On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

New views on the identification of the various cell types in the pancreatic islets of the rat. An ultrastructural and morphometrical study

Islets of Langerhans taken from different parts of the pancreas have been studied ultrastructurally in adult rats. Five different islet cell types were identified in each islet with the aid of morphometrical analysis of their specific secretory granules. Previous immunohistochemical findings concerning the amount and location of insulin-, glucagon-, somatostatin- and pancreatic-polypeptide-containing cells and their ultrastructurally recognizable counterparts were compared, and it was possible to identify four main islet cell types with the electron microscope. Moreover, cells quite similar to the enterochromaffine cells described elsewhere in the exocrine pancreas and in the gastrointestinal tract were found to normally occur in the pancreatic islets of the rat.     

Electron-microscopical evidence for heterogeneity of micronodular argyrophil cell growth in man

Hypergastrinaemia-associated changes of non-antral argyrophil cells in man are of increasing interest, because of the development of potent inhibitors of gastric acid secretion. Using an antibody against chromogranin A, we identified micronodular endocrine cell hyperplasia of the oxyntic mucosa in gastric biopsy specimens of patients with hypergastrinaemia of different backgrounds. Consecutive ultrathin sections were examined at the electron-microscopical level. Endocrine cell types within the (extraepithelial) micronodules closely resembled those in the adjacent mucosa. Micronodules were classified into two groups. The first group was composed of endocrine cells only and predominated in patients with drug-induced hypergastrinaemia and/or chronic gastritis, and in a gastrinoma/MEN I patient. The second group represented “neuroendocrine complexes”, showing a close intermingling of non-myelinated nerve fibres with endocrine cells, and was found predominantly in pernicious anaemia. Micronodular argyrophil cell growth in man is therefore heterogeneous and depends on the background of the hypergastrinaemia.     

Histochemistry of the pancreatic islets in golden hamsterMesocricetus auratus waterhouse 1839

Summary The histological picuture of the Langerhans' islets in hamster pancreas is quite similar to that in white rat pancreas, i.e. the B-cells are located in the middle of the islet, while the A-cells in its periphery. Very often the argyrophil cells (D-cells) are located between the A- and B-cells forming a peculiar “barrier”. The histochemical studies reveal differences between the endocrine tissue and exocrine parenchyma. In general, the islet cells are richer in enzymes, as compared with the acini. The histochemical characteristic of hamster pancreas is closest to that of white rat pancreas. Like in rat, alkaline phosphomonoesterase reaction is very strong in the A-cells, while G-6-P reaction is negative. But, concerning zinc localization, there are differences between hamster and rat. Zinc reaction is very strong in the peripheral A-cells in white rat pancreas, while in hamster this reaction is much stronger in the B-cells (the reaction is negative in the A-cells). The D-cells can not be differentiated from the other endocrine pancreatic cells by means of hystochemical studies. But these studies permit certain conclusion on the possible role of the enzymes and substances investigated in cytophysiology of the islet cells.     

Light and electron microscopic identification of several types of endocrine cells in the gastrointestinal mucosa of the cat

Summary The following histological methods, previously proved to be useful in selective light microscopic detection of endocrine cells, were applied to the cat gastrointestinal mucosa: for the identification of biogenic amines, diazonium, ammoniacal silver and xanthydrol methods; for granules identification, methyl green-red acid dyes, toluidine blue, HCl-basic dye, lead-haematoxylin, phosphotungstic haematein and argyrophil methods. Results were compared with those of an extensive electron microscopic investigation.Five types of endocrine cells were identified in the gastric mucosa. Three types were found in the pyloric mucosa: the previously described 5-hydroxytryptamine-producing enterochromaffin cell, the gastrin producing G cell and a cell with an unknown function, labelled in this paper the X cell. Four types were found in the fundic mucosa: enterochromaffin cells (rarely observed), enterochromaffin-like cells secreting a 5-hydroxyindole but showing some ultrastructural and staining differences from true enterochromaffin cells (numerously present), A-like cells (few), resembling A cells of the pancreatic islets, and X cells, resembling those in the pyloric mucosa.In the intestinal mucosa, at least three endocrine cell types were distinguished in its duodenal part: enterochromaffin cells and two types of polypeptide-producing cells — some with smaller granules (S cells) and others with larger granules (L cells). Only two types were found in the mucosa of terminal ileum: enterochromaffin cells and numerously-occurring cells with large granules resembling in part duodenal L cells. The possibility of a relationship between S and L cells and the production respectively of the intestinal hormones secretin and cholecystokinin-pancreozymin was discussed.This investigation was supported by a grant N. 115/1139/0/4715 of the Italian Consiglio Nazionale delle Ricerche.     

Direct communication of homologous and heterologous endocrine islet cells in culture

The transfer of 6-carboxyfluorescein between islet cells in monolayer culture was observed by fluorescence microscopy, and the endocrine cells involved in this transfer were identified by immunohistochemistry and electron microscopy. The results show that carboxyfluorescein was directly exchanged between homologous B-cells and also between B- and A- or D-cells. Successive microinjections of the probe into different cells of the same cluster showed the existence of separate territories, each formed by 2-8 communicating cells. Intercellular communication was not observed after every dye microinjection, and communicating and noncommunicating islet cells were found to coexist within the same cluster. The data indicate that the exchange of exogenous cytoplasmic molecules occurs between different types of endocrine islet cells. However, within a single cluster, all islet cells are not metabolically coupled to one another, at a given time.     

Close association of centroacinar/ductular and insular cells in the rat pancreas

Close contacts between endocrine insular cells and exocrine acinar, centroacinar and ductular cells occur frequently in the rat pancreas as seen by both light and electron microscopy. Islets of Langerhans are surrounded incompletely by a thin connective tissue capsule or mantle but numerous exocrine-endocrine cell contacts occur at the periphery, which is irregular with considerable "intermingling" of the two cell types. Centroacinar and ductular cells are seen to be in contact with all endocrine cell types but most commonly insulin-secreting B-cells. The basal surface of centroacinar cells in the region of contact may be extensive, sometimes with overlap of basal processes of these cells and their lateral extension between acinar and insular cells. The areas of contact contain no connective tissue or basal lamina and show no surface specializations. The presence of both the "open" and "closed" type of enteroendocrine cells within acini is confirmed, some also being in contact with centroacinar cells. The functional significance of these exo-endocrine cell contacts is discussed in terms of the endocrine-acinar portal system, possible direct paracrine secretion, compartmentalization within the islet, and the known effects of islet hormones on exocrine secretion. Also relevant is the developmental origin of islets from ductal tissue and the cellular origin of some tumours, e.g., insulinomas, from duct cells.     

Light and electron microscopic study of crocodile islets of Langerhans

The tissue of the islets of Langerhans was studied in Alligator mississippiensis and Caiman niger. The distribution of the insular tissue in the pancreas was described from the picture in the light microscope, together with the incidence of characteristic islets, which frequently surround the duct tress, and of scattered insular cells. Five types of endocrine cells, distinguishable by their staining properties and typical electron microscopic image, were described in the insular tissue. In addition to A-, B- and D-cells, others which very probably correspond to EC- and PP-cells are described. Mixed cells were also found. The pancreatic interstitial connective tissue contains large numbers of nerve fibre bundles; isolated nerve fibres may penetrate to the insular cells. At the site of contact of a nerve ending with an endocrine cell, characteristic thickening of the membranes is sometimes found.     

Endocrine cells in oxyntic mucosa of a dog 5 years after pancreatectomy.

Immunofluorescence shows that the oxyntic mucosa of a dog depancreatized for 5 years and having a poorly-controlled diabetes has more glucagon- and somatostatin-containing cells than the mucosa of a control dog. At the ultrastructural level, 4 endocrine cell types are identified: A-, A-like, D- and enterochromaffin-like (ECL) cells, with increased numbers of A-, A-like and D-cells in gastric glands of the depancreatized dog, together with a higher concentration of immunoreactive glucagon in the gastric mucosa. The increase in A-, A-like and D-cells is compatible with: a) a change induced by the diabetic state itself; b) a hyperplasia secondary to the loss of corresponding pancreatic cells. At any rate, the fact that A-, A-like and D-cells increase parallely may indicate that these three cell types are functionally related one with another.     

The islets of Langerhans in ducks and chickens with special reference to the argyrophil reaction

Summary The pancreas of birds is a suitable object for studying the A and B cells separately, since the two cell systems are topografically almost entirely segregated in the form of light (= B cells) and dark (= A cells) islets of Langerhans.On the whole in the chicken and duck the actual distribution of the light islets into different size classes followed the same regular pattern previously found in the rat and man. In the body of the pancreas, containing the great majority of islets, the volume distribution curves thus appeared symmetrical.With the silver impregnation method used a distinct argyrophil reaction in both types of islets was obtained on paraffin sections of the pancreas. According to the presence or absence of blackening, the cells of the dark islets could be divided into two distinct fractions. Especially in the duck the silver-positive cells were grouped in a characteristic way along the walls of the capillaries. Ducks and chickens are not the only animals in which it is possible to identify an argyrophil fraction in what the usual granule stains had shown to be A cells. Parallel studies of various mammals are in complete agreement with these observations. It is, however, still uncertain whether we are here dealing with differences in function, age etc. in one and the same type of cell or with two completely different kinds. No correlation between the argyrophil reaction in the dark islet cells and their content of SH and SS groups or tryptophane could be established.This work was aided by grants from the Swedish Medical Research Council.     

Regulation of amylin release from cultured rabbit gastric fundic mucosal cells

Background  

Amylin (islet amyloid polypeptide) is a hormone with suggested roles in the regulation of glucose homeostasis, gastric motor and secretory function and gastroprotection. In the gastric mucosa amylin is found co-localised with somatostatin in D-cells. The factors regulating gastric amylin release are unknown. In this study we have investigated the regulation of amylin release from gastric mucosal cells in primary culture. Rabbit fundic mucosal cells enriched for D-cells by counterflow elutriation were cultured for 40 hours. Amylin and somatostatin release over 2 hours in response to agonists were assessed.     

The ultrastructure of the islets of Langerhans in normal and obese-hyperglycemic mice

Summary The islets of Langerhans in normal and obese-hyperglycemic mice were studied by electron microscopy. Two different types of islet cells with ultrastructural features well distinguished from the majority of B cells were observed. While one of them was provided with complete cell membranes, the other appeared to be arranged in a syncytium. It was postulated that these islet cells may be identified with the argyrophil A₁ cells and the non argyrophil A₂ cells demonstrated in different species with light microscopy. In the normal mouse the fine structure of the islet B cells corresponds in the main to what has been observed previously in the rat. However, in addition to the great majority of spherical B cell granules some appear rectangular, i.e. show similar morphological characteristics as described for the granules in the dog. The long termada ptation of the B cells to increased functional demands in the obese-hyperglycemic syndrome was associated with a pronounced degranulation with margination of the granules and with obvious changes of some organelles. The mechanisms for formation and liberation of insulin from the B cells are discussed in the light of the ultrastructural appearance of these cells in the obese-hyperglycemic mice.Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.     

On the nature of the metachromatic cells of pancreatic islets

Summary Islet cells with cytoplasmic granules metachromatically stained by toluidine blue are found in the pancreas of dog, cat, pig, rabbit, teleostian fish, monkey and man, but not in the rat, mouse and guinea pig; dubious results are obtained on duck and ox islets. These cells do not react with the staining methods for the demonstration of A- and B-cells; conversely, they are silver-impregnated by a modification of Davenport's method, and, at least in dog islets, can be readily identified with dark-field microscopy and stained blue with the Mallory-Heidenhain trichrome stain. No obvious changes of this cell type are found either in synthalintreated dogs or in diabetic men.The Authors suggest that the islet argyrophil-metachromatic cells (A₁-cells of Swedish Authors) are identical with D-cells and very likely represent a morphologically and functionally indipendent cell type.     

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.     

Morphology of the endocrine part of the pancreas of birds]

Methods of light and electron microscopy were used to study the structural organization of the endocrinous part of the pancreas in birds (domestic ducks and chickens). It has been established that the endocrinous part is formed by three types of pancreatic islands: dark, light and mixed islands. "Dark" islands consist of A- and D-cells, "light" ones--of B and D-cells and "mixed" islands--of A-, B- and D-cells. The index of B/A-cell ratio is equal to 0,39--0,4. This shows that each B-cell corresponds to 4 A-cells. On this basis one can say that the counterinsular apparatus of birds became stronger in the process of evolution and due to this a comparatively high level of glycemia is sustained in them. Availability of "dark" and "light" pancreatic islands also points out that in birds there occurred a partial disconnection of insular and counterinsular components of the endocrinous part of the pancreas, necessary for adaptation to principally new conditions of the environment.     

Evidence of three cell types in the islets of Langerhans in sand rats (Psammomys obesus).

The islets of Langerhans of sand rats were examined light- and electron microscopically with respect to the presence of various cell types. Apart from A- and B-cells, already known in sand rats, the presence of the third type of cells was established in the islet organ. According to their topography, argyrophilia (Hellman and Hellerstr?m technique) and electron microscopical appearance of their granules they should be considered as D-cells.     

Electron dense plaques (calcium binding sites) in the plasma membrane of the endocrine cells in rodent pancreatic islets and parathyroid glands

Summary Small electron dense plaques (EDP) were found in the plasma membrane of pancreatic islet A-, B- and D-cells and parathyroid chief cells of mice and gerbils. The identification of the EDP was facilitated by the use of special fixation techniques. The EDP may represent sites of calcium binding in the cell membranes.Supported by grants from the Swedish Medical Research Council (Project No. B76-12X-00718-11B)     

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B- and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.     

Cysteamine exerts multiple effects on the endocrine cells of the rat pancreas

We have studied the effects by cysteamine in vitro and in vivo on hormone production and islet cell metabolism in isolated pancreatic islets and perfused pancreas of the rat. In isolated islets, cysteamine dose-dependently depleted somatostatin immunoreactivity by 50% after 60 min exposure to 1 mmol/l of the compound. This effect appeared to be independent of interaction of the drug with secretion of somatostatin from the pancreatic D-cells. Cysteamine, however, interacted acutely not only with the D-cells, but also markedly suppressed glucose-induced insulin release. Moreover, cysteamine inhibited islet glucose oxidation, an effect which reflects interference with the metabolism mainly of the B-cells. The effect of cysteamine on glucose-induced insulin release was prolonged, since it was still observed in the isolated rat pancreas perfused 24 h after in vivo treatment with cysteamine. In contrast to the effects on glucose-induced insulin release, the response to glibenclamide remained unaffected by a previous exposure to cysteamine in vivo. However, both glucose- and glibenclamide-induced somatostatin secretion was reduced by 50%, whereas basal glucagon secretion was significantly enhanced in pancreata from cysteamine-treated rats vs. control rats. We conclude that (1) cysteamine does not specifically affect the D-cells of the islets, and (2) the multiple effects by cysteamine on islet cell function, particularly on B-cell metabolism and secretion, renders the compound unsuitable for the study of paracrine interactions in the islets.