Processing of normal and non-glycosylated forms of toad pro-opiocortin by rat intermediate (pituitary) lobe pro-opiocortin converting enzyme activity

The influence of glycosylation of a prohormone, pro-opiocortin, on its processing by intermediate (pituitary) lobe converting enzyme activity in vitro was studied. [3H]-arginine-labeled glycosylated and non-glycosylated pro-opiocortins were isolated from untreated, and tunicamycin treated toad neurointermediate lobes, respectively, after pulse-labeling in [3H]-arginine containing incubation media. These labeled precursors were then incubated at 37 degrees C in the presence of pro-opiocortin converting enzyme activity derived from rat intermediate lobe (pituitary) secretory granule lysates. The rates of conversion of the glycosylated and nonglycosylated pro-opiocortins to smaller peptide products, in vitro, were similar. Analysis of the peptide products by immunoprecipitation with ACTH and beta-endorphin antisera, and subsequent electrophoresis on acid-urea gels, indicate a comparable processing in vitro of the two forms of pro-opiocortin substrate. The only difference was that the normally glycosylated peptide products derived from glycosylated pro-opiocortin (i.e., 13K ACTH, 21K ACTH, and the 16K glycopeptide) differed in their gel electrophoretic mobilities from their counterparts derived from nonglycosylated prohormone, in a manner consistent with the absence of carbohydrate on the latter's peptides. These data show that glycosylation of the prohormone does not influence its processing in vitro by the converting enzyme activity.     

Biosynthetic fate of the amino-terminal fragment of pro-opiomelanocortin within the intermediate lobe of the mouse pituitary

All the biosynthetic derivatives of pro-opiomelanocortin (POMC) were purified from an extract of 300 mouse neurointermediate pituitaries. Inspection of the amino acid composition of these peptides indicated that cleavage at all available dibasic processing sites within POMC was essentially complete except for -Arg49-Lys50- within the 1 to 74 amino-terminal sequence. Only about 50% of the 1 to 74 fragment was processed to the 1 to 49 sequence and Lys1 gamma 3MSH (i.e., the 50 to 74 sequence). The existence of these derivatives of the 1 to 74 fragment was confirmed by pulse-labelling explant cultures of mouse neurointermediate pituitaries with tritiated amino acids. Pulse/chase biosynthetic experiments indicated that the cleavage of the 1 to 74 sequence takes place 3 to 6 hours post-translation. This time course of biosynthesis suggests that the cleavage of the 1 to 74 sequence is a secretory granule event. Time course studies revealed that the minimum time required for newly synthesized derivatives of POMC to emerge from the intermediate lobe tissue was approximately 3 hours.     

Beta-endorphin in pituitary intraglandular colloid of intermediate lobe origin

Radioimmunoassay (RIA) detected the presence of beta-endorphin in the intraglandular colloid (IGC) of bovine pituitary intermediate lobe origin. The amount of beta-endorphin recovered in each of twelve samples ranged from 0.15 to 218.30 pmol/mg protein. A second group of assays [amino acid analysis, high performance liquid chromatography (HPLC) and mass spectral analysis] confirmed the RIA findings in another series of colloid samples. Approximately 75 pmol was collected from eight pooled glands. beta-Endorphin is an addition to the list of peptide hormones (e.g., methionine-enkephalin, adrenocorticotropin, arginine-vasopressin, alpha-melanocyte-stimulating hormone, beta-lipotropin and somatostatin) previously discovered in IGC by this laboratory.     

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.     

Immunoreactive calcitonin in the intermediate lobe of the pituitary gland.

Immunoreactive-calcitonin was observed in all cells of the intermediate lobe of the rat pituitary gland. It may thus be a fragment of the ≥31, 000 daltons precursor molecule of adrenocorticotropin and β-lipotropin.     

Adrenocorticotropin production by the intermediate lobe of the rat pituitary

Summary Tissue from the four chambers of the heart of the plaice (Pleuronectes platessa, L.) has been examined in the electron microscope in order to describe the morphology of the heart at a fine structural level.The sinus venosus is a thin walled chamber between 60–90 thick consisting of a connective tissue matrix in which are situated the plexus of the parasympathetic cardiac ganglion and localised bundles of myocardial cells. The myocardial cells do not form a continuous layer but are associated in particular with the region of the cardiac ganglion and are innervated by it.The sino-auricular junction has hitherto been described as a pacemaker region but the myocardial cells in this region are identical in morphology to myocardial cells in other parts of the heart. There is a large complex of nerves, derived from the cardiac plexus, that runs around the junction before branching to innervate the auricle.The myocardial tissues consist of an outer layer of myocardium forming the wall of the heart and a profusion of trabeculae. The endocardium invaginates into the endocardium to divide up the cells into populations of approximately 25 cells in profile. There is no well-defined coronary blood supply although capillaries are occasionally seen. The myocardial cells themselves are small in diameter (3.5–5.5 ) and show some primitive features which are: a short sarcomere (1.4–2.0 ), the absence of any sarcoplasmic reticulum, and very scarce fasciae occludentes. In the atrium in particular, there are many groups of 1500 Å membrane-bound, dense-cored vesicles in the myocardial cells. Ventricular cells contain more myofilaments and mitochondria than do atrial cells and have many vesicles of 0.1–0.3 diameter whose function and contents are unknown.Connective tissue is very evident in the plaice heart, being an integral part of the sinus venosus and the auriculo-ventricular junction and being the sole constituent of the auriculoventricular valve and the bulbus arteriosus.This investigation was carried out during the tenure of an S. R. C. studentshyp awarded to R. M. S.     

Evidence for an opiomelanotropin acetyltransferase in the rat pituitary neurointermediate lobe

Results of this study demonstrate two distinct forms of acetyltransferase activity which will acetylate α-MSH. These acetyltransferases are distinguished by pH optima, subcellular distribution and sensitivity to magnesium and several solubilizing detergents. A general acetyltransferase, as characterized in the rat pituitary anterior lobe and lens, has a pH optima of 7.4 and is inhibited by magnesium. Subcellular fractionation of anterior and neurointermediate lobes revealed that this acetyltransferase is primarily localized in the cytosol fraction of these tissues. An α-MSH acetyltransferase (MAT) and a β-endorphin acetyltransferase (EAT) have a pH opitma of 6.0–6.6, are inhibited by detergents, and are specifically localized in the secretory granules of the neurointermediate lobe. Comparative studies of MAT and EAT suggest a single enzyme responsible for the acetylation of opioid and melanotropin peptides, and we term this enzyme opiomelanotropin acetyltransferase (OMAT).     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

The dopaminergic innervation of the intermediate lobe and of the neural lobe of the pituitary gland

The intermediate and the neural lobe of the pituitary gland are innervated by two, virtually independent, groups of dopaminergic neurons which, until recently, were considered as a uniform system and referred to as the "tubero-hypophyseal dopamine system". Some aspects of the separate physiological functions of these neurons in the intermediate and in the neural lobe, of their microanatomy and biochemistry as well as of dopamine release from their terminals are discussed in this review.     

Secretory morphology of the intermediate lobe of the rat pituitary incubated in vitro

Summary The ultrastructure of the incubated intermediate lobe of the rat pituitary and the morphological effect of isoproterenol stimulation on its cells were studied under in vitro conditions. The general structure of isolated neurointermediate lobes maintained for 2–3 h in vitro was well preserved, and the presence of intact nerve terminals establishing synaptic contacts with the glandular cells of the intermediate lobe was confirmed. Removal of the intermediate lobe from central inhibition leads to increased hormonal secretion, which was reflected by large Golgi areas and the appearance of secretory images. However, no obvious degranulation or peripheral migration of the secretory granules after 2–3 h in vitro was seen. The secretory granules varied in electron density; totally electron-lucent granules were regularly observed and exocytotic phenomena were shown. In addition, more extensive invaginations suggesting secretion by compound exocytosis were seen. A three-fold increase in the -endorphin secretion during a 4-min stimulation with 10^-6 M isoproterenol did not induce any morphometrically detectable changes in the incubated cells. This indicates that only a minor fraction of the total granule content is mobilized during an acute increase in secretory activity.     

DNA synthesis in immunreactive glandular cells in pituitary intermediate lobe cultures

Summary Cell suspensions were prepared from adult rat pituitary intermediate lobes and grown in medium TC 199 supplemented with foetal calf serum. 7 or 8 days old cultures were pulse labelled with tritiated thymidine and thereafter processed for immunostaining using an antiserum against a synthetic -1-28-ACTH-analogue.Immunreactivity was mainly confined to epithelial cells, but some spindle-shaped cells were also immunpositive. Thymidine incorporation was observed in some immunopositive cells which shows that cells committed to hormone production may enter the mitotic cycle.     

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     

Presence of arginine-vasopressin in bovine pituitary intraglandular colloid of intermediate lobe origin

In order to determine whether pituitary intraglandular colloid might contain enough hormonal material to be considered a transport medium for intermediate lobe peptides, arginine-vasopressin was assayed in three pools of colloid collected from nearly 100 steers. Colloid samples taken from the pituitary glands of freshly slaughtered cattle were pooled, freed of extraneous tissue, and lyophilized. Three such pools were further extracted on octadecylsilyl-silica and assayed. The radioimmunoassay showed that arginine-vasopressin was indeed present in the bovine intraglandular colloid at levels ranging from 0.18 to 6.95 ng/mg dry weight. These results indicate that the intraglandular colloid contains a sufficient amount of arginine-vasopressin to be considered as a transport medium for this and other intermediate lobe peptides.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Effects of the monovalent ionophore monensin on the intracellular transport and processing of pro-opiomelanocortin in cultured intermediate lobe cells of the rat pituitary

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.     

Electron microscope studies on the intermediate lobe of the embryonic mouse

The development of the intermediate lobe of the hypophysis was studied in the embryonic C3H mouse; at least four glands from embryos of every gestational day from 15 to 19 were examined. In the 16 day-old embryo prospective secretory cells proliferate at the centre of the intermediate lobe anlage. At the same stage cylindrical cells bordering the hypophyseal cleft begin to reorganize into marginal cells. By the end of fetal life marginal cells are well differentiated. In the 17 day-old embryo a few granular inclusions appear in some centrally located cells. Secretory cells increase in number during the following two embryonic days. Some of these cells contain polymorphic populations of granular and vesicular inclusions by gestational day 19. The possibility of a dual formation of secretory inclusions is discussed. The result implies that the onset of granule-formation by these cells is not contemporaneous with the start of production of melanophore-expanding substances, the presence of which has been detected by earlier biological assays.