Regulation of cellular alpha-MSH and beta-endorphin during stimulated secretion from intermediate pituitary cells: involvement of aspartyl and cysteine proteases in the control of cellular levels of alpha-MSH and beta-endorphin

The regulation of cellular levels of alpha-melanocyte stimulating factor (alpha-MSH) and beta-endorphin in response to stimulated secretion from intermediate pituitary cells in primary culture was investigated in this study. Regulation of the cell content of alpha-MSH and beta-endorphin occurred in two phases consisting of (a) initial depletion of cellular levels of these peptide hormones during short-term secretion (3 h) induced by isoproterenol, forskolin, or phorbol myristate acetate (PMA) which was followed by (b) long-term (24 h) increases in cellular levels of alpha-MSH and beta-endorphin in response to stimulated secretion induced by isoproterenol and PMA. In short-term experiments (3 h), cellular levels of alpha-MSH and beta-endorphin were reduced by 30-50% during stimulated secretion of these peptide hormones by isoproterenol (agonist for the beta-adrenergic receptor), forskolin that activates protein kinase A (PKA), and PMA that activates protein kinase C (PKC). Moreover, dopamine inhibited isoproterenol-induced depletion of cellular alpha-MSH and beta-endorphin. During long-term incubation of cells (24 h) with isoproterenol, cellular alpha-MSH and beta-endorphin were increased to twice that of controls (unstimulated cells). Treatment with PMA for 24 h also increased cellular levels of alpha-MSH and beta-endorphin. Moreover, cellular levels of alpha-MSH and beta-endorphin were decreased during long-term treatment of cells with an aspartyl protease inhibitor, pepstatin A, and with the cysteine protease inhibitor E64c. These results implicate aspartyl and cysteine proteases in the cellular production of alpha-MSH and beta-endorphin that requires proteolytic processing of their common precursor proopiomelanocortin (POMC). These findings demonstrate the parallel regulation of cellular levels of alpha-MSH and beta-endorphin during their cosecretion, which may involve aspartyl and cysteine proteases in the metabolism of these peptide hormones.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.     

Tumor promoters enhance basal and growth hormone releasing factor stimulated cyclic AMP levels in anterior pituitary cells

Tumor promoters, such as phorbol esters and teleocidin, amplified the ability of growth hormone releasing factor to increase pituitary cyclic AMP levels. This effect of tumor promoters was concentration-dependent, could be observed in 5 minutes, and was over by 4 hours. Inactive tumor promoters (i.e., 4-alpha-didecanoate) had no effect on this system, whereas a synthetic diacylglycerol (i.e., 1-oleoly-2-acetyl glycerol), mimicked the action of tumor promoters. Due to the known stimulation of protein kinase C by both tumor promoters and diacylglycerols, we suggest that this calcium and phospholipid dependent protein kinase C can enhance the ability of the growth hormone releasing factor receptor to activate the cyclic AMP generating system.     

Localization of sialic acid-containing hormones in GTH cells and ACTH cells of the rat anterior pituitary

Summary The localization of sialic acid-containing substances in the rat anterior pituitary gland has been studied by light and electron microscopy, using a peroxidase-labeled lectin (Limulus polyphemus agglutinin: LPA) which binds specifically to sialic acid residues. LPA stains two types of anterior pituitary cells: (1) round or ovoid cells which are also positively stained with anti-hCG (GTH cell), and (2) small, stellate cells which are unstained with anti-hCG (ACTH cell). All of the LPA-positive cells can be distinguished from TSH cells which are identified by the use of anti-hTSH. On ultrathin sections directly stained with LPA using the postembedding method, the reaction is confined to the secretory granules in GTH cells, and ACTH cells. Of two types of secretory granules in GTH cells, the larger one is intensely stained, whereas the smaller type shows only weak staining with LPA. Since follicle-stimulating hormone (FSH) is known to have high sialic acid contents, the results suggest possible detection of FSH with a technique other than immunohistochemistry. Furthermore, if the sialic acid-containing substances in GTH cells represents FSH, then these results support the hypothesis that LH cells and FSH cells are one cell type.This research was supported by grants from the Ministry of Education of Japan     

β-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of β-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), β-lipotropin and 3.5 kDa β-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of β-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10⁻⁸ M CRF, the levels of intracellular β-endorphin and ACTH immunoreactivity were reduced by 15–30% and there was a 4–5-fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa β-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10⁻⁷ M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10⁻⁶ M dexamethasone the basal secretion of ACTH and β-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.β-EndorphinACTHPituitary cell cultureProcessingCRFEpinephrine     

ACTH in bovine pituitary intraglandular colloid, the holocrine secretion of intermediate lobe cells

The presence of bovine pituitary intermediate lobe peptides in intraglandular colloid, the holocrine secretion of intermediate lobe cells, is explored by ELISA. Intraglandular colloid collected immediately after sacrificing the animal, is placed in phosphate buffered saline, pH 7.6. This material is homogenized, centrifuged to remove extraneous tissue, lyophilized and stored at -20 degrees C. ACTH in intraglandular colloid is measured by competitive ELISA. Human ACTH (1-24) is used in the preparation of the solid phase antigen and as the standard for competition. The antibody is rabbit anti-human ACTH (1-24), and the alkaline phosphatase conjugate is goat anti-rabbit IgG with p-nitrophenyl phosphate as substrate. It is concluded that ACTH is present in bovine pituitary intraglandular colloid of intermediate lobe origin and that the colloid may serve as a transport medium for intermediate lobe materials.     

Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.     

Stimulation of prolactin secretion from turkey anterior pituitary cells in culture

Prolactin (PRL) secretion by monolayer cultures of turkey anterior pituitary cells was significantly increased (up to 44-fold) by vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), and by an extract of turkey hypothalami (HE). Several other neuropeptides (including thyrotropin-releasing hormone) and neurotransmitters were ineffective in influencing PRL secretion at doses up to 10(-6) M. The dynamic PRL response to HE and VIP was studied using superfused pituitary cells attached to microcarrier beads. HE, administered in 30-min pulses, resulted in a significant, dose-related increase in PRL secretion from a basal secretion rate of 2.32 ng/min/10(7) cells to a peak secretion rate of 127.13 ng/min/10(7) cells at the highest dose of HE tested (1 mg tissue-equivalent weight/ml). VIP significantly increased PRL secretion at all doses studied (from 10(-10) to 10(-6) M), with 10(-8) M VIP producing a response similar to that observed with 1 mg/ml HE. A highly significant (P less than 0.001) linear relationship was demonstrated between the log-dose of VIP administered and peak PRL secretion rate. These studies suggest that VIP, but not TRH, may be a physiological stimulus for PRL release in the turkey.     

Dynamics and regulation of TSH secretion by superfused anterior pituitary cells

Superfused dispersed cells respond rapidly to 2- to 10-min pulses of TRH (10(-10) to 10(-7) M) in a dose-dependent manner. The effects of decreasing the stimulus duration can be overcome by a proportional increase in concentration of TRH. A TRH stimulus of 10 min or greater duration results in a sharp peak in TSH secretion followed by a lower plateau. Somatostatin (10(-8) M inhibits the response to TRH (t X 10(-9) M). T3 (2.0 microgram/dl) inhibits TRH-induced TSH secretion by superfused pituitary fragments, but not by dispersed cells. Corticosterone (50 microgram/dl), however, inhibits crude CRF-induced ACTH secretion by such cells.     

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.     

Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release

Apelin is a bioactive peptide recently identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. The presence of apelin-immunoreactive nerve fibers, together with the detection of apelin receptor mRNA in the parvocellular part of the paraventricular nucleus and the stimulatory action of apelin on corticotropin-releasing hormone release, indicate that apelin modulates adrenocorticotropin (ACTH) release via an indirect action on the hypothalamus. However, a direct action of apelin in the anterior pituitary cannot be excluded. Here, we provided evidence for the existence of an apelinergic system within the adult male rat pituitary gland. Double immunofluorescence staining indicated that apelin is highly coexpressed in the anterior pituitary, mainly in corticotrophs (96.5 +/- 0.3%) and to a much lower extent in somatotropes (3.2 +/- 0.2%). Using in situ hybridization combined with immunohistochemistry, a high expression of apelin receptor mRNA was also found in corticotrophs, suggesting a local interaction between apelin and ACTH. In an ex vivo perifusion system of anterior pituitaries, apelin 17 (K17F, 10(-6) M) significantly increased basal ACTH release by 41%, whereas apelin 10 (R10F, 10(-6) M), an inactive apelin fragment, was ineffective. In addition, K17F but not R10F induced a dose-dependent increase in K(+)-evoked ACTH release, with maximal increase being observed for a 10(-6) M concentration. Taken together, these data outline the potential role of apelin as an autocrine/paracrine-acting peptide on ACTH release and provide morphological and neuroendocrine basis for further studies that explore the physiological role of apelin in the regulation of anterior pituitary functions.     

Regulation of cysteine proteinases during different pathways of differentiation in cellular slime molds

Cysteine proteinase activities have been determined using gelatin-SDS-PAGE analysis and assays based on peptide nitroanilides. Vegetative myxamoebae of all species examined contain high levels of cysteine proteinase activity present in multiple forms. In both Dictyostelium discoideum and Polysphondylium pallidum the proteinase content is dependent on whether the cells are grown axenically or in association with bacteria. In all instances development is accompanied by a decreased intracellular cysteine proteinase activity. This occurs during the formation of fruiting bodies in D. discoideum, microcysts in P. pallidum, and macrocysts in Dictyostelium mucoroides. Significant quantities of proteinase activity are always secreted by myxamoebae immediately on starvation. In D. mucoroides this leads to an almost total depletion of intracellular cysteine proteinases by the aggregation stage. As a consequence of this depletion it has been relatively easy to detect a developmentally regulated accumulation of cysteine proteinases at the enzyme activity level, something which has not yet proved possible with D. discoideum. Three cysteine proteinases are produced as D. mucoroides macrocysts develop and mature. In the case of microcyst formation in P. pallidum the proteinase contents of the developing cells and of the microcysts are dependent on how the myxamoebae are grown. In this developmental pathway at least, there is no absolute requirement for specific proteinases to be present (or absent) at a particular stage. The diversity of cysteine proteinases found in cellular slime molds and the variety of features apparent in their regulation suggest that they will prove to be very useful for investigating features of the structure/function relationships in this important group of enzymes.