Guanylin, a bioactive peptide, has recently been isolated from the intet; this peptide activates intestinal guanylate cydase (i.e., guanylate cyclase C) and thus is poten-
Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polydonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority ofcells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like I Supported by Deutsche Forschungsgemeinschaft (Gr 681/2-2) and by Rrschungsschwerpunkt No. 24 of the State of Baden-Wurttemberg.
The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the brain. Furthermore, by acting through pituitary GABA A -, GABA B -, and GABA C receptors, it also plays an important role in the regulation of pituitary function. Although it has generally been assumed that the source of this pituitary GABA is the hypothalamus, here we provide evidence that GABA synthesis also occurs within the pituitary gland itself. Using RT-PCR, in situ hybridization histochemistry, immunohistochemistry, and immunoelectron microscopy, were detected the GABA synthesizing enzyme glutamic acid decarboxylase (GAD 67), the vesicular GABA transporter (VIAAT/VGAT), and GABA in the rat and rhesus monkey pituitary. Although these proteins were found in all of the endocrine cells of the intermediate lobe, we found these proteins in the growth hormone (GH) producing endocrine cells in the anterior lobe only, as well as in a rat GH producing cell line (GH3). In addition, GAD enzyme activity was readily detectable in the rat pituitary and GH3 cells. Many endocrine cells in the anterior pituitary, including GH-cells as well as PRL-, TSH-, ACTH-, and LH/FSH-cells were found to contain GABA A and or GABA B receptors, as shown by double-immunofluorescence staining. Therefore the identification of a novel site of synthesis of GABA within the pituitary, namely POMC-cells of the intermediate lobe and GH cells in the anterior lobe and the presence of pituitary GABA receptors, imply unexpected auto/paracrine actions of the neurotransmitter GABA to occur within the pituitary. These results suggest a new basic mechanism in the regulation of pituitary function by GABA.
Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.
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