs as APUD cells

Summary Cytochemical techniques, performed sequentially on single sections, have confirmed the identity of human and murine pituitary corticotrophs as APUD cells. These same methods indicate that in both species the acidophil somatotroph, identified as such by immunofluorescence using anti-human GH, possesses APUD qualities not only with respect to amine-handling, but also in terms of enzyme content.Inclusion of the somatotroph in the APUD series of cells which produce polypeptide hormones has far-reaching implications.     

The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Proliferation of pituitary corticotrophs following adrenalectomy as revealed by immunohistochemistry combined with bromodeoxyuridine-labeling

Proliferation of corticotrophs following adrenalectomy (ADX) was studied by a combination of bromodeoxyuridine (BrdU)-labeling and immunohistochemistry. Rats were adrenalectomized, allowed to survive for 1, 3, 7, and 14 days and given 100 mg/kg body wt BrdU 3 h before sacrifice. BrdU and adrenocorticotropic hormone (ACTH) were detected in the same sections of the anterior pituitary using double-labeling immunohistochemistry. BrdU-labeled cells in the pituitary showed a tendency to increase until 1 week after ADX and slightly decreased at 2 weeks. Corticotrophs were increased to about 1.5 times of the control level 1–2 weeks after ADX. The number of cells double-labeled with both BrdU and ACTH increased markedly after ADX, suggesting active mitosis of existing corticotrophs. On the other hand, the ratios of these double-stained cells to all BrdU-labeled cells and to all corticotrophs were 5–7% and 0.9–1.3%, respectively, even after ADX, suggesting that the majority of corticotrophs which were increased after ADX were recruited from some other type of immature cells. The extent to which the two mechanisms are involved in hyperplasia of corticotrophs after ADX remains to be elucidated.     

Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized,orchidectomized hamsters

Summary We investigated the effects of corticotrophin-releasing hormone (CRH) on the percentage of anterior pituitary gland (APG) cells which are corticotrophs as well as the size and shape of corticotrophs. Pituitary glands were removed from 7-week-old male hamsters and placed beneath the renal capsules of hamsters that had been hypophysectomized and orchidectomized 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 g CRH or vehicle for 16 days. Six hosts in each group were decapitated 16 h after the last injection. Sections of anterior pituitary tissue were stained for ACTH and with hematoxylin. The percentage of corticotrophs among APG cells was greater in allografts exposed to exogenous CRH (20%) than in allografts exposed to vehicle (15%). Exposure to exogenous CRH increased the cross-sectional area of corticotroph cells in allografts to values greater than those measured for corticotrophs in allografts exposed to vehicle, without altering the shape of cells. Results of subsequent studies suggested that hamsters with allografts injected with vehicle do not release ACTH and that exogenous CRH causes an abrupt release of ACTH from allografts. These results indicate that CRH releases ACTH from ectopic corticotrophs and that administration of CRH can increase corticotroph size and the percentage of APG cells that are corticotrophs.     

Opposing actions of the enantiomers of BAY-K-8644 on calcium currents and ACTH secretion in clonal pituitary corticotrophs

BAY-K-8644 in low concentrations is known to stimulate, and in higher concentrations, to depress calcium-dependent ACTH secretion from mouse clonal (tumor) pituitary corticotrophs, AtT-20/D16-16 (AtT-20). In the present study, voltage-dependent inward calcium currents in these cells were potentiated by low concentrations of this compound and depressed by higher concentrations consistent with its actions on ACTH secretion. A similar relationship was demonstrated for a different but related compound, CGP 28,392. Each of BAY-K-8644's enantiomers, BAY-R(-)5417 and BAY-R(+)4407, had opposing effects upon these inward calcium currents and ACTH secretion. The (+)isomer antagonized both inward calcium currents and ACTH secretion. In contrast, the (-)enantiomer was responsible for the stimulatory effects of BAY-K-8644. Nevertheless, some antagonistic properties were noted with high concentrations of this latter enantiomer. The stimulation of ACTH secretion in AtT-20 cells by low concentrations of BAY-K-8644 can be attributed to a potentiation of voltage-activated calcium currents by one of its enantiomers, BAY-R-(-)5417. In contrast, the depression of secretion that occurs at higher concentrations is likely to be the result of the reduction of these currents by the other enantiomer (BAY-R(+)4407).     

Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs. Evidence of post-translational control of cell differentiation

Using gel and ion-exchange chromatography monitored by radioimmunoassays specific for sequences essential in the processing of preprogastrin and preprocholecystokinin, the products were characterized in extracts of porcine pituitary lobes before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. The intermediate and neural lobes contained only fully activated (i.e. alpha-amidated) preprogastrin products (component I, gastrin-34, and gastrin-17). In contrast, the anterior lobe contained, in addition to traces of alpha-amidated gastrin (2 pmol/g), hundredfold higher amounts of a nonamidated progastrin (189 pmol/g; Mr approximately 7000) and two nonamidated procholecystokinin fragments (75 pmol/g; Mr approximately 7000 and 5000). These results show that hormone genes, in spite of translation of their mRNA, are not necessarily expressed in functional peptides in cells outside the principal production regions. Hence, the study indicates that differentiation of endocrine cells may be controlled at the post-translational level.