Immunohistochemical study of the adenohypophysis of the monkey Macaca irus with the use of antibodies anti 1-24-ACTH, anti 17-39-ACTH, anti alpha-and beta-endorphins, anti beta-LPM]

Indirect immunofluorescence technique with anti1-24- and anti17-39 ACTH, anti alpha- and anti beta-endorphins, anti beta-LPH sera has allowed us to detect a cellular type in the anterior lobe of the hypophysis of Macacus irus which react simultaneously with these five antisera. These cells are especially localized in the ventro-medial zone, but there are also present in the pars distalis, under the glandular capsule, and in the lateral lobes, amid the other cellular types. The cells of the intermediate lobe react on the whole with anti1-24-, these antisera are also immunoreactive with the anti alpha- and anti17-39ACTH and anti beta-LPH ; SOME CELLS, WHich react with anti beta-endorphin antisera. The adenohypophysis of Macacus irus contains therefore two categories of cells reacting with the above mentioned antisera : one of this type, localized in the anterior lobe and in the intermediate lobe, react simultaneously with the five antisera, the other type, localized only in the intermediate lobe does not react with the antiendorphins antisera.     

Cyto-immunological study of the Pleurodele pituitary gland]

In the pituitary of an Amphibian (Urodela), various cell types were localized by cytoimmunological techniques: corticotrophs located in a rostral zone near the median eminence, ventral orangeophilic cells secreting a prolactin-like hormone and erythrosinophilic cells in a more dorso-caudal situation secreting growth hormone. Alpha-MSH and beta-MSH produceng cell were identified in the intermediate lobe; these cells were also labelled with antibodies against 1-24 ACTH and 17-39 ACTH. These data are in accordance with results obtained after an experimental study of the prolactin cells.     

Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.     

Comparative immunocytochemical localization of prolactin and somatotropin in the pituitaries of Lepidosiren paradoxa,Rana temporaria and Ambystoma mexicanum

Summary The cellular binding sites of anti-oPRL IgG and anti-bSTH IgG were demonstrated in the pituitary glands of Lepidosiren paradoxa, Rana temporaria and Ambystoma mexicanum by means of the unlabeled antibody enzyme method by light and electron microscopy (the latter only in Lepidosiren). With the light microscope PRL or PRL-like substances and STH or STH-like substances were revealed in two different cell types in the distal lobe corresponding to the acidophils. However, as a result of the insufficient differentiation of the acidophils in Lepidosiren after staining with Brookes' procedure it was not possible to distinguish the two types of acidophils in this animal. Treatment with low dilutions of both anti-oPRL and anti-bSTH IgG revealed simultaneous immunocytochemical staining in both types of acidophils in Lepidosiren and in Rana. These results, indicating that there is antigenic cross-reaction between anti-oPRL and anti-bSTH IgG and both PRL and STH in these animals, are discussed.The electron microscopic investigations of Lepidosiren revealed that the specific anti-oPRL IgG reactive cells contain granules ranging in size from 200 to 300 nm, while the specific anti-bSTH IgG reactive cells contain smaller immunoreactive granules ranging from 80 to 160nm.     

Existence of "endorphin cells" in the adenohypophysis of the cat; comparison with "corticotropin cells." Immunocytochemical study]

Indirect immunofluorescence technique with anti-17-39ACTH and anti beta-endorphin sera has allowed us to detect "corticotropic cells" in the anterior and intermediate lobes of the adenohypophysis of the male cat. The corticotropic cells of the anterior lobe are localized in the median zone; they are PAS-positive and appeared intensively coloured in dark blue with the Herlant's tetrachrome. All the cells of the intermediate lobe react with the anti-17-39ACTH serum. Using an anti-beta-endorphin serum, we have observed that all the corticotropic cells of the anterior lobe react; but in the intermediate lobe, only a part of "corticotropic cells" react with the anti-beta-endorphin serum.     

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.     

Biosynthesis and characterization of adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone, and an NH2-terminal fragment of the adrenocorticotropic hormone/beta-lipotropin precursor from rat pars intermedia.

Isolated intermediate lobe cells from 40 rat pituitaries were incubated for 3 h with [35S]methionine + [3H]-phenylalanine, [35S]methionine, [3H]valine, and [3H]leucine. The cell extracts were purified by carboxymethyl-cellulose chromatography (CMC) and the fraction eluting with ovine adrenocorticotropic hormone (ACTH) was further purified either by another CMC under the same conditions or by high performance liquid chromatography (HPLC). Microsequencing of the product from the second CMC allowed the identification of a peptide containing methionine 4 and phenylalanine 7, as expected for the NH2 terminus of ACTH. Purification by HPLC of a similar peptide obtained from the three other incubations gave three main raoactive peaks which were further characterized by their migration rates on polyacrylamide gels, molecular weight, and microsequencing. Results indicated that intact ACTH (residues 1-39) is present in extracts of rat intermediate lobe, but in very small quantities (less than 1% of the beta-endorphin content). ACTH is probably broken down into smaller fragments, e.g. alpha-melanocyte-stimulating hormone (alpha-MSH) (ACTH, 1-13) and corticotropin-like intermediate lobe peptide (CLIP) (ACTH, 18-39). These studies also revealed with existence of a peptide having identical sequence with the (N-1) terminus of the ACTH/lipotropin (LPH) precursor.     

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.     

The role of a low pH intracellular compartment in the processing, storage, and secretion of ACTH and endorphin

To determine whether a low pH intracellular "sorting" step is required to route peptides into secretory granules, the effects of pH altering drugs on the biosynthesis and secretion of peptides by AtT-20 mouse corticotrope tumor cells and rat intermediate pituitary cells were examined. Doses of each drug maintaining normal protein synthesis and cell morphology, while obliterating the intracellular pH gradients detected by acridine orange fluorescence, were experimentally determined. Regions of the cell rich in secretory granules were localized by immunocytochemistry and were found to coincide with organelles with a low internal pH. Biosynthetic labeling experiments were coupled with immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel analyses to examine the biosynthesis and secretion of corticotropin (ACTH(1-39], alpha-melanotropin, ACTH(18-39), beta-endorphin, gamma-melanotropin, alpha-amidated joining peptide, and the NH2-terminal region of pro-ACTH/endorphin. Chloroquine (20-40 microM) and a mixture of NH4Cl and methylamine (2-5 mM each) dissipated pH gradients but had no effect on the synthetic rate of pro-ACTH/endorphin, the extent and rate of precursor processing to smaller peptides, the rate of basal secretion of the various peptides, or the extent to which secretion of each of the peptides could be stimulated by secretagogues. Monensin (0.1-1 microM) had no discernible effect on intracellular pH gradients yet totally blocked proteolytic processing of pro-ACTH/endorphin. Thus, a monensin-blockable step occurs in peptide processing, presumably in the trans Golgi region; however, a low pH chloroquine-sensitive sorting step is not required for processing or for routing peptides to a stable storage form which can be released in response to secretagogues.     

Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary

Summary The question is examined whether -melanocyte stimulating hormone (-MSH), adrenocorticotropic hormone (ACTH), met-enkephalin and -endorphin are detectable by enzyme immunocytochemistry in the cells of the intermediate lobe (PI) of the rat pituitary. By applying antibodies against MSH, ACTH and -endorphin on light microscopic sections, intense immunostaining was found in all PI-cells. At the ultrastructural level, after treatment of consecutive serial sections with these three antibodies the immunoreactivity was localized in the same secretory granules. No specific metenkephalin immunoreactivity could be detected in the cells of the intermediate lobe.Supported by Deutsche Forschungsgemeinschaft SFB 87/B2     

Identificaton of UV,green and red receptors,and their projection to lamina in the cabbage butterfly,Pieris rapae

Summary The photoreceptors in the compound eye of a cabbage butterfly, Pieris rapae, were examined by conventional and intracellular-labeling electron microscopy by the use of the cobalt(III)-lysine complex as an ionized marker. Five types of spectral sensitivity were recorded intracellularly in electrophysiological experiments. They peaked at about 340, 380, 480, 560 and 620 nm, respectively. One of the distal retinula cells (R2) was a UV receptor, whereas the R4 distal retinula cell was a green receptor. The basal retinula cell, R9, was found to be a red receptor; it was localized near the basement membrane, having a bilobed cell body with an individual nucleus in each lobe. A small number of rhabdomere microvilli were present in a narrow cytoplasmic bridge connecting the two lobes. The axons of six retinula cells (R3–R8) in each ommatidium terminated at the cartridge in the lamina (short visual fiber), whereas those of the other three retinula cells, R1, R2 and R9, extended to the medulla (long visual fiber). The information from the UV and red receptors is therefore probably delivered directly to the medulla neurons, independent of that from the other spectral receptor types.     

Vasopressin and "CRF" in the regulation of ACTH secretion by the anterior and intermediate lobes of the pituitary gland (author's transl)

The aim of this review was to summarize the present state of knowledge concerning the mode of action of vasopressin (VP) and the putative corticotropin releasing factor (CRF) on ACTH secretion from the anterior and intermediate lobes of the pituitary gland. In vitro data show that although both CRF and VP enhanced release of anterior pituitary ACTH, the pattern of hormonal release, based on kinetical and dose-dependent studies, appeared to be different. Also, the effect of VP most probably was mediated by specific putative receptor sites. In contrast, VP was found not to alter ACTH secretion from the intermediate lobe; that secretion seems to be regulated by CRF-like material and neurotransmitters. The importance of VP as a corticotropin agent is discussed.     

Demonstration by immunofluorescence of adenohyphysis corticotropin and melanotropin cells in Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas]

Using antibodies against synthetic corticotropic hormones (1-24 ACTH and 17-39 ACTH), and melanotropic hormones (alpha-MSH and beta-MSH), it is possible to identify corticotropic and melanotropic cells in the adenohypophysis of three species of monkeys : Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas. The corticotropic cells are numerous in the anterior lobe in both the adult and infant male and female monkeys of these three species. The intermediate lobe reacts with antibodies against ACTH and also with antibodies against the two MSH. In the anterior lobe, the corticotropic cells react also with anti-beta MSH antibody but not with the anti-alpha MSH antibody.     

Plasminogen activators of the pituitary gland: enzyme characterization and hormonal modulation

We studied plasminogen activator (PA) of the rat pituitary gland in organ and cell monolayer culture. Both anterior and intermediate lobes contain, synthesize and secrete a mixture consisting of the two known types of PA: urokinase and so-called tissue PA. Both enzymes were formed essentially by all PA secreting cells, and PA was identified specifically in mammotrophs, corticotrophs, and luteinizing hormone containing gonadotrophs. Pituitary PA production was modulated on exposure to a variety of biological effectors: anterior lobe PA secretion was stimulated by agents that raised intracellular cAMP concentration; his process depended on de novo enzyme synthesis. Enzyme production was repressed by androgens and glucocorticoids. When anterior lobe cultures were maintained in plasminogen-free media, the extracellular, secreted forms of ACTH consisted almost exclusively of the high molecular weight forms (31,000 and 23,000); the smaller forms (13,000 and 4,500) were also found in the extracellular medium of cultures supplemented with plasminogen. In contrast, the size distribution of intracellular ACTH species was unaffected by the presence of plasminogen. These results resemble those previously obtained with pancreatic islets and are consistent with the possibility that plasmin, generated by PA secretion, participates in prohormone processing. PA synthesis in intermediate lobe explants was stimulated by exposure to dibutyryl cAMP, and repressed by hydrocortisone. In accordance with the dopaminergic control of intermediate lobe function in some vertebrates, apomorphine strongly repressed PA synthesis in intermediate, but not anterior lobe cultures.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Gastro-intestinal and neurohormonal peptides in the alimentary tract and cerebral complex ofCiona intestinalis (Ascidiaceae)

Summary Polypeptide-hormone producing cells were localized in the alimentary tract and cerebral ganglion ofCiona intestinalis using cytochemical, immunocytochemical and electron-microscopical methods.Antisera to the following peptides of vertebrate type were employed: bombesin, human prolactin (hPRL), bovine pancreatic polypeptide (PP), porcine secretin, motilin, vasoactive intestinal polypeptide (VIP),-endorphin, leu-enkephalin, met-enkephalin, neurotensin, 5-hydroxytryptamin (5-HT), cholecystokinin (CCK), human growth hormone (GH), ACTH, corticotropin-like intermediate lobe peptide (CLIP) and gastric inhibitory peptide (GIP).Immunoreactive cells were found both in the alimentary tract epithelium and in the cerebral ganglion for bombesin, PP, substance P, somatostatin, secretin and neurotensin. Additionally, in the cerebral ganglion only, there were cells immunoreactive for-endorphin, VIP, motilin and human prolactin. 5-HT positive cells, however, were restricted to the alimentary tract.No immunoreactivity was obtained either in the cerebral ganglion or in the alimentary tract with antibodies to leu-enkephalin, met-enkephalin, CCK, growth hormone, ACTH, CLIP and GIP. Prolactin-immunoreactive and pancreatic polypeptide-immunoreactive cells were argyrophilic with the Grimelius' stain and were found in neighbouring positions in the cerebral ganglion.At the ultrastructural level five differently granulated cell types were distinguished in the cerebral ganglion. Granules were present in the perikarya as well as in axons. The possible functions of the peptides as neurohormones, neuroregulators and neuromodulators are discussed.     

Cytoimmunological and histochemical study of cat adenohypophyseal cells, made fluorescent by the Falck and Hillarp technic]

In the Cat, after Falck and Hillarp method, all the fluorescent cells of the PI and the anterior lobe of adenohypophysis can be revealed with specific anti-sera to ACTH(1-24), ACTH(17-39), bovine beta-MSH and porcine beta-LPH. With the lead hematoxyline staining method, two types of cells are recognizable in the anterior lobe, in which the non hormonal constituents of the granules must be different.     

Electron-microscopic investigation of the innervation of the pars distalis of the hypophysis in adult Rana temporaria L.

Summary In the pars distalis of the hypophysis of adult Rana temporaria, three types of nerve-fiber profiles were found at two distinct sites, in both lateral parts of the bordering regions of the anterior lobe with the intermediate lobe of the hypophysis. The first type of nerve-fiber profile consists of bundles of very fine axonal elements (diameter: <0.7 m). The second type is formed by larger nerve fibers (diameter up to 4 m) containing a few neurosecretory granules of approximately 100 nm. The third type of nervefiber profile resembles the second type but these nerve fibers make synaptoid contacts on at least two different types of glandular cells. The possible functional significance of these nerve fibers in the pars distalis is discussed.No nerve fibers were found (1) in the central part of the bordering region of the pars distalis with the intermediate lobe, (2) at the bordering region with the median eminence and (3) with the neurohypophysial stalk, and (4) in all other parts of the pars distalis.     

Regulated Secretion of Pro-Opiomelanocortin Converting Enzyme and an Aminopeptidase B-Like Enzyme from Dispersed Bovine Intermediate Lobe Pituitary Cells

Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro-opiomelanocortin converting enzyme (PCE) and an aminopeptidase B-like enzyme (APBE)] and alpha-melanotropin (alpha-MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8-bromo-cyclic AMP produced significant increases in levels of immunoreactive alpha-MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2-bromo-alpha-ergocryptine resulted in significant decreases in secretion of alpha-MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal beta-glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro-opiomelanocortin primarily to 23,000-Mr corticotropin (ACTH), 13,000-Mr ACTH, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Argo-[Met]-enkephalin to [Met]-enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of alpha-MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro-opiomelanocortin processing in vivo.