Stimulation of pituitary corticotrophs in the rat--ultrastructural studies

The ultrastructural changes occurring in the corticotrophs of adult male and female Sprague-Dawley rats at 2 and 6 weeks after bilateral adrenalectomy were assessed on both a qualitative and quantitative basis. Qualitative changes were similar to those previously described but at both time points, female rats showed more marked changes than males. Corticotroph hypertrophy reached a plateau in male animals between 2 and 6 weeks, but continued to increase in females. There was an increase in mean granule diameter in both sexes at 2 weeks after adrenalectomy. The changes induced by the daily administration of CRF for 2 weeks by intraperitoneal injection were also examined in male rats. CRF induced corticotroph hypertrophy at both 25 micrograms/Kg and 50 micrograms/Kg body weight and increased the granule content. The addition of vasopressin (VP) to the higher dose of CRF induced a further increase in cell area and reduction in granule content. Low dose CRF was associated with an increase in mean granule diameter, whereas a decrease was seen after high dose.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

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).     

Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

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.     

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)     

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.     

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.     

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.     

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.     

Extracellular calcium sensing and signalling

Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+--the extracellular calcium-sensing receptor--has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular calcium-sensing receptor.     

Pituitary cell type-specific electrical activity, calcium signaling and secretion

All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.     

Atherosclerosis and calcium signalling in endothelial cells

The link between atherosclerosis and regions of disturbed flow and low wall shear stress is now firmly established, but the causal mechanisms underlying the link are not yet understood. It is now recognised that the endothelium is not simply a passive barrier between the blood and the vessel wall, but plays an active role in maintaining vascular homeostasis and participates in the onset of atherosclerosis. Calcium signalling is one of the principal intracellular signalling mechanisms by which endothelial cells (EC) respond to external stimuli, such as fluid shear stress and ligand binding. Previous studies have separately modelled mass transport of chemical species in the bloodstream and calcium dynamics in EC via the inositol trisphosphate (IP(3)) signalling pathway. We review existing models of these two phenomena, before going on to integrate the two components to provide an inclusive new model for the calcium response of the endothelium in an arbitrary vessel geometry. This enables the combined effects of fluid flow and biochemical stimulation on EC to be investigated and is the first time spatially varying, physiological fluid flow-related environmental factors have been combined with intracellular signalling in a mathematical model. Model results show that low endothelial calcium levels in the area of disturbed flow at an arterial widening may be one contributing factor to the onset of vascular disease.     

Atrial cardiomyocyte calcium signalling

Whereas Ca²⁺ signalling in ventricular cardiomyocytes is well described, much less is known regarding the Ca²⁺ signals within atrial cells. This is surprising given that atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. Disruption of the rhythmic beating of atrial cardiomyocytes can lead to life-threatening conditions such as atrial fibrillation. Atrial and ventricular myocytes have many structural and functional similarities. However, one key structural difference, the lack of transverse tubules (“T-tubules”) in atrial myocytes, make these two cell types display vastly different calcium patterns in response to electrical excitation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that originate around the periphery of the cells. Under resting conditions, such Ca²⁺ signals do not propagate towards the centre of the atrial cells and so do not fully engage the contractile machinery. Consequently, contraction of atrial myocytes under resting conditions is modest. However, when atrial myocytes are stimulated with a positive inotropic agonist, such as isoproterenol, the peripheral Ca²⁺ signals trigger a global wave of Ca²⁺ that propagates in a centripetal manner into the cells. Enhanced centripetal movement of Ca²⁺ in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.