Gut hormones in Salamandra salamandra

Summary Histological, cytochemical and immunocytochemical methods were used in light and electron microscopical studies to demonstrate the presence of a neuroendocrine system in the gut of the urodele, Salamandra salamandra.Cytochemical stains capable of detecting peptide-producing endocrine cells demonstrate cells reacting with Masson's silver (argentaffin) method, Grimelius' argyrophil silver method, masked metachromasia method and the lead haematoxylin stain.Using antisera raised to a variety of mammalian gut peptides, cells containing bombesin-, gastrin-, somatostatin-, substance P- and glucagon-like immunoreactivity were identified; vasoactive intestinal polypeptide- and substance P-like immunoreactivities were found in nerve fibres in the submucous and myenteric plexus. No immunoreactivity was detected for motilin, gastric inhibitory polypeptide, cholecystokinin or secretin.The ultrastructure of the immunoreactive cells and nerves was revealed by the semithin/thin method. All the cells identified contained numerous electrondense secretory granules, which varied in their chracteristic morphological structure from one cell type to another.The evidence collected in this study indicates that a complex neuroendocrine system regulating gut function is present in this amphibian and may have developed prior to the emergence of the phylum.     

Catecholamine-containing pancreatic islet cells of the domestic fowl

Summary In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Chromogranin A in the pancreatic islet: cellular and subcellular distribution

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal 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 of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.     

Distribution and partial characterisation of immunoreactivity to the putative C-terminus of rat pancreastatin

The presumptive C-terminal nonapeptide of rat pancreastatin was synthesised based upon the sequence of rat chromogranin A (CGA) analogous to that of porcine pancreastatin as contained within porcine CGA. Antisera were produced which were used to determine the qualitative and quantitative distribution of pancreastatin-like immunoreactivity in rat tissues by immunocytochemistry and radioimmunoassay respectively. Pancreastatin-like immunoreactivity was most abundant in pituitary, adrenal, gastric corpus and thyroid with considerably lower levels detected in the remainder of the gastroentero-pancreatic system and brain. Immunoreactivity was localised exclusively in endocrine cells and the relative abundance of immunoreactive cells paralleled the levels obtained radioimmunometrically. Chromatographic characterisation of pancreastatin-like immunoreactivity revealed molecular heterogeneity. Immunoreactive peptides of similar size to synthetic rat pancreastatin were present in gastrointestinal tissues and thyroid. These data indicate a tissue specific processing of CGA in the rat.     

Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract

 Colocalisation of synaptophysin has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum and colon) using double-immunofluorescence stainings. Numerous synaptophysin immunoreactive cells were seen in the antrum, while a smaller number were found in the intestinal tract. Synaptophysin immunoreactivity was strong in the antrum but weak in the intestine. In the intestinal colocalisation studies the synaptophysin immunoreactivity was enhanced by using the tyramide amplification method. Synaptophysin and chromogranin A were colocalised but the latter occurred mainly basally, whereas synaptophysin was found to occur diffusely throughout the cytoplasm. Synaptophysin immunoreactivity occurred in the serotonin cells throughout the gastrointestinal tract, and in the antral gastrin and somatostatin cells. In the intestinal tract only a small fraction of somatostatin, gastrin, cholecystokinin, enteroglucagon, enteroglucagon/ peptide tyrosine tyrosine displayed synaptophysin immunoreactivity. In the gastrointestinal tract (except the antrum), chromogranin A is a better general neuroendocrine marker than synaptophysin. The functional role of synaptophysin is unclear but it may be involved in the intracellular transport and release of hormones. Based on the distribution background of synaptophysin, it seems to be of greater importance in the antrum than in the intestinal tract as a whole. Accepted: 3 September 1998     

Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

Immunocytochemical localization of cyclooxygenase-1 and cyclooxygenase-2 in the rat stomach

Summary Prostaglandins are considered to play important roles in gastric mucosal protection. The rate-limiting enzyme involved in the biosynthesis of prostaglandins is cyclooxygenase (COX), also known as prostaglandin H synthase. Two forms of COX are known: a constitutively expressed form (COX-1) and a newly-characterized, inducible form (COX-2). In the present study, the immunocytochemical localization of COX-1 and COX-2 was examined in the rat gastrointestinal tract. A strong immunoreactivity for COX-1 was localized in the mucous neck cells of gastric gland. A weak reactivity for COX-1 was also found in the mucous cell types in the cardiac gland and pyloric gland of the stomach as well as in the Brunner's gland of duodenum. Ultrastructurally, the immunoreactivity was localized to the apical cytoplasm of these cells. On the other hand, immunoreactivity for COX-2 was distributed in the surface mucous cells in both the fundic and pyloric regions of stomach. These results suggest that a subset of mucous cells is the primary site for production of prostaglandins in the rat gastrointestinal tract, and that two forms of COX are expressed in distinct types of mucous cell.     

Ontogeny of the distribution and colocalization of calbindin D28K within neural and endocrine cells of the gastrointestinal tract of fetal and neonatal sheep.

Using immunocytochemical techniques we have demonstrated that Calbindin D28K (CaBP) is present in the gastrointestinal tract of ovine fetuses early in development (by day 45). At day 45, CaBP was limited to neuronal elements in the developing intestine. By day 100, CaBP immunoreactivity was abundant in both epithelial endocrine cells and nerves of the submucous and myenteric ganglia. The location of CaBP containing cells and fibers was similar in duodenal sections taken from day 100 and term (145 days), as well as those taken from 24-48 h postnatal lambs. CaBP is colocalized in endocrine cells containing gastrin, glucagon, somatostatin and neurotensin, but not glucose dependent insulinotrophic peptide (GIP). Furthermore, it is extensively colocalized in nerve fibers and cells containing neurotensin but not somatostatin or vasoactive intestinal peptide. The colocalization of CaBP within various endocrine and nerve cells does not change in fetal sheep over the last one-third of gestation and there is no difference between fetal and neonatal sheep.     

The Modified Steiner Stain: a New Use for an Old Stain? Staining Cytomegalovirus-infected Cells in Gastrointestinal Biopsies

The modified Steiner stain is a non-specific silver stain for identifying bacteria in formalin-fixed, paraffin-embedded tissues. The principle behind its use is that bacteria are first sensitized using uranyl nitrate solution, making them able to precipitate silver from a silver nitrate solution. It is used routinely for staining gastric biopsies to identify Helicobacter pylori. Upon staining a gastric biopsy from a patient with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus gastritis, we recognized that this technique also stains the viral inclusions of cytomegalovirus-infected cells. We then proceeded to stain 43 consecutive cytomegalovirus-positive gastrointestinal biopsies from 33 immunocompromised patients based on positive cytomegalovirus immunohistochemistry (DAKO-cytomegalovirus monoclonal antibody, clones DDG9 and CCH2). We also stained eight cytomegalovirus-infected, non-gastrointestinal tissue s, including lung, adrenal gland, ovary, skin and neural tissue, to ensure that the stain was staining the cytomegalovirus-infected cells and not argyrophilic or argentaffin neuroendocrine cells of the gastrointestinal tract. In 40 of the 43 cytomegalovirus-infected gastrointestinal biopsies, we saw positive staining with the modified Steiner stain (93% sensitivity). The cytomegalovirus-infected, non-gastrointestinal tissues all stained positively with the modified Steiner stain. Because the modified Steiner stain is frequently used to identify Helicobacter pylori in gastric biopsies, we propose that it be studied further for possible use either as a screen or as a confirmatory tool, or both, for cytomegalovirus inclusions in gastrointestinal biopsies.     

PC12 cells show immunoreactivity to a number of proteins and peptides, including vasostatin

N-terminal chromogranin A (CGA) contains peptides with vasoinhibitory properties, called vasostatin I (VST) and II [CGA(1–76) and (1–113) in human and bovine; (1–128) in rat]. Three fragments of VST were synthesized and antisera raised: human CGA(68–76) (VST I), rat CGA(121–128) (VST II fragment 2), and bovine/human CGA(83–91) (VST II, fragment 3). Strong immunoreactivity was observed in PC12 cells with antisera to VST II, fragment 3, VST I, and neuron-specific enolase. Little or no immunoreactivity was observed using antisera to synaptophysin, whole molecule CGA, pancreastatin, protein gene product 9.5, somatostatin, pancreatic polypeptide, or with antibodies 875 and 876 to VST II, fragment 2. Most of the VST antisera cross-reacted, with a species of molecular weight, 61 kDa but one, 874, cross-reacted with two species of molecular weights, 7.2 and 12 kDa. Our results show the presence of N-terminally processed CGA in PC12 cells.     

白yuan胃肠道内分泌细胞的免疫组织化学定位

为了解白yuan的消化生理过程,利用ABC免疫组织化学方法,对白yuan胃肠道5种内分泌细胞：5-羟色胺(5-HT)、脑啡肽、P物质、胃动素和抑胃素的分布进行了研究。结果表明：5-HT细胞主要分布于十二指肠及其以下部位,尤其是盲肠和直肠;而腺胃、肌胃和幽门中均未发现该类细胞。脑啡肽和P物质细胞数量明显较5-HT少,它们稀疏分布于十二指肠及其以下部位,无显著的分布积聚区。抑胃素和胃动素在整个胃肠道中均无分布。分析表明白yuan胃肠道5种内分泌细胞的分布与其他鸟类相似,但与其他脊椎动物明显不同。     

白鹮胃肠道内分泌细胞的免疫组织化学定位

为了解白的消化生理过程 ,利用ABC免疫组织化学方法 ,对白胃肠道 5种内分泌细胞 :5 -羟色胺 (5 HT)、脑啡肽、P物质、胃动素和抑胃素的分布进行了研究。结果表明 :5 HT细胞主要分布于十二指肠及其以下部位 ,尤其是盲肠和直肠 ;而腺胃、肌胃和幽门中均未发现该类细胞。脑啡肽和P物质细胞数量明显较 5 HT少 ,它们稀疏分布于十二指肠及其以下部位 ,无显著的分布积聚区。抑胃素和胃动素在整个胃肠道中均无分布。分析表明白胃肠道 5种内分泌细胞的分布与其他鸟类相似 ,但与其他脊椎动物明显不同。     

Expression of the ammonia transporter proteins Rh B glycoprotein and Rh C glycoprotein in the intestinal tract

Ammonia metabolism is important in multiple aspects of gastrointestinal physiology, but the mechanisms of ammonia transport in the gastrointestinal tract remain incompletely defined. The present study examines expression of the ammonia transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) in the mouse gastrointestinal tract. Real-time RT-PCR amplification and immunoblot analysis identified mRNA and protein for both RhBG and RhCG were expressed in stomach, duodenum, jejunum, ileum, and colon. Immunohistochemistry showed organ and cell-specific expression of both RhBG and RhCG. In the stomach, both RhBG and RhCG were expressed in the fundus and forestomach, but not in the antrum. In the forestomach, RhBG was expressed by all nucleated squamous epithelial cells, whereas RhCG was expressed only in the stratum germinativum. In the fundus, RhBG and RhCG immunoreactivity was present in zymogenic cells but not in parietal or mucous cells. Furthermore, zymogenic cell RhBG and RhCG expression was polarized, with apical RhCG and basolateral RhBG immunoreactivity. In the duodenum, jejunum, ileum, and colon, RhBG and RhCG immunoreactivity was present in villous, but not in mucous or crypt cells. Similar to the fundic zymogenic cell, RhBG and RhCG expression in villous epithelial cells was polarized when apical RhCG and basolateral RhBG immunoreactivity was present. Thus the ammonia transporting proteins RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they function cooperatively to mediate gastrointestinal tract ammonia transport.     

The occurrence of serotonin-containing cells in the esophageal epithelium of the bullfrog Rana catesbeiana: a fluorescence histochemical and immunohistochemical study

Fluorescence histochemical examination of biogenic amines of the frog esophageal mucosa revealed that a serotonin-like monoamine exhibiting an yellow fluorescence was present in a certain type of cells. The new type of cells was specifically stained by the immunohistochemical method using anti-serotonin antiserum. From these observations, it is suggested that the new cell type in the esophageal mucosa probably contains 5-hydroxytryptamine (serotonin). The serotonin-containing cells were argentaffin, but negative for Grimelius' silver stain.     

The occurrence of serotonin-containing cells in the esophageal epithelium of the bullfrog Rana catesbiana

Summary Fluorescence histochemical examination of biogenic amines of the frog esophageal mucosa revealed that a serotonin-like monoamine exhibiting an yellow fluorescence was present in a certain type of cells. The new type of cells was specifically stained by the immunohistochemical method using anti-serotonin antiserum. From these observations, it is suggested that the new cell type in the esophageal mucosa probably contains 5-hydroxytryptamine (serotonin). The serotonin-containing cells were argentaffin, but negative for Grimelius' silver stain.     

Immunohistochemical localization of bombesin-like peptides in the digestive tract of the bowfin,Amia calva

1. The distribution of bombesin-like immunoreactivity was determined in the gastrointestinal tract of the primitive holostean fish, the bowfin (Amia calva) using immunocytochemistry.2. Immunostaining using two different antisera raised against frog skin bombesin revealed a population of apparent endocrine cells containing bombesin-like immunoreactivity in the epithelium of the antral mucosa in the stomach.3. No bombesin-containing endocrine cells were present in any other segment of the gut. Bombesinergic nerves were not observed anywhere in the bowfin gastrointestinal tract.4. The antral bombesin endocrine cells were of the open type and were distributed diffusely from the base to the tips of antral glands, with some tendency to cluster near the base of the glands.5. These results suggest that a bombesin-like peptide may play an endocrine role in control of gastric functions such as regulation of acid secretion and gastric motility. These results support the hypothesis that bombesin serves a role in bony fish analogous to the role of antral cholecystokinin in amphibia and antral gastrin in amniotes.     

Immunocytochemical studies on endothelin in mast cells and macrophages in the rat gastrointestinal tract

 To reveal the distribution of endothelin (ET)-containing stromal cells (mast cells and macrophages), we investigated the rat gastrointestinal tract immunohistochemically using antibodies to Big ET-1, Big ET-2, Big ET-3, and mature ETs. In all the regions of the gastrointestinal tract, immunoreactivity for all the antibodies used was found in stromal cells that were located mainly in the lamina propria (not in the submucosa). The number of these cells was largest in the small intestine and smallest in the colon. Moreover, Big ET-2, which was originally identified in the gastrointestinal tract, was also found in many stromal cells, but Big ET-3-containing cells, unexpectedly, were found in almost the same number as Big ET-2-containing cells, while Big ET-1-containing cells were few. These immunopositive stromal cells seemed to be mast cells and macrophages from their histological features. Double-immunohistochemical staining revealed that 92% of the mature ETs-positive cells were mast cells; the rest were macrophages. Furthermore, we confirmed that mature ETs coexisted with ET-A or ET-B receptors in identical cells. Hence, we presume that ETs are synthesized in and secreted from stromal cells in the rat gastrointestinal tract, that their main isotypes are not only ET-2 but also ET-3, and that ETs may act in an autocrine/paracrine fashion. Accepted: 4 November 1997     

Peptide hormone-like immunoreactivity in the gastrointestinal tract and endocrine pancreas of eleven teleost species

Summary The distribution of peptide hormone-like immunostaining in the gastrointestinal tract of 11 teleost species was investigated by immunofluorescence.Cells immunoreactive for somatostatin were found in the glandular epithelium of the stomach of four species and in the epithelium of the pyloric appendage of one species. The mid-gut epithelium contained cells reactive with antibodies to glucagon (three species), gastrin (five species), pancreatic polypeptide (five species), and substance P (two species). Cells immunoreactive for met-enkephalin were found in the epithelium of both the mid-gut and the stomach of six species.In six species in which the endocrine pancreas was investigated, insulin-, glucagon-, and somatostatin-like immunoreactivity was observed. Pancreatic polypeptide was definitely localised by immunostaining in cells of the endocrine pancreas of only one out of three species examined.Vasoactive intestinal polypeptide-, neurotensin-, bombesin-, and enkephalin-like immunoreactivity was identified in the gastrointestinal nerve fibres in various species.In view of the considerable species variation found, caution should be exercised in generalising about the peptides present in the gastrointestinal tract of fish.