Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Ultrastructural localization of immunoreactive corticotropin, β-lipotropin, α- and β-endorphin in Cells of the human fetal anterior pituitary

Summary Cells immunoreactive with anti--(17–39) ACTH, -(1–24) corticotropin, -LPH, - and -EP were identified in the human fetal anterior pituitary at the ultrastructural level using the peroxidase-antiperoxidase complex method on ultrathin sections.Only one definite cell type was revealed by all these antisera. All granules of each individual immunostained cell reacted regardless of the antiserum used. The immunostained cells occurred in groups and were sometimes located in the wall of the follicle-like structures commonly observed in the fetal anterior pituitary. The cells revealed two main aspects: 1) The largest elements were rich in organelles, and their numerous secretory granules showed significant variations in size (250–500 nm in diameter), electron density of their content and stain-deposit intensity. The ergastoplasm, consisting of irregular tubules, was poorly developed. In the vicinity of the conspicuous Golgi apparatus, organelles related to the GERL complex were commonly observed. Multivesicular bodies were frequent. Some of these cells showed bundles of microfilaments (60 nm in thickness). 2) The smaller cells had an electron-lucent hyaloplasm with sparse organelles; they contained fewer granules and never showed microfilaments.The immunocytological results are consistent with the synthesis of a molecule similar to pro-opiocortin by this type of endocrine cell in human fetuses. Morphological evidence for the maturation process of this precursor and for the secretory activity of these cells and its possible regulation is presented and discussed.Abbreviations used ACTH corticotropin (39 amino acid polypeptide) - -MSH -melanotropin (ACTH [1–13]) - CLIP corticotropin-like intermediate lobe peptide (ACTH [18–39]) - -LPH -lipotropin (91 amino acid polypeptide) - -MSH -melanotropin (-LPH [41–58]) - -EP -endorphin (-LPH [61–91]) - -EP -endorphin (-LPH [61–76]) - PTA phosphotungstic acid Acknowledgements: The authors would like to thank Professors P. Magnin and J. Liaras, Hôpital Edouard Herriot; M. Dumont, Hôpital de la Croix-Rousse; A. Notter and R. Garmier, Hôtel Dieu; M. Bethenod, Hôpital Debrousse, Lyon, and the entire staff whose cooperation enabled samples to be taken under optimal conditions. The authors also thank Professor L. Graf, Research Institute for Pharmaceutical Chemistry (Budapest), and Professor R. Guillemin (Salk Institute, La Jolla) for their generous gift of antigensThis work was supported by a grant from I.N.S.E.R.M., ATP 46.77.78 (P.M. Dubois)     

Evidence for the occurrence of two forms of β-lipotropin in rat pituitary

A peptide isolated from porcine gut according to its glucagon-like activity in liver (bioactive enteroglucagon) has been characterized immunologically, biologically and chemically: its potency relative to pancreatic glucagon in interacting with an antiglucagon antibody, hepatic glucagon-binding sites and hepatic adenylate cyclase was ～100%, 20% and 10%, respectively. In contrast, it is ～20-times more potent than glucagon in oxyntic glands, justifying the term ‘oxyntomodulin’. Chemically, it consists in the 29 amino acid-peptide glucagon elongated at its C-terminal end by the octapeptide Lys—Arg—Asn—Lys—Asn—Asn—Ile &;—Ala; accordingly, it is called ‘glucagon-37’     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.     

Presence of β-endorphin-like immunoreactivity in the anterior pituitary gland of rat and man and evidence for the differential localization with ACTH

Summary Two antisera, Y-10 and Y-18 were raised in rabbits against synthetic human -endorphin conjugated to bovine serum albumin and keyhole limpet haemocyanin respectively. Antiserum Y-10 has been shown by radioimmunoassay to be highly specific for human -endorphin with minimal or no cross-reactivity against other pituitary peptides whilst antiserum Y-18 crossreacted on an equimolar basis against -endorphin and -lipotropin. When used in the immunohistochemical procedure, both antisera specifically stained the corticotrophs in human anterior pituitary tissue. A similar effect was observed when antiserum Y-18 was applied to rat anterior pituitary tissue in the immunohistochemical procedure. Y-10 antiserum, on the other hand, stained not only rat corticotrophs but also somatotrophs. The somatotrophin staining could not be attributed to the enkephalins reported to be present in these cells.The non-specific -endorphin antiserum Y-18 was used to stain anterior pituitaries from dehydrated and adrenalectomized rats as well as rats of the Brattleboro strain. In tissues from the three experimental animals, cells that stained positively for -endorphin did not give a positive immunoreaction for ACTH and vice versa in some other sections. It is concluded that under the physiological conditions, formalin fixation of the tissue causes the proopiocortin molecule to be trapped in a conformation such that either ACTH or -endorphin-like determinants are available for reacting with the appropriate antiserum.This work was financed by the Medical Research Council of New Zealand, NIH Research Program Project Grant HD-12303 and by U.S.P.H.S. Grant NS-16304 from NIH. We thank Drs Guillemin, Bloom and Ling for samples of -endorphin and -endorphin antisera     

Radioimmunoassay for β-endorphin: Presence of immunoreactive “big-big” β-endorphin (“big” β-lipotropin) in human and rat pituitaries

A sensitive and specific radioimmunoassay for β-endorphin has been developed with an antiserum obtained in a rabbit immunized with β-endorphin contained in crude porcineACTH preparations. The minimal detectable quantity was 5 pg. The antiserum used reacted slightly with ovine β-lipotropin (5.5 %), but showed negligible cross-reactivity with other fragments of β-lipotropin, α-MSH and ACTH. Using this radioimmunoassay we have observed the presence of “big-big” β-endorphin (“big” β-lipotropin) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitaries respectively, in addition to β-lipotropin and β-endorphin, by Sephadex gel-chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Reinvestigation of the N-terminal amino acid sequence of β-lipotropin from human pituitary glands

The N-terminal sequence of the first 60 amino acid residues of human β-LPH was reinvestigated by automated Edman degradation on the intact peptide. The result shows some differences with the recently proposed data. We have established the amino acid sequence of residues 9–26 to be Glu-Gly-Asp-Gly-Pro-Asp-Gly-Pro-Ala-Asp-Asp-Gly-Ala-Gly-Ala-Gln-Ala-Asp. From residues 1–8 and 27–60 the sequence is exactly identical with that of recently reported data.     

The regulation of growth hormone secretion from the isolated rat anterior pituitary in vitro. The role of adenosine 3′:5′-cyclic monophosphate

1. The release of growth hormone from isolated fragments of rat anterior pituitary tissue incubated in vitro was studied by employing a double-antibody radioimmunoassay. 2. In the absence of added stimuli, two phases of hormone release could be distinguished, an early phase of 2h duration and a subsequent late phase. In the early phase, hormone release was rapid but could be significantly decreased by calcium depletion and by 2,4-dinitrophenol whereas the rate of release in the late phase was uninfluenced by these incubation conditions. These results have been interpreted as indicating the existence of a secretory component in the early phase of release. 3. In subsequent experiments, the effects of various agents on the rate of hormone output during the late phase of incubation were investigated. Hormone release was increased by theophylline and by dibutyryl cyclic AMP (N(6)-2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate), the response to both of these agents being related to the concentration of the stimulant employed. 4. The stimulation of growth hormone output by theophylline was significantly decreased by calcium deprivation and by 2,4-dinitrophenol. The response to dibutyryl cyclic AMP was diminished by 2,4-dinitrophenol, iodoacetate and 2-deoxyglucose but not by malonate or colchicine. 5. Arginine, beta-hydroxybutyrate, albumin-bound palmitate and variation in the glucose concentration of the incubation medium over a wide range were without any statistically significant effect on the rate of hormone release from either control pituitary fragments or those subject to secretory stimulation by dibutyryl cyclic AMP. 6. It is suggested that the regulation of growth hormone secretion is mediated by cyclic AMP (adenosine 3':5'-cyclic monophosphate). The secretion observed in response to cyclic AMP requires the presence of ionized calcium and a source of metabolic energy but is independent of pituitary protein synthesis de novo. The integrity of the glycolytic pathway of glucose metabolism appears to be essential for cyclic AMP-stimulated growth hormone secretion to occur.     

Immunohistochemical localization of γ-melanocyte-stimulating hormone in the rat pituitary gland

A third melatropin fragment named γ-MSH has been described in the N-terminal portion of the common precursor of bovine ACTH and β-LPH by Nakanishi et al. (Nakanishi, S., Inoue, A., Kita, K., Nakamura, M., Chang, A.C.Y., Cohen, S.N. and Numa, S., Nature, 278 (1979) 423–427). In order to determine if immunoreactive γ-MSH was present in the rat pituitary gland and to accurately localize this peptide, an immunocytochemical localization of γ-MSH was conducted at both light and electron microscopic levels. Specific immunostaining was detected in stellate cells scattered throughout the pars distalis and in all the cells of the pars intermedia. At the ultrastructural level, immunoreactive γ-MSH was only observed in the lipocorticotrophs. Using serial ultrathin sections, it was shown that the secretory granules which contain ACTH were also labeled for γ-MSH. These results suggest that fragment(s) of the common precursor of ACTH and β-LPH and/or the whole common precursor is released with peptides of known biological activity.     

Serotonergic mechanism in the control of β-endorphin and acth release in male rats

The role of the serotonergic mechanism in the regulation of β-endorphin (β-EP) and adrenocorticotropin (ACTH)-like immunoreactivity in plasma was investigated. Increases in β-EP and ACTH-LI produced by quipazine maleate (QPZ), a serotonergic agonist, 1 hr after injection could be completely prevented by the serotonin (5-HT) antagonist, cinanserin (CIN), which when injected alone, decreased basal plasma concentrations of both β-EP-LI and ACTH-LI. Concurrent injections of L-5-HTP with the 5-HT reuptake inhibitor, fluoxetine, produced an additive increase in plasma β-EP-LI 1 hr after injection. Injection of the 5-HT antagonist, cyproheptadine, significantly decreased plasma β-EP-LI. Stress by immobilization for 30 min or exposing the rats to 40° ± 1°C for 30 min produced an approximate 4-fold increase in plasma β-EP-LI and ACTH-LI, which was potentiated by I.P. injections of fluoxetine. Furthermore, the stress induced increases in plasma concentrations of β-EP-LI and ACTH-LI were significantly reduced by the serotonin antagonists metergoline and cinanserin. These results suggest that 5-HT is a potent stimulator of both β-EP and ACTH release and the increase in plasma concentrations of ACTH and β-EP induced by stress are probably mediated, at least in part, by central serotonergic mechanisms.     

Isolation,characterization and opiate activity of β-endorphin from human pituitary glands

The isolation of a 31-amino acid peptide from human pituitary glands has been described. Its amino acid sequence has been proposed to be identical to the sequence of the carboxyl-terminal 31 amino acids of human β-lipotropin. The peptide, designated as β_h-endorphin, possesses significant opiate activity.     

The effect of PGP on β-hexosaminidase and histamine secretion in rat peritoneal mast cells in vitro

The investigation of the effect of peptide prolyl-glycyl-proline (PGP) on β-hexosaminidase and histamine secretion by mast cells in primary culture has shown that incubation of mast cells with PGP (6 × 10⁻⁵ M) before their activation by synacten significantly decreased the amount of secreted histamine and β-hexosaminidase in comparison with the action of synacten only. The peptide in investigated concentration had no influence on the level of spontaneous secretion. Incubation of cells with PGP did not prevent their activation by compound 48/80. Therefore, PGP can have a direct effect on isolated rat mast cells in vitro and diminish their secretory activity under activation by synacten.     

Avian β-endorphin: Alterations in immunoreactive forms in plasma and pituitary of embryonic and adult chickens

1. A heterologous radioimmunoassay for β-endorphin (β-endo) was established. Plasma and/or extracts of pituitaries from embryonic (days 14.5 and 17.5 of incubation), newly hatched, and adult chickens were chromatographed on a Sephadex G-75 column with 0.1 M acetic acid.2. Embryonic plasma had only a single immunoreactive peak that eluted similar to a β-lipotropin (β-LPH) standard. In contrast, adult plasma had 2 peaks, co-eluting with β-LPH (34%) and β-endo (66%).3. Chromatography of pituitary extracts demonstrated two immunoreactive peaks in both embryonic and adult birds. Although 70% of immunoreactivity eluted with β-endo for embryonic birds, 80% eluted with β-LPH from adults.4. The smaller proportion of β-endo in adult pituitaries may reflect a higher rate of secretion of this hormone into the blood.