Influence of fasting and stimulation on the rat gastric endocrine cells

Summary The ultrastructure and certain cytochemical parameters of endocrine cells of the rat gastric mucosa during 168 h of fasting were investigated. To some of the fasting animals peroral food or alcohol was administered before decapitation.The EC (enterochromaffin cells) the ECL (enterochromaffin-like cells), D₁ cells, AL (A-like cells) and G cells were identified by means of electron microscopy. Only the EC, ECL, and G cells could be identified by means of light microscopy by an adequate histochemical technique.The ultrastructural picture of the ECL and of the EC cells did not change markedly during the fasting. In the D₁ cells there occurred an agglomeration of secretory granules. Some of them disintegrated and disappeared. In the AL cells an agglomeration of granules during the fasting was also observed. Granules engulfed in lysosomes were often found. The participation of lysosomes in the degradation of granules during the fasting was more marked in the AL cells than in the G cells. The participation of lysosomes was questionable in the EC and D₁ cells, and in the ECL cells no lysosomes were observed. In contradistinction to the G cells of the non-fasting animals, where more than one half of the gastrin granules were empty, the G cells during the fasting were filled with agglomerated dense granules and contained lysosomes with fragments of engulfed secretory granules.Following the administration of food (Larsen's diet) 3 h before sacrificing the dissolution of the content of granules with well preserved membranes was observed (emiocytosis did not take place). The administration of food did not lead to changes in the ultrastructural appearance of the EC cells. The peroral administration of alcohol did not lead to any changes in the ultrastructural appearance of the AL and G cells.     

Light and electron microscopic identification of the histamine-storing argyrophil (ECL) cell in murine stomach and of its equivalent in other mammals

Summary A peculiar type of cell, the ECL cell, accounts for a large portion of nonenterochromaffin endocrine cells in the gastric oxyntic glands of several mammals, including rat, mouse, guinea pig, rabbit, cat, dog, pig, and man. The ECL cell is characterized mainly by its secretory granules with irregular cores heavily reacting to Grimelius' silver, Sevier-Munger silver and phosphotungstic acid, while failing to react, in all species but the cat and rabbit, to Masson's argentaffin method. In the rat and mouse, the ECL cell seems to correspond exactly to the argyrophil non-argentaffin histamine-storing enterochromaffin-like cell of Håkanson and Owman (1967); in the remaining species, ECL cells seem to account for only a part of the gastric argyrophil enterochromaffin-like cells described by Håkanson and coworkers. Besides sporadic amines, ECL cell granules store non-amine components, whose possible nature is discussed.This investigation was supported in part by Farmitalia S.p.A., Milano.     

The endocrine cells of the pyloric glands of adult ox

As part of a project to identify the endocrine cells ("EC" and "APUD" series) of the gastroenteric apparatus of ruminants, the ultrastructure of the mucosa of the pyloric glands of adult ox was studied morphologically and cytochemically, in parallel with a light microscope histochemical analysis. The results show that: the "EC" cells (producing 5-HT) are recognizable by their secretory granules which are heavily osmiophilic, argentaffin ("Masson") and argyrophilic ("Grimelius"). A further distinction is possible on the basis of their morphological features: the "EC" cells of the gastric type (which belong to the "ECn" group) contain granules fairly homogeneous in shape and size, while the "EC" cells of the intestinal type (or "EC1") show granules which are more pleiomorphic and variable in size. Of particular interest is the presence in some cells of granules typical of the "EC" cells of the intestinal type, in the vicinity of a few others, which appear quite similar to those of the adjoining exocrine cells; the "G" cells (gastrin producing) contain medium sized granules, which are unreactive to "Masson" and poorly argyrophilic. Their morphology is rather diverse; some of them (these are the "typical" cells) have a granular and weakly electron dense content, others (which we consider "atypical") show a homogeneous and heavily osmiophilic core, with an eccentrical empty area. Also present are granules whose appearance is intermediate and empty vesicles; the "D cells" (somatostatin producting) show round, medium sized granules which have a granular, moderately osmiophilic core, tightly encircled by the membrane. These granules are unreactive to "Masson" and to "Grimelius"; the "D1" cells (whose function is yet unclear) contain small, round granules whose core is variously but discretely electron dense and not always homogeneous; they are unreactive to "Masson" and fairly argyrophilic. These granules may be numerous and packed, or scarce; in this latter instance the few granules are intermingled with variously running tufts of parallel filaments, thus resembling the "P" cells, whose function is still undefined. These data show therefore that the types of endocrine cells we have identified in the pyloric glands of adult ox correspond to those described in other mammals; "X" and "F" or "PP" cell appear to be lacking.     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Ultrastructural identification of argyrophil and argentaffin cells of the gastric mucosa in the rat

Ultrastructure of endocrine cells impregnated in the rat gastric mucosa by Grimelius method (identification of argyrophilia) and by Masson--Hamperl method (identification of argentaffinity) and influence of various fixatives on the structure and properties of the secretory granules in these cells have been studied. Fixation of the material in paraformaldehyde or glutaraldehyde varies in its effect on the granule structure of EC-, D1- and ECL-cells, while its influence on the granule structure of G-, D- and AL-cells is identical. The granules of EC-, ECL- and G-cells are argyrophil, and only those of EC-cells are argentaffin. Weak argyrophilia, which is evidently not appearant at the light-optical level, is specific for granules of D1- and AL-cells. Fixation in paraformaldehyde and especially the subsequent treatment in osmium tetroxide results in increasing argyrophilia of the endocrine cells, as compared to fixation in glutaraldehyde. Varieties in the effect of the fixatives do not prevent ultrastructural and histochemical identification of the endocrine cell types.     

Histochemical and ultrastructural studies on the enterochromaffin-like cell in the gastric mucosa of the opossum Didelphis albiventris (Marsupialia)

Summary An ultrastructural study of enterochromaffin-like (ECL) cells in the gastric mucosa of the white-belly opossum Didelphis albiventris (Marsupialia) was carried out. In parallel, histochemical methods were used at the light-microscopical level to demonstrate argentaffin cells, argyrophilic cells, and serotonin- and histamine-immunoreactive elements. Argentaffin and serotonin-immunoreactive cells were scattered, and argyrophilic cells were numerous, within the full thickness of the mucosa. Argyrophilic cell distribution was similar to that of histamine-immunoreactive elements. At the electron-microscopical level, the oxyntic mucosa of D. albiventris presented endocrine cells with secretory granules morphologically similar to those of the ECL cell of eutherian mammals. However, in this marsupial, the ECL cell exhibited a variable mixture of two distinct types of secretory granules: (1) granules with the morphological appearance of the eutherian ECL cell, and (2) granules morphologically similar to those of the eutherian enterochromaffin (EC) cells. Based on this morphological pattern of the ECL cell granules, it is proposed that in the oxyntic mucosa of the opossum D. albiventris, the EC and ECL cells represent distinct steps in the same line of cell differentiation; the ECL cell should also be a site of histamine storage.     

The endocrine cells of the rectum of adult ox.

In order to identify the endocrine cell types in various parts of the Ruminant gut, we have applied ultrastructural, both morphological and cytochemical, techniques, in parallel to the histochemical ones, to study the rectal mucosa of the adult Ox. In these studies we show that: "EC" cells, of the intestinal type, contain predominantly pleiomorphic granules, which are very electron dense and heavily reactive to "Masson" and "Grimelius" methods; "L" cells are recognizable by their numerous granules, which are fairly homogeneous in shape and osmiophilia. They do not react with "Masson" and are weak or negative to Grimelius s reaction. These granules occur near to others that are less dense, unreactive to "Masson", and that contain an argyrophilic matrix, with an eccentric electron dense core, which does not react with silver; "F-like" cells contain granules which are variable in shape, size and osmiophilia. They are unreactive to "Masson" and weak or unreactive to Grimelius silver; "H" cells contain few, small and uniformly osmiophilic granules. These are unreactive to "Masson" and uniformly reactive to "Grimelius". Our data suggest that the morphology, frequency and distribution of the cell types we have identified in the mucosa of the bovine rectum correspond with those reported in large intestine and rectum of Monogastrics, as by other authors described.     

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells

Summary The thiocarbohydrazide-silver proteinate (TCH-SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D₁ cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D₁ cells failed to react. In most experiments granules of X, A and O cells showed peripheral staining, while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.     

Regeneration of endocrine cells in the stomach

A mucosal defect was produced by cryosurgery in the antral and the fundic wall of the rat stomach, and regeneration of gastric endocrine cells was studied 50, 100 and 200 days after operation. Fifty days after the operation, the mucosal defect was completely covered with regenerated epithelium. The regenerated mucosa both in the antral and in the fundic region consisted of mucinous glandular structures. The regenerated mucosa in the corpus remained pseudopyloric in type even 200 days after operation. Regardless of the time after operation, regeneration of endocrine cells was always observed. We could identify G cells and EC cells in the regenerated mucosa of the antrum, and EC cells, A cells and AL cells in the regenerated mucosa of the corpus, respectively. By electron microscopy, endocrine-exocrine cells were frequently encountered. These cells had two different types of intra-cytoplasmic granules; one was an endocrine-specific, small electron-dense granule, and the other a large, lucent mucin droplet-like granule. These findings indicate that the endocrine cells of the stomach are formed from endodermal precursor cells.     

Eight types of endocrine cells in the abomasum of sheep

In the ovine abomasum, 8 types of endocrine cells were classified at ultrastructural level. The gastric-type EC cells contained oval and pleomorphic granules with high electron density. The intestinal-type EC cells were filled with oval, irregular and highly dense granules. ECL cells contained irregular granules with high density and wide clear spaces. D cells were filled with round granules showing low to moderate density and finely granular matrix D1 cells contained round or oval granules with variable, low to moderate density and finely granular content. G cells showed round and oval granules with moderate density, densely packed or flocculent content. F cells were filled with oval or elliptic granules showing low density with a finely granular and flocculent matrix. X cells contained round granules with high density and homogeneous material. Gastric-type EC cells, intestinal-type EC cells, D cells, and D1 cells were represented in the cardiac, fundic and pyloric gland areas of the ovine abomasum. ECL cells and F cells were confined to the fundic glands, G cells and X cells to the pyloric glands.     

Endocrine cells of the gastric mucosa of Rana temporaria L

The endocrine cells of the gastric mucosa of Rana temporaria have been studied according to the ultrastructure, the staining properties of the granules with Masson Fontana's and Grimelius' silver methods, silver impregnation of Davenport on deplasticised semithin sections and immunocytochemical techniques. Seven different types of endocrine cells have been described. Six were regarded as belonging to known types: G, A, EC, ECL, D and P cells. One type was considered as unclassifiable.     

Effects of background adaptation on α-MSH and β-endorphin in secretory granule types of melanotrope cells of Xenopus laevis

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including -MSH and N-acetyl-endorphin. In this study three types of secretory granules, electron-dense(130 nm Ø), moderately electron-dense ( 160 nm Ø) and electronlucent ( 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl--MSH and N-acetyl--endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl--MSH and N-acetyl--endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl--endorphin, probably by stimulating intragranular acetylation of -endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl--MSH which takes place near the plasmalemma at the time of granule exocytosis.     

Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 g/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.     

Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion

Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg^-1·h^-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.     

Histochemical investigations on the secretory cells in the oesophagogastric tract of the Eurasian green toad,Bufo viridis

The secretory cells of the oesophagogastric tract of the Eurasian toad, Bufo viridis, were examined using standard histochemical methods and lectin histochemistry. Two goblet cell types were found in the oesophageal epithelium, differing in their morphology and the histochemical features of the secretory granules. These contained mainly acidic glycoconjugates, both sulphated and carboxylated, and a small amount of pepsinogen. Type I goblet cells contained stable class-III mucosubstances, which were absent in Type II. No pluricellular oesophageal glands were found. The oesophagogastric junction had a superficial epithelium similar to that of the oesophageal epithelium, with alveolar pluricellular glands, secreting stable class-III mucins, and few oxynticopeptic cells. The gastric mucosa presented secretory cells both in the surface epithelium and in the gastric glands. Superficial and foveolar cells produced neutral mucins with Gal1,3GalNAc residues. Neck cells, oxynticopeptic cells and endocrine cells were found in the gastric glands. Neck cells produced stable class-III mucosubstances. A functional gradient was observed in the oxynticopeptic cells from the oral to the aboral fundus, with a decrease in pepsinogen secretion towards the aboral fundus and a possible increase in HCl secretion. In the pyloric mucosa, the oxynticopeptic cells disappeared and the glands produced only neutral mucins, without stable class-III mucosubstances.     

An electron microscopical investigation of the follicle gland (cerebral gland) and of some neurosecretory cells in the lateral lobe of the cerebral ganglion of the pulmonate gastropod Lymnaea stagnalis L.

Summary The ultrastructure of the follicle gland and of some Gomori-positive neurosecretory cells in the lateral lobes of the cerebral ganglia of Lymnaea stagnalis is described.The follicle wall consists of epithelial cells containing secretion granules, and of processes of lateral lobe nerve cells bearing cilia, including the processes of bipolar neurosecretory B-cells. The ultrastructure of the follicle of Ancylus fluviatilis and Planorbarius corneus appeared to be very similar to that of L. stagnalis. From the ultrastructure of the follicle wall and lumen, no conclusions can be drawn as to its formerly proposed possible functions (sense organ, endocrine organ, neuroendocrine organ). Of three neurosecretory cell types (B-cells, canopy cells, droplet cells), the positivity to chrome-haematoxylin and paraldehyde-fuchsin appeared to be due to elementary granules of different size and appearance. This finding supports in a particular sense the view that results obtained with the neurosecretory stains, should be judged carefully. Also in ordinary neurons elementary granules can be found, at times in great numbers. Several types of elementary granules (at least 6) could be distinguished. Their character and function (neurohormones, neurotransmitters) are not clear.     

Mast cells in rat dermis and jejunal lamina propria show a five-fold difference in unit granule volume

Summary The cytoplasmic granules of mast cells have a periodic multimodal size distribution in which the volumes of individual granules are integral multiples of the intermodal distance, a volume defined as the unit granule or ₁. In this study, we used two 3-month-old male rats to analyze two classical mast cell subpopulations, dermal connective tissue-type mast cells and jejunal lamina propria mucosal mast cells, for the morphometric characteristics of their cytoplasmic granules. Both and the mean volume of individual cytoplasmic granules were much smaller in dermal than in jejunal mast cells (ratios of 1:5.5 and 1:4.2, respectively), but dermal mast cells contained 150% more granules per cell than did jejunal mast cells. The two types of mast cells did not differ significantly in total cell volume, nucleus volume, aggregate volume of cytoplasmic granules per cell or numbers of unit granules comprising a granule of mean volume. These findings add unit granule volume to the list of phenotypic characteristics which express significant variation in anatomically distinct populations of mast cells.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Summary Certain disseminated endocrine-like cells have reviously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (paraxons). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.This work was reported in part at the 29th Congress of the International Union of Physiological Sciences, Sydney, Australia, August 28–September 3, 1983     

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.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.