Ultrastructural studies of endocrine cell populations showing an argentaffin reaction and/or serotonin immunoreactivity in the rat antral mucosa

Summary On the basis of staining results in closely related semi-thin sections from rat antral mucosa immunostained with polyclonal serotonin antibodies and silver-stained for the argentaffin reaction, respectively, three different cell populations could be distinguished. One of these cell populations showed both serotonin immunoreactivity and an argentaffin reaction, a second one serotonin immunoreactivity alone, and a third one only an argentaffin reaction.These cell populations were studied electron microscopically in ultra-thin sections located between the stained semi-thin sections. The cell population displaying an agentaffin reaction and serotonin immunoreactivity showed secretory granules of the enterochromaffin cell type. A similar granular appearance was observed in cells which only exhibited an argentaffin reaction. Serotonin immunoreactivity in the absence of an argentaffin reaction was evident in some G (gastrin) cells. and in some D₁ and possibly also some D (somatostatin) cells; but not all the endocrine cells of the non-enterochromaffin type displayed serotonin immunoreactivity. The significance of the different reactions in the three cell populations is discussed.     

Bipolarity of duodenal enterochromaffin cells in the rat

Summary Enterochromaffin cells of the rat duodenum have been studied immunocytochemically by use of a specific antiserum to serotonin. At the light-microscopic level serotonin immunoreactivity was observed in enterochromaffin cells located in the epithelium of the duodenal mucosa. Most of the serotonin-immunoreactive material was localized to the basal portion of the enterochromaffin cells, but small amounts of immunoreactive material were regularly observed in the apical portion. At the electron-microscopic level serotonin immunoreactivity in enterochromaffin cells was found to be concentrated over the dense cores of the cytoplasmic granules. The majority of these granules was located in the basal cytoplasm of the enterochromaffin cells, but serotonin-immunoreactive granules were also observed in the apical cytoplasm immediately beneath the microvilli. These observations indicate that duodenal enterochromaffin cells are bipolar and that they secrete serotonin both basally, to the circulation, and apically, to the gut lumen. Rat duodenal enterochromaffin cells thus appear to have an exocrine as well as an endocrine function.     

Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

Summary In the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.     

Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study

Electron microscopic immunocytochemistry using protein A-gold labelling of ultrathin sections revealed immunoreactive glicentin (gut-type glucagon) and peptide YY (PYY) in virtually all secretory granules in a population of L-type endocrine cells in feline colon and human rectum. The granules of the human glicentin/PYY cells were considerably smaller in size than those in the cat. In both species the results indicate co-existence of glicentin and PYY in the same secretory granules, despite the probable derivation of the two peptides from two different precursors.     

Gamma-aminobutyric acid immunoreactivity in the enterochromaffin cells of the rat stomach.

The present immunocytochemical study revealed gamma-aminobutyric acid (GABA) immunoreactivity in the oxyntic and pyloric mucosa of the rat stomach at light- and electron-microscopic levels. GABA-immunoreactive endocrine cells were numerously seen in the lower half portion of the pyloric mucosa but rarely in the oxyntic mucosa. These cells were round or oval in shape and sometimes had a short cytoplasmic process. Serotonin-immunoreactive enterochromaffin (EC) cells were also observed in the oxyntic and pyloric mucosa of the stomach. The distribution and shapes of the immunoreactive cells were similar to those of the GABA-immunoreactive cells. With a double immunolabeling technique using anti-GABA and antiserotonin serum, GABA-immunoreactive endocrine cells showed serotonin immunoreactivity and were identified as EC cells. At the electron-microscopic level the GABA-immunoreactive cells contained round or oval, spindle-like, pear-shaped granules in EC cells. The immunoreaction product in the EC cells was generally confined to the granular cores. These findings suggest that GABA may be synthesized in the EC cells and be released from the granules of the cells after adequate stimuli.     

Serotonin-storing cells of the chicken duodenum: light,fluorescence and electron microscopy,and immunohistochemistry

Summary In an attempt to identify duodenal endocrine cells emitting formaldehyde-induced fluorescence (FIF), chicken duodena were studied by combined fluorescence, ultrastructural, silver impregnation and immunohistochemical methods in the same or consecutive sections. Our results show that: (1) Almost all the cells emitting yellow fluorescence by both the Falck-Hillarp and the Furness methods exhibit an immunohistochemical reaction with serotonin (5-HT) antiserum. (2) Almost all cells radiating yellow fluorescence by the Furness method stain with toluidine blue in Epon-embedded sections but, by high-voltage electron microscopy, can be subdivided into two types of cell containing either small round or polymorphous types of granules. (3) In the sections from which resin had been removed, all the cells emitting yellow FIF show argentaffinity by the Singh method, but not all cells display argyrophilia with the Grimelius method. (4) Cells exhibiting both argyrophil and argentaffin reactions in deresined serial sections are also separated into two types of cell, containing either small spherical or polymorphous types of granules by conventional electron microscopy in thin sections. Therefore, chicken enterochromaffin cells emit yellow FIF, store 5-HT, show both argentaffinity and argyrophilia, but are ultrastructurally classified into two types of granule-containing cells which may be related to polypeptides coexisting with 5-HT.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut

Summary The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut. An immunohistochemical study.

The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Immunoelectron microscopic study of the luminal release of chromogranin A from rat enterochromaffin cells

 Recently we found that raising the intraluminal pressure caused an increase in the luminal release of serotonin from enterochromaffin (EC) cells and serotonin immunoreactivity normally restricted within the secretory granules was diffusely scattered over the extragranular matrix. In the present study we investigated the intracellular localization of chromogranin A, a protein co-stored with serotonin in the EC cells, after stimulating the luminal release of serotonin. In situ vascularly and luminally perfused rat duodenum was exposed to intraluminal pressure and fixed for immunoelectron microscopic study. For immunoelectron microscopy, the pre-embedding DAB reaction for serotonin combined with the postembedding immunogold reaction for chromogranin A was used. Results showed that a number of secretory granules labeled with immunogold chromogranin A immunoreactivity located close to the apical plasma membrane. Some EC cells showed that one part of the apical cytoplasm was protruded into the lumen and a number of secretory granules with immunogold labeling were included in the protruded cytoplasm. These results suggest that EC cells may release chromogranin A into the intestinal lumen together with serotonin, by means of a different manner of secretion from that in serotonin. Received / Accepted: 9 December 1998     

Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure

 Since definitive morphological studies showing the luminal release of serotonin have not been reported, we used a perfused system which allows physiological monitoring and biochemical as well as morphological evidence indicating release of serotonin from enterochromaffin cells. Isolated vascularly and luminally perfused rat duodenums exposed to 5–35 cmH₂O of luminal pressure were measured for release of serotonin into the blood vessels and intestinal lumen. Immediately after raising the luminal pressure, the duodenum was fixed for immunoelectron microscopic localization of serotonin. Peristaltic contraction and serotonin content of the perfusates were continuously measured. The luminal release of serotonin increased with elevated intraluminal pressure, but the vascular release of serotonin was not altered. Tetrodotoxin had no effect on the pressure-stimulated luminal serotonin release. Enterochromaffin cells in control animals without increased luminal pressure contained immunogold-labeled secretory granules in the apical and basal cytoplasm. After intraluminal pressure increased, many apical secretory granules were no longer dense and immunogold particles were localized over the cytoplasmic matrix and microvilli. These findings indicate that luminal serotonin release is increased after raising the intraluminal pressure and serotonin, normally stored in the secretory granules of enterochromaffin cells, appears to be released into the cytoplasmic matrix and then diffuses or is transported into the intestinal lumen. Accepted: 15 January 1997     

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.     

Serotonin storage and chromogranins: an experimental study in rat gastric endocrine cells.

Chromogranins (Cg) and secretogranins (Sg) are acidic proteins localized in the secretory granules of a large variety of endocrine cells collectively named APUD cells (amine precursor uptake and decarboxylation). To examine the possible function of Cg/Sg as amine storage proteins, enteroendocrine cells of the rat gastric antral mucosa, i.e., serotonin-containing enterochromaffin (EC)-cells, gastrin (G)-, and somatostatin (D)-cells, were investigated immunohistochemically in serial semi-thin sections of controls and after intervention in serotonin synthesis. CgA and CgB immunoreactivity was determined semiquantitatively by optical density measurements. Experiments included inhibition of serotonin synthesis by p-chlorophenylalanine (pCPA), exogenous application of the serotonin precursor 5-hydroxytryptophan (5-HTP), and a combination of both treatments. The cellular distribution of Cg and the density of its immunoreactivity were closely related to the primary content of serotonin and the ability to store serotonin after 5-HTP application. Thus, Cg may act as amine-binding proteins in enteroendocrine cells, binding most probably being due to ionic interactions between Cg and the biogenic amines. EC- and G-cells, however, differed in their amine-handling properties and in the response of their Cg immunoreactivity after intervention in serotonin synthesis. We conclude, therefore, that the physiological function of Cg as amine storage proteins is restricted to endocrine cells with an endogenous content of amines. In other endocrine cells, exhibiting only a potential amine production, APUD may be considered as a kind of supravital staining without physiological significance.     

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.     

Biogenic amines in the guinea pig endocrine pancreas.

Pancreata of guinea-pigs were investigated for the presence and cellular distribution of biogenic amines. Out of the established endocrine cell types only insulin (B-) cells contained immunoreactivity for serotonin and noradrenaline. However, the B-cells' content of both amines was quite variable. Serotonin was also confined to enterochromaffin (EC-) cells. No immunoreactivity for dopamine or histamine was present in any islet cell. Treatment of guinea-pigs with Ro-4-4602 led to a marked decrease of serotonin and noradrenaline in pancreatic endocrine cells. The present findings suggest that serotonin and noradrenaline are involved in the function of the endocrine pancreas, particularly of islet B-cells.     

Ultrastructure of enterochromaffin cells and associated neural and vascular elements in the mouse duodenum

Summary Enterochromaffin cells of adult mouse duodenum were studied with light- and electron-microscopical techniques. They were distinguished from other enteroendocrine cells by their pleomorphic, electron-dense secretory granules in the basal cytoplasm. At the apices of enterochromaffin cells, tufts of short microvilli bordered the gut lumen. At their bases, irregular cytoplasmic extensions were either in contact with or passed through the basal lamina. The presence of cytoplasmic extensions in close proximity to fenestrated capillaries and subepithelial nerves suggested an endocrine or paracrine function. Electron micrographs of serial thin sections were used to reconstruct an enterochromaffin cell from the crypt epithelium in three dimensions and to determine its relationship with the underlying neural plexus. Although extensions from the serially sectioned and reconstructed cell and other enterochromaffin cells studied in crypt epithelia protruded through the basal lamina, no synaptic contacts were seen. Evidence of a synaptic contact between a neurite and another type of enteroendocrine cell (possibly an intestinal A cell), suggested a neurocrine role for some of the basally-granulated cells. Possible functions of enterochromaffin cells are discussed in the light of recent literature on the system of enteroendocrine cells, also known as APUD (amine precursor uptake and decarboxylation) cells and/or paraneurons.     

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells

Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

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.     

Immunocytochemical identification of polypeptide YY (PYY) cells in the human gastrointestinal tract

Various parts of the human gastrointestinal tract were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). PYY-immunoreactive cells were observed in the lower part of the ileum, in the colon and in the rectum, and PP-immunoreactive cells were found in the colon and rectum. Both cell types were of the open type, i.e. they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were seen to emit cytoplasmic processes to the neighbouring goblet cells. This latter observation suggests that PYY cells may exert a paracrine action on the mucus-secreting goblet cells. Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immunoreactive cells. On the ultrastructural level PYY-immunoreactivity was localized in basal granulated endocrine cells. These cells contained round or slightly oval electron dense granules with a mean diameter of 150 nm (range 100-300 nm).     

Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.