Immunocytochemical identification and localization of peptide hormones in the gastro-entero-pancreatic (GEP) endocrine system of the mouse and a stomachless fish,Barbus conchonius

Summary A large number of antisera mainly raised against mammalian hormones are tested immunocytochemically on the GEP-endocrine system of mouse and fish (Barbus conchonius). The endocrine pancreas of mouse and fish appeared to contain the same four endocrine cell types; insulin-, glucagon-, PP- and somatostatin-immunoreactive cells.In mouse about 13 GEP endocrine cell types are distinguished 1. insulin-, 2. somatostatin-, 3. glucagon-, 4. PP-, 5. (entero)glucagon-/PP-like, 6. CCK-like, 7. substance P-, 8. neurotensin-, 9. VIP-, 10. gastrin-, 11. secretin-, 12. -endorphin-, 13. serotonin-immunoreactive cells.Based on this and a previous study at least 13 GEP endocrine cell types seems to be present in stomachless fish: 1–9 as described for mouse, 10. (entero)glucagon-like, 11. met-enkephalin, 12. VIP-like, 13. unspecific immunoreactive endocrine cells.Coexistence of glucagon and PP-like peptides is found in the gut and pancreas of mice and in the gut of B. conchonlus. In mouse pancreas and fish gut, endocrine cells showing only PP-or glucagon-like immunoreactivity are found too. In mouse stomach some endocrine cells, showing only PP-immunoreactivity are demonstrated. In the same region coexistence of C-1-gastrin-and FMRF-amide-immunoreactivity is found in endocrine cells. The importance of these phenomena are discussed.Enteric nerves immunoreactive with antisera raised against substance P and GRP are found in mouse, against somatostatin and met-enkephalin in both mouse and fish and against VIP in fish.In honour of Prof. P. van Duijn     

Immunocytochemical identification and localization of peptide hormones in the gastro-entero-pancreatic (GEP) endocrine system of the mouse and a stomachless fish, Barbus conchonius

A large number of antisera mainly raised against mammalian hormones are tested immunocytochemically on the GEP-endocrine system of mouse and fish (Barbus conchonius). The endocrine pancreas of mouse and fish appeared to contain the same four endocrine cell types; insulin-, glucagon-, PP- and somatostatin-immunoreactive cells. In mouse about 13 GEP endocrine cell types are distinguished: 1. insulin-, 2. somatostatin-, 3. glucagon-, 4. PP-, 5. (entero)glucagon-/PP-like, 6. CCK-like, 7. substance P-, 8. neurotensin-, 9. VIP-, 10. gastrin-, 11. secretin-, 12. beta-endorphin-, 13. serotonin-immunoreactive cells. Based on this and a previous study at least 13 GEP endocrine cell types seems to be present in stomachless fish: 1-9 as described for mouse, 10. (entero)glucagon-like, 11. met-enkephalin, 12. VIP-like, 13. unspecific immunoreactive endocrine cells. Coexistence of glucagon and PP-like peptides is found in the gut and pancreas of mice and in the gut of B. conchonius. In mouse pancreas and fish gut, endocrine cells showing only PP- or glucagon-like immunoreactivity are found too. In mouse stomach some endocrine cells showing only PP-immunoreactivity are demonstrated. In the same region coexistence of C-t-gastrin- and FMRF-amide-immunoreactivity is found in endocrine cells. The importance of these phenomena are discussed. Enteric nerves immunoreactive with antisera raised against substance P and GRP are found in mouse, against somatostatin and met-enkephalin in both mouse and fish and against VIP in fish.     

Urotensin I-like immunoreactivity in the midgut endocrine cells of the insects Gryllus bimaculatus and Periplaneta americana

Summary The occurrence of urotensin I (UI)-like immunoreactivity is demonstrated in the midgut of the cricket Gryllus bimaculatus and the cockroach Periplaneta americana. The immunoreactivity is restricted to endocrine cells dispersed in the epithelium of the midgut. The UI-positive endocrine cells are numerous in the cricket, but much fewer in the cockroach. These UI-positive cells are different from bovine pancreatic polypeptide (BPP)-immunoreactive cells which represent a dominant cell type in the midgut of both insects. This study suggests the possibility that the neuropeptide UI or a closely related substance may also occur in animals other than fishes.     

Localization of Allatostatin-Producing Cells in Larval Gut of the Potato Worm Agrius convolvuli

ABSTRACT This study has been carried out to investigate localization of allatostatin-producing cells in the gut of Agrius convolvuli using an immunocytochemical method. Allatostatin producing cells could not be found in the foregut and hindgut, but they were abundantly distributed only in the posterior part of midgut. These endocrine cells had typically columnar shape, with secretory surface positioned to muscular layer of midgut wall and also showed different intensity of immunoreactivity to AST, mainly with strong or moderate intensity.     

Presence of Locusta diuretic hormone in endocrine cells of the ampullae of locust Malpighian tubules

This is an investigation of an endocrine cell type in the midgut of the migratory locust Locusta migratoria. This cell type is found in the posterior region of the midgut and is especially common in the ampullae through which Malpighian tubules drain into the gut at the midgut-hindgut junction. Strong Locusta diuretic hormone-like immunoreactivity in these cells was colocalized with FMRFamide- and substance P-like immunoreactivities. At the ultrastructural level, immunoreactivity for Locusta diuretic hormone was found in spherical granules (mean diameter of 450 nm), the contents of which showed variable electron density. Fractionation of a methanolic extract of the ampullae by reversed-phase high performance liquid chromatography revealed the presence of two peaks of Locusta diuretic hormone-like immunoreactive material, both of which stimulate cyclic AMP production by isolated Malpighian tubules. The more hydrophobic material is most likely Locusta diuretic hormone, which has the same retention time when chromatographed under identical conditions. Received: 15 September 1995 / Accepted: 16 February 1996     

Immunohistological localization of regulatory peptides in the midgut of the female mosquitoAedes aegypti

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

Localization of allatostatin-immunoreactive material in the central nervous system,stomatogastric nervous system,and gut of the cockroach Blattella germanica

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH₂ C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed. Arch. Insect Biochem. Physiol. 37:269–282, 1998. © 1998 Wiley-Liss, Inc.     

Light and electron microscopical study of two types of endocrines cell in the midgut of the adult worker honeybee (apis mellifera)

The occurrence, development and ultrastructure of two types of gut endocrine cell have been studied in the midgut of adult honeybees. These cells, one of a basal granular type and one of a vesicular type, are evenly distributed throughout the posterior three-quarters of the midgut. Each crypt complex contains one of each cell type, both of which may be derived from the same stem cells as the enterocytes. They already contain their respective secretory product while still in the nidus. Both reach the midgut lumen by a narrow apex and are therefore of the open type. The granular cells release their secretory granules at the cell base in a typical endocrine way. In young vesicular cells the secretory vesicles are released at the cell base and in the intercellular spaces. Old cells are still filled with vesicles when they are shed in the midgut lumen. This seems to indicate that these cells have both an endocrine (or paracrine) and an exocrine function, the latter apparently by holocrinc release.     

Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust,Schistocerca gregaria (Forskal)

Summary The endocrine cells of the midgut epithelium of the desert locust are found dispersed among the digestive cells and are similar to those of the vertebrate gut. According to their reactivity to silver impregnation techniques and the ultrastructural features of the secretory granules (shape, electron-density, size, and structure) 10 types of endocrine cell have been identified, of which seven are located in the main segment of the midgut or in the enteric caeca, and the other three seem to be present only in the ampullae through which the Malpighian tubules drain into the gut. The endocrine cells have a slender cytoplasmic process that reaches the gut lumen, a feature that supports the receptosecretory nature postulated for this cellular type in insects as well as vertebrates. Antisera directed against mammalian gastrin, CCK, insulin, pancreatic polypeptide and bombesin reacted with some of the endocrine cells. This is the first time that insulin- and bombesin-like immunoreactive cells have been described in the midgut of an insect.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

A novel tissue in an established model system: the <Emphasis Type="Italic">Drosophila</Emphasis> pupal midgut

The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body.” The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects.     

A comparative ultrastructural and physiological study on melanophores of wild-type and periodic albino mutants of Xenopus laevis

Summary The central and visceral nervous systems of the cockroach Periplaneta americana were studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibody to bovine pancreatic polypeptide (PP). PP-like immunoreactive neuron somata are most numerous in the brain; at least 6 pairs of cell groups occur in clearly defined regions. Three pairs of cells each are also present in the suboesophageal ganglion and the thoracic ganglia, one pair of a single cell each in the first abdominal and the frontal ganglia, and 4 to 6 pairs of single cells in the terminal ganglion. No reactive cells were found in the retrocerebral complex and the second to the fifth abdominal ganglia. The axons containing PP-like immunoreactivity issue many branches that are distributed in the entire brain-retrocerebral complex, ventral cord, and visceral nervous system. PP-like immunoreactive material produced in the brain seems to be transported by three routes: protocerebrum to corpora cardiaca (-allata) through the nervi corporis cardiaci, tritocerebrum to visceral nervous system through frontal commissures, and to ventral cord through circumoesophageal connectives.A possible homology between the mammalian brain-GEP (gastro-enteropancreatic) system and the brain-midgut system of this insect is discussed.     

Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: Preliminary immunocytochemical observations

Summary Pancreatic polypeptide (PP) is a candidate hormone of unknown physiological significance. It is produced by a population of endocrine cells in the pancreas. In the present study a PP-like peptide was found to occur in the mammalian and avian central and peripheral nervous systems. Immunoreactive nerve fibres and nerve cell bodies were widely distributed in the brain. Dense accumulations of nerve fibres occurred in the following areas: nucleus accumbens, interstitial nucleus of the stria terminalis, para- and periventricular hypothalamic nuclei, and medial preoptic area. In addition, nerve fibres were regularly seen in cortical areas. Immunoreactive perikarya were observed in the following regions: cortex, nucleus accumbens, neostriatum and septum. In the gut, immunoreactive nerve fibers were distributed in the myenteric plexus, in smooth muscle, around blood vessels, and in the core of the villi. Immunoreactive perikarya occurred in the submucosal and myenteric plexus, suggesting that PP immunoreactive nerves are intrinsic to the gut.In the species examined, the neuronal PP-like peptide could be demonstrated with an antiserum raised against avian PP, but not with those raised against bovine or human PP. Thus, neuronal PP is distinct from the PP that occurs in pancreatic endocrine cells.     

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.     

Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L.

Summary Substance P-like immunoreactivity in the alimentary canal of the frogRana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

A Study of the Alimentary Canal of the Brachyopterygian Fish Polypterus senegalus with Electron Microscopy and Immunohistochemistry

The gastro-intestinal tract of Polypterus senegalus was investigated by means of electron microscopy and immunohistochemistry. Cilia-bearing cells can be observed over the whole length of the intestine. All enterocytes along the intestinal tract are characterized by apical pinocytotic vesicles. However, a typical intestinal region, which in other fish is characterized by large supranuclear vacuoles, is lacking. By means of electron microscopy, four types of endocrine cells and three types of nerve cell processes can be identified. By means of immunohistochemistry, endocrine cells with immunoreactivity for bombesin-, enkephalin-, G/CCK-, 5-HT-, somatostatin- and substance P-antisera can be found. Nerve cell processes show immunoreactivity for bombesin-, enkephalin-, 5-HT-, substance P- and VIP-antisera. The number of immunoreactive endocrine cells, nerve cells and nerve cell processes is different for each part of the gut.