An histological and immunocytochemical study of the neuroendocrine cells in the intestine of Podarcis hispanica Steindachner, 1870 (Lacertidae)

Summary Numerous endocrine cells can be observed in the gut of the lizard Podarcis hispanica after application of the Grimelius silver nitrate technique. The argyrophilic endocrine cells are usually tall and thin in the small intestine but short, basal, and round in the large intestine. Eleven types of immunoreactive endocrine cells have been identified by immunocytochemical methods. Numerous serotonin-, caerulein/gastrin/cholecystokinin octapeptide-and peptide tyrosine-tyrosine-immunoreactive cells; a moderate number of pancreatic polypeptide-, neurotensin-, somatostatin-, glucagon-like peptide-1-and glucagon-immunoreactive cells, and few cholecystokinin N-terminal-and bombesin-immunoreactive cells were found in the epithelium of the small intestine. Coexistence of glucagon with GLP-1 or PP/PYY has been observed in some cells. In the large intestine a small number of serotonin-, peptide tyrosine-tyrosine-, pancreatic polypeptide-, neurotensin-, somatostatin-and glucagon-like peptide-1-immunoreactive cells were detected. Vasoactive intestinal peptide immunoreactivity was found in nerve fibers of the muscular layer. Substance P-immunoreactive nerve fibers were detected in lamina propria, submucosa and muscular layer. Chromogranin A-immunoreactive cells were observed throughout the intestine, although in lower numbers than argyrophilic cells.     

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.     

Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunner's duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.     

Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.     

Colocalization of glucagon-like peptide and glucagon immunoreactivities in pancreatic islets and intestine of salmonids

Summary Pancreatic islets of salmon contain at least two peptides of the glucagon family: 29-amino acid glucagon and 31-amino acid glucagon-like peptide (GLP). Both peptides were recently isolated from the pancreatic islets of coho salmon and sequenced (Plisetskaya et al. 1986). Antibodies generated against these two peptides and against human glucagon were used as immunocytochemical probes to investigate whether glucagon and GLP are processed in the same, or in different cell types in the pancreatic islets and the gut of salmon. Two salmonid species, rainbow trout and coho salmon, were studied. All islet A-cells in the two species were immunoreactive toward both anti-salmon (s)-glucagon and anti-s-GLP. Similar colocalization of glucagon and GLP immunoreactivities was found in open-type endocrine cells in mucosae of the small intestine (including the pyloric coecae) and the large intestine close to the vent of rainbow trout. None of the antibodies stained mucosal cells of the body of the stomach. These results suggest that in the pancreas and the gut of salmonid fish the same cells produce both glucagon and GLP. These peptides are most likely the products of a single gene coding for the preproglucagon sequence.     

Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei)

Summary The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemicaly, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33-49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense "halo" of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophys. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Evolution of receptors for proglucagon-derived peptides: isolation of frog glucagon receptors

The mammalian proglucagon gene encodes three glucagon-like sequences, glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Each of these three functionally distinct proglucagon-derived peptides has a unique, but related, receptor. To better understand the origin of the unique physiological functions of each proglucagon-derived glucagon-like sequence we have cloned glucagon-like receptors from two species of frogs, Xenopus laevis and Rana pipiens. The cloned glucagon-like receptor sequences were found to be most closely related to glucagon receptors. To determine whether the evolutionary history of the receptors for proglucagon-derived peptides was the same as that inferred for the peptide hormones, we conducted a phylogenetic analysis using both parsimony and distance methods. We show that the evolutionary history of the receptors for glucagon-like sequences differ from the history of the glucagon-like sequences. The phylogeny of receptors for proglucagon-derived peptides is not monophyletic (i.e. they are not each other's closest relatives), as the receptor for the hormone glucose-dependent insulinotropic peptide (GIP) is more closely related to the glucagon receptor than either the GLP-1 or GLP-2 receptors. In contrast to the evolutionary origin of glucagon-like sequences, where glucagon is of most ancient origin, we found that the GLP-2 receptor has the most ancient origin. These observations suggest that the diversification of the glucagon-like sequences encoded by the proglucagon gene and of the receptors for these peptides occurred independently, and that either these hormones or their receptors have been recruited for new functions.     

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.     

Cell-specific post-translational processing of preproglucagon expressed from a metallothionein-glucagon fusion gene

Glucagon is a peptide hormone of 29 amino acids encoded by a preprohormone which contains in tandem the sequences of glucagon and two additional glucagon-like peptides (GLPs) structurally related to glucagon and separated by intervening peptides. Glucagon arises by cleavage from the prohormone within the A cells of the pancreatic islets but in the intestine remains as part of a partially processed precursor (glicentin). To determine whether additional glucagon-like peptides are processed from preproglucagon and to analyze for potential cellular specificity in the processing of preproglucagon, we introduced and expressed a metallothionein-glucagon fusion gene in a fibroblast and two endocrine (pituitary and pancreatic islet) cell lines. Chromatographic analyses of cell extracts utilizing specific radioimmunoassays to chemically synthesized peptides demonstrate the liberation of intact glucagon, glicentin, GLP-I(1-37), GLP-I(7-37), GLP-II, and an intervening peptide amidated at its carboxyl terminus. The peptides were present in distinct yet different patterns in the two endocrine but not the fibroblast cell lines. The cell-specific liberation of the glucagon-like and intervening peptides suggests their potential as new bioactive peptides. The cellular specificity in the processing of preproglucagon indicates that the genetic determinants of the processing activity are complex and are expressed in a cell-specific manner.     

Effect of truncated glucagon-like peptide 1 on cAMP in rat gastric glands and HGT-1 human gastric cancer cells

We tested the truncated 7-37 glucagon-like peptide 1 (TGLP-1), a naturally occurring porcine intestinal peptide, and other members of the glucagon family, including pancreatic glucagon (G-29), GLP-1 and GLP-2 for their ability to activate the cAMP generating system in rat gastric glands and HGT-1 human gastric cancer cells. In rat fundic glands, TGLP-1 was about 100 times more potent (EC50 = 2.8 X 10(-9) M) than GLP-1 of G-29, and 10 times more potent than G-29 in the HGT-1 cell line. Our results support the notion that TGLP-1 plays a direct role in the regulation of acid secretion in rat and human gastric mucosa.     

Biological activities of catfish glucagon and glucagon-like peptide

The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Immunocytochemical demonstration of glucagon-like peptides in Mytilus edulis cerebral ganglia and an in vitro effect of vertebrate glucagon on glycogen metabolism

Immunological detection of glucagon-like peptides was performed in the cerebral ganglia of the mussel Mytilus edulis using an anti-vertebrate glucagon antibody. Two clusters of positive neurosecretory cells were observed, as well as stained nervous fibers. The effect of vertebrate glucagon on glucose incorporation into glycogen of reserve cells was tested using an in vitro microplate bioassay. Optimal incubation conditions were previously defined and an inhibitory effect of porcine glucagon was obtained for concentrations ranging from 10(-6) to 10(-9)M. It is postulated that the glucagon-like peptide may be implicated in the regulation of glucose metabolism in bivalves.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Summary Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33–49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense halo of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophs. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Expression of epidermal growth factor in salivary adenoid cystic carcinoma

This study used an immunohistochemical technique to assess the expression of epidermal growth factor (EGF) in 40 specimens of salivary adenoid cystic carcinoma (ACC), 7 specimens of labial glands adjacent to mucocele, and 5 specimens of normal submandibular glands. In normal submandibular glands, immunohistochemically detectable EGF was demonstrated in all ductal segments, including intercalated, striated, and excretory duct cells. No EGF positive staining was found in acinar compartments. including serous and mucous acinar cells. In degenerated labial glands adjacent to mucocele, no EGF staining was detected in the remaining acinar and ductal cells. In salivary ACCs, positive EGF immunostaining was observed in one of the 5 (20%) ACCs with a solid pattern and in 13 of the 35 (37.1%) ACCs with a tubular-cribriform pattern. The overall EGF expression rate in 40 salivary ACCs was 35%. Positive EGF staining was predominantly found in tubular structures in the tubular ACCs and in duct-like structures in large cribriform patterns or in the stroma of the cribriform ACCs. There was no significant correlation between EGF expression in salivary ACCs and any of the clinicopathological parameters including patient age and sex, cancer location, TNM status, clinical stage, histologic type, perivascular or perineural invasion, focal necrosis of tumor, and cellular atypia. We conclude that the duct segments of the normal submandibular gland are the sites of EGF synthesis and secretion. In degenerated labial glands adjacent to mucocele, EGF synthesis is completely inhibited. Furthermore, EGF is mainly biosynthesized in cells forming tubular or duct-like structures in tubular or cribriform salivary ACCs, and EGF may play a biologic role, particularly as a mitogen in salivary ACC growth.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Monoclonal antibodies against rat saliva and salivary gland antigens

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.