Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.     

Connexin43 in rat pituitary: localization at pituicyte and stellate cell gap junctions and within gonadotrophs

Immunohistochemical methods were employed to investigate the cellular and ultrastructural localization of the gap junction protein connexin43 (Cx43) in rat pituitary. Western blots of pituitary homogenates probed with anti-Cx43 antibodies showed the presence of Cx43 in both anterior and posterior pituitary lobes. By light microscopy (LM), Cx43-immunoreactive (Cx43-IR) puncta were found in all areas of the posterior lobe, but at greater concentrations in peripheral regions of this structure. By electron microscopy (EM), immunogold labelling for Cx43 was seen at gap junctions between thin cytoplasmic processes of pituicytes. No immunoreactivity was detected in the intermediate lobe. The anterior lobe contained puncta similar to but more sparsely scattered than those in the posterior lobe, and by EM analysis these were demonstrated to correspond to labelled gap junctions between stellate cells. In addition, anti-Cx43 antibodies produced intracellular labelling in a small percentage of endocrine cells, which were distributed throughout the anterior lobe and determined by double immunostaining methods to be cells containing luteinizing hormone. By EM, labelling within these cells was associated with predominantly large secretory granules and other loosely organized organelles. The results indicate that gap junctions in the pituitary are composed of Cx43 and that this or a related protein may have a novel intracellular function within gonadotrophs.     

β-Endorphin-like immunoreactivity in plasma,pituitaries and hypothalamus of rats following treatment with opiates

β-Endorphin was measured using a radioimmunoassay (RIA) in plasma, pituitary lobes and hypothalamus of rats following treatment with the opiate agonist morphine and the antagonist naloxone. β-Endorphine-like immunoreactivity (β-ELI) in plasma was found to be increased after high doses of morphine (50 mg/kg i.p.). A high increase of β-ELI in plasma was further observed in morphine tolerant/dependent rats after precipitated withdrawal by naloxone. This release of β-ELI into plasma was accompanied by a significant reduction of β-ELI content in the anterior lobe of the pituitary and the hypothalamus but not in the intermediate/posterior lobe of pituitary. Chronic treatment of the rats by the s.c. implantation of morphine pellets (each containing 75 mg morphine; 6 within 10 days) did not alter β-ELI levels in plasma and in the pituitary lobes. A long term administration of morphine (21 pellets within 1 month), however, causes a significant reduction of the β-ELI content of anterior lobe and intermediate/posterior lobe of pituitary without changing the β-ELI levels in plasma.     

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in the rat and mouse pituitary gland.

The distribution of the octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephalin A-derived opioid peptide, in the rat and mouse pituitary gland was studied using the indirect immunofluorescence technique and immunoelectron microscopy. The anterior lobe contained a few MEAGL-immunoreactive cells but no nerve fibers. A previously unknown enkephalin-immunoreactive nerve fiber system was revealed in the intermediate lobe. These fibers originated in a dense MEAGL-immunoreactive plexus located along the border between the intermediate and posterior lobes and were distributed throughout the lobe. In the posterior lobe, MEAGL immunoreactivity was found in a very dense network of varicose fibers that was evenly distributed over the entire lobe. These results provide a morphological correlate for previous chemical studies and together with them suggest that MEAGL-immunoreactive innervation regulates endocrine functions of the intermediate and posterior lobes directly at the pituitary level.     

The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.     

Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma-aminobutyric acid (GABA)

Summary The GABAergic innervation of the mouse pituitary, including the median eminence, was studied at light microscopic and ultrastructural levels by use of a pre-embedding immunocytochemical technique with antibodies directed against GABA. In the median eminence, a high density of GABA-immunoreactive fibers was found in the external layer where the GABAergic varicosities were frequently observed surrounding the blood vessels of the primary capillary plexus. In the internal and subependymal layers, only few fibers were immunoreactive. The intense labeling of the external layer was observed in the entire rostro-caudal extent of the median eminence. In the pituitary proper, a dense network of GABA-immunoreactive fibers was revealed throughout the neural and intermediate lobes, entering via the hypophyseal stalk. The anterior and tuberal lobes were devoid of any immunoreactivity. The GABA-immunoreactive terminals were characterized in the median eminence, and in the intermediate and posterior lobes at the electron-microscopic level. They contained small clear vesicles, occasionally associated with dense-core vesicles or neurosecretory granules. In the intermediate lobe they were seen to be in contact with the glandular cells. In the posterior lobe and in the median eminence, GABA-immunoreactive terminals were frequently located in the vicinity of blood vessels. These results further support the concept of a role of GABA in the regulation of hypophyseal functions, via the portal blood for the anterior lobe, directly on the cells in the intermediate lobe, and via axo-axonic mechanisms in the median eminence and posterior lobe.     

Modulation of in vitro release of beta-endorphin from the separate lobes of the rat pituitary.

The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10^?7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe.Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue.These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

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.     

Cholinesterase in the posterior and intermediate lobes of the pituitary

Summary Posterior and intermediate lobes of pituitary glands of cat, rabbit, beef, and rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase by light and electron microscopy. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW 284C51 to demonstrate BuChE. Glandular cells of the intermediate lobe of cat, rabbit and rat contained variable amounts of AChE, whereas those of beef contained BuChE. In the posterior pituitary, AChE was detected in the cat, BuChE in the beef and rat, and both AChE and BuChE in the rabbit. In the posterior lobe of all species examined, cholinesterase, whether true or pseudo enzyme, as the case may be, was localized to certain pituicytes and pituicyte-neuron junctions. These histochemical studies failed to identify cholinergic neurons in the posterior pituitary. Large blood vessels of the pituitary were innervated apparently by adrenergic nerves only. Speculations on the role of pituicyte cholinesterase in posterior pituitary secretion are presented.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.     

High-Protein Carboxymethylase Activity and Low Endogenous Methyl Acceptor Proteins in Posterior Pituitary Lobe of Rats Lacking Neurophysin-Vasopressin (Brattleboro Rats)

Abstract: The activity of protein carboxymethylase and the endogenous protein methyl acceptor capacity were examined in the posterior, intermediate, and anterior lobes of the pituitaries of homozygous Brattleboro rats with diabetes insipidus and in heterozygous Brattleboro and Long-Evans control rats. Protein carboxyl methylation is selectively altered in the posterior pituitary lobes of homozygous Brattleboro rats. Protein carboxymethylase activity is higher (+40%) and endogenous methyl acceptor protein capacity is lower (-80%) with respect to heterozygous Brattleboro and Long-Evans control rats. This latter change is correlated with decreased methylation of proteins of a molecular weight of approximately 11K daltons, is selective for the posterior pituitary lobe, since it does not occur in the intermediate and anterior lobes, and probably reflects the absence of vasopressin-associated neurophysin in homozygous Brattleboro rats. Our results support a physiological role of protein carboxyl methylation in the neurosecretory process in the posterior pituitary gland.     

Localization of multiple forms of ACTH- and β-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Lectin-labeled membrane is transferred to the Golgi complex in mouse pituitary cells in vivo

Labeling of the Golgi complex with the lectin conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP), which binds to cell surface membrane and enters cells by adsorptive endocytosis, was analyzed in secretory cells of the anterior, intermediate, and posterior lobes of mouse pituitary gland in vivo. WGA-HRP was administered intravenously or by ventriculo-cisternal perfusion to control and salt-stressed mice; post-injection survival times were 30 min-24 hr. Peroxidase reaction product was identified within the extracellular clefts of anterior and posterior pituitary lobes through 24 hr but was absent in intermediate lobe. Endocytic vesicles, spherical endosomes, tubules, dense and multivesicular bodies, the trans-most saccule of the Golgi complex, and dense-core secretory granules attached or unattached to the trans Golgi saccule were peroxidase-positive in the different types of anterior pituitary cells and in perikarya of supraoptico-neurohypophyseal neurons; endoplasmic reticulum and the cis and intermediate Golgi saccules in the same cell types were consistently devoid of peroxidase reaction product. Dense-core secretory granules derived from cis and intermediate Golgi saccules in salt-stressed supraoptic perikarya likewise failed to exhibit peroxidase reaction product. The results suggest that in secretory cells of anterior and posterior pituitary lobes, WGA-HRP, initially internalized with cell surface membrane, is eventually conveyed to the trans-most Golgi saccule, in which the lectin conjugate and associated membrane are packaged in dense-core secretory granules for export and potential exocytosis of the tracer. Endoplasmic reticulum and the cis and intermediate Golgi saccules appear not to be involved in the endocytic/exocytic pathways of pituitary cells exposed to WGA-HRP.     

The endorphins of the epithelial and subepithelial structures of the rat small intestine and the evolutionary hypotheses of the formation of the mechanisms of the negative regulation of their synthesis]

The effects of the agonist of the glucocorticoid hormones dexamethasone and dopamine antagonist--haloperidol on the concentration of immunoreactive alpha-, beta- and gamma-endorphins in duodenum, ileum, and jejunum of rats were studied. Besides the extracts of the intestines, the immunoreactive endorphins were measured in the extracts of their mucosa-submucosa and muscle-serous layers, that allowed to separate the endorphin-producing cells of the nervous system (muscle-serous layer) from endorphin producing cells of endocrine and immune systems (mucosa-submucosa layer). The injection of dexamethasone (0.2 mg per rat, daily for 6 days) caused the reliable decrease in concentrations of all three types of endorphins in mucosa-submucosa and muscle-serous layer of duodenum, ileum, and jejunum. Under the action of haloperidol (0.6 mg per rat, daily for 6 days) the reliable increase of beta-endorphin concentration was noticed only in jejunum. The suggestion is made that two distinct subpopulations of endorphin-producing cells exist in the intestine: in one cells endorphin synthesis is regulated by glucocorticoids, as in the anterior lobe of pituitary, in the other cells the synthesis of endorphins is regulated by dopamine, as in the cells of the intermediate lobe of pituitary. It is suggested that both glucocorticoid and dopamine types of regulation of endorphins synthesis were formed in the intestine or even in the gastric cavity. In process of evolution the cells with glucocorticoid type of regulation gave rise to the anterior lobe of pituitary, the cells with the dopamine type of regulation--to the intermediate lobe.     

N-cadherin loss in POMC-expressing cells leads to pituitary disorganization

Pituitary tumors are the third most common intracranial tumor in humans and can cause altered hormone secretions leading to hypercortisolism, acromegaly, and infertility. Reduced expression of the cell adhesion molecule N-cadherin has been linked with the formation of pituitary tumors, but its role in normal pituitary gland physiology or tumor initiation is unknown. In the murine pituitary, N-cadherin expression is detected in virtually all cells of the posterior, intermediate, and anterior lobes. N-cadherin may function to initiate important cues such as controlling proliferation, directing cell placement, and promoting formation of cell networks that coordinately release hormones into the bloodstream. To address this, we generated mice lacking N-cadherin in proopiomelanocortin-expressing melanotrope and corticotrope cells of the intermediate and anterior lobes of the pituitary. We observed that intermediate lobe cells can aberrantly displace SOX2-containing progenitor cells in the N-cadherin conditional knockout mice at postnatal d 1. By postnatal d 30, although a reduction in α- and β-catenin membrane staining occurs, there is little effect on intermediate lobe architecture with N-cadherin loss. Also, despite these changes in adherens junction molecules, no alterations in cell proliferation occur. In contrast, loss of N-cadherin in the corticotropes leads to aberrant cell clustering and a reduction in Pomc mRNA. Taken together, our data reveal important roles of N-cadherin in pituitary cell placement and that loss of N-cadherin alone does not lead to pituitary tumor formation.     

Quantitative autoradiographic localization of neuropeptide Y (NPY) binding sites in rat posterior pituitary lobe

1. We have localized and quantified neuropeptide Y (NPY) binding sites in the rat pituitary gland after incubation of tissue sections in the presence of 125I-Bolton-Hunter NPY followed by autoradiography, computerized microdensitometry, and comparison to 125I-standards. 2. In the rat, NPY binding sites are localized exclusively to the part of the posterior pituitary lobe closer to the pituitary stalk. No NPY binding sites could be found in the intermediate or the anterior pituitary lobes. 3. Our results suggest a role for NPY in the regulation of pituitary function and, in particular, that of the neural lobe.