Immunoelectron cytochemical localization of motilin and substance P in rabbit bile duct enterochromaffin (EC) cells.

Using an immunoreactive technique the two peptides, motilin and Substance P, have been localized at the ultrastructural level in enterochromaffin (EC) cells. Motilin occurs in cells containing a mixed population of biconcave and round secretory granules whereas Substance P is found in cells with exclusively round granules. These observations confirm the existence of at least two functionally and morphologically different types of EC cell in rabbit bile duct, both of which contain 5-hydroxytryptamine. Classification of the endocrine cells of the gut on a purely morphological basis is clearly impossible, however.     

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.     

Ultrastructural identification of round-granule EC cells in rabbit fundic mucosa

In endocrine (EC) cells of rabbit fundic mucosa, it is practically impossible to obtain unequivocal ultrastructural identification of all cells found in order to perform morphometric analysis. In addition to classic EC cells with pleomorphic granules, a cell type with entirely round granules is encountered which can be confused with non-EC cells. To solve this problem, all EC cells in our study were first identified by their 5-HT (immunocytochemistry) and argentaffinity. Examination of the fine structures of reactive cells then revealed that the round granules of EC cells were differentiated from those of non-EC cells by the existence of a dense core surrounded by a less dense halo, a feature providing unequivocal ultrastructural identification. EC cells with round granules showed less argentaffinity and less immunoreactivity to 5-HT as compared with classic EC cells. After labelling with [3H]L-dopa, EC cells with round-granules displayed an overall staining index higher than that of classic EC cells and comparable with that of D cells; however, the nuclear staining index was higher than that of D cells.     

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.     

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.     

Carboxy groups as essential residues in beta-lactamases.

Purified rat peritoneal mast cells have a 10-20-fold higher dipeptidyl peptidase II (DPP II) activity as compared with that of macrophages from the same source. Upon stimulation with the secretagogue Compound 48/80, DPP II is released from peritoneal-lavage cells and from purified mast cells, but not from purified macrophages, in a dose-dependent manner. Maximally, about one-third of the DPP II present in peritoneal-lavage cells is released. Substance P and the antigen/IgE system probably produce a similar effect. Both histamine and Zn2+, two ingredients of mast-cell granules, strongly inhibit DPP II at concentrations reported to occur in the granules. A possible role of mast-cell DPP II in the remodelling of connective tissue is discussed.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Endocrine cells of the APUD-system in the human lung (electron microscopy characteristics)

Lungs of 4 human fetuses (11-, 13-, 22-, 28-week-old), of 1 stillborn and of 3 mature persons, operated in connection with pulmonary cancer, have been investigated. In the fetal lungs apudocytes and neuroepithelial bodies (NEB) have been revealed. The apudocytes differ from each other by structure and size of endocrine granules. In the 11-week-old fetus P1 cells with two types of granules occur most often. Among P1 cells there are several subgroups, differing in their granule dimensions. P2 apudocytes possess granules of one type with a round core and a narrow rim of cytoplasm. P3 cells are characterized with still larger granules, a very dense core and a narrow rim. In large bronchi some groups are found, consisting of two and more endocrine cells of all three types. In the lungs of the 13-week-old fetus P1 cells are defined and a new type of cells, that contain homogenous granules, characterizing by their small size. In 22 weeks of development in the intrapulmonary bronchi apudocytes with granules specific for Ec-cells are found. NEB consists of cells and islands, possessing polymorphous granules. Various types of apudocytes are defined in large bronchi of the 22-week-old fetus. In the stillborn infant apudocytes in the lung are found very seldom. In lung of the mature persons the morphology of apudocytes is unitypical. Thus, during embryogenesis and after birth there are variable types of endocrine cells and NEB.     

Effects of Substance P on Nicotinic Acetylcholine Receptor Function in PC 12 Cells

The effects of substance P on the functioning of nicotinic acetylcholine receptors in PC12 cells were examined. Carbachol-stimulated 22Na+ uptake was used to assess the functional state of the nicotinic receptor. We found that incubation of the cells with substance P alone caused a loss of receptor function. Receptors recovered from this effect with a t1/2 of 0.94 +/- 0.10 min. Since receptors recovered from carbachol-induced desensitization at a significantly slower rate (t1/2, 1.77 +/- 0.21 min), it was concluded that the two inactive states are not kinetically equivalent. The effects of substance P on carbachol-induced loss of receptor activity were also examined. Substance P had no effect on a component of carbachol-induced loss of activity that was nonrecoverable (inactivation). However, substance P had several effects on the recoverable loss of activity induced by carbachol (desensitization). Substance P caused a shift to the left in the EC50 for carbachol-induced desensitization at equilibrium. If cells were simultaneously incubated with carbachol and substance P7-11, a low-potency analog of substance P, an increase in the rate of formation of a state of the receptor that was kinetically indistinguishable from the state induced by carbachol alone was observed. However, not all inhibition of nicotinic cholinergic function could be explained by an increased rate of formation of a desensitized receptor and it is concluded that substance P causes both enhanced desensitization and block of the nicotinic receptor-linked channel.     

Enterochromaffin cells as the endocrine source of gastrointestinal Substance P

Summary Immunoreactive Substance P is localized in the intramural neural plexuses of mammalian intestine, and in endocrine cells in the intestinal mucosa which have now been identified as enterochromaffin (EC). The presence of a neurotransmitter peptide in these cells favours the hypothesis of their neuroectodermal origin.On leave from the Department of Pathology, University of Basle; in receipt of a grant from the Swiss Academy of Medical Sciences     

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.     

Morphology of the air-breathing stomach of the catfish Hypostomus plecostomus

Histological and ultrastructural investigations of the stomach of the catfish Hypostomus plecostomus show that its structure is different from that typical of the stomachs of other teleostean fishes: the wall is thin and transparent, while the mucosal layer is smooth and devoid of folds. The epithelium lining the whole internal surface of the stomach consists of several types of cells, the most prominent being flattened respiratory epithelial cells. There are also two types of gastric gland cells, three types of endocrine cells (EC), and basal cells. The epithelial layer is underlain by capillaries of a diameter ranging from 6.1-13.1 microm. Capillaries are more numerous in the anterior part of the stomach, where the mean number of capillary sections per 100 microm of epithelium length is 4, compared with 3 in the posterior part. The cytoplasm of the epithelial cells, apart from its typical organelles, contains electron-dense and lamellar bodies at different stages of maturation, which form the sites of accumulation of surfactant. Small, electron-dense vesicles containing acidic mucopolysaccharides are found in the apical parts of some respiratory epithelial cells. Numerous gastric glands (2 glands per 100 microm of epithelium length), composed of two types of pyramidal cells, extend from the surface epithelium into the subjacent lamina propria. The gland outlets, as well as the apical cytoplasm of the cells are Alcian blue-positive, indicating the presence of acidic mucopolysaccharides. Zymogen granules have not been found, but the apical parts of cells contain vesicles of variable electron density. The cytoplasm of the gastric gland cells also contains numerous electron-dense and lamellar bodies. Gastric gland cells with electron-dense cytoplasm and tubulovesicular system are probably involved in the production of hydrochloric acid. Fixation with tannic acid as well as with ruthenium red revealed a thin layer of phospholipids and glycosaminoglycans covering the entire inner surface of the stomach. In regions of the epithelium where the capillaries are covered by the thin cytoplasmic sheets of the respiratory epithelial cells, a thin air-blood barrier (0.25-2.02 microm) is formed, thus enabling gaseous exchange. Relatively numerous pores closed by diaphragms are seen in the endothelium lining the apical and lateral parts of the capillaries. Between gastric gland cells, solitary, noninnervated endocrine cells (EC) of three types were found. EC are characterized by lighter cytoplasm than the surrounding cells and they contain dense core vesicles (DCV) with a halo between the electron-dense core and the limiting membrane. EC of type I are the most abundant. They are of an open type, reaching the stomach lumen. The round DCV of this type, with a diameter from 92-194 nm, have a centrally located core surrounded by a narrow halo. EC of type II are rarely observed and are of a closed type. They possess two kinds of DCV with a very narrow halo. The majority of them are round, with a diameter ranging from 88-177 nm, while elongated ones, 159-389 nm long, are rare. EC of type III are numerous and also closed. The whole cytoplasm is filled with large DCV: round, with a diameter from 123-283 nm, and oval, 230-371 nm long, both with a core of irregular shape and a wide, irregular halo. EC are involved in the regulation of digestion and probably local gas exchange. In conclusion, the thin-walled stomach of Hypostomus plecostomus, with its rich network of capillaries, has a morphology suggesting it is an efficient organ for air breathing.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates

Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by β2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and β2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1)·s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.     

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.     

Enteroglucagon and substance P-like immunoreactivity in argentaffin and argyrophil rectal carcinoids.

A specific immunofluorescence for enteroglucagon or substance P or for both hormones was demonstrated in nine out of 12 examined rectal carcinoids. One tumor was argentaffin, contained ultrastructurally pleomorphic granules of the entero-chromaffin cell type, and showed immunofluorescence for substance P. The rest were non-argentaffin but were argyrophil with the Grimelius technique and contained round granules. The argyrophil carcinoids were immunoreactive to one or both hormones in eight cases and not fluorescent in three cases. In two of the non-argentaffin carcinoids a small number of argyrophil cells was found with the method of Sevier-Munger.     

Substance P Protects Against Desensitization of the Nicotinic Response in Isolated Adrenal Chromaffin Cells

Substance P, a peptide endogenous to the splanchnic nerve, is known to inhibit the acetylcholine-and nicotine-induced release of catecholamines from isolated adrenal chromaffin cells. In the present study the effect of substance P on desensitization of catecholamine release from these cells was examined. Substance P (10(-5) M) completely protected against desensitization of catecholamine release produced by acetylcholine at 37 degrees C or 23 degrees C and by nicotine at 23 degrees C; substance P also afforded appreciable protection against nicotine-induced desensitization at 37 degrees C. The peptide had no effect on K+-induced desensitization of catecholamine release. Like substance P, d-tubocurarine also prevented nicotinic desensitization. Substance P prevented both of two components of nicotinic desensitization, i.e. the Ca2+-dependent component and the Ca2+-independent, depletion-independent component of desensitization. Substance P had little effect on subsequent catecholamine uptake, indicating that substance P's protection against desensitization is a result of facilitation of catecholamine release rather than inhibition of catecholamine reuptake. Nicotine-induced catecholamine release and nicotinic desensitization of catecholamine release were Na+-independent, although substance P's inhibition of nicotine-induced catecholamine release was reduced by extracellular Na+. These in vitro studies suggest a similar role for substance P in vivo: substance P's protection against nicotinic desensitization may ensure a maintained output of adrenal catecholamines during stress, when the splanchnic nerve releases large amounts of acetylcholine.     

Ultrastructural and immunohistochemical study of endocrine cells in the midgut of the cockroach Blaberus craniifer (Insecta,Dictyoptera)

Summary The midgut of Blaberus craniifer is principally made up of columnar epithelial cells which are derived from small regenerative cells found grouped in nidi. Between them, small sparsely granulated cells with clear cytoplasm can be observed lying on the basal lamina. Mainly based on the size, shape and texture of their secretory granules, at least ten types of such endocrine cells have been identified. Five cell types contain a uniform population of dense granules: (1) medium-sized, round to oval granules; (2) small elongated granules; (3) large irregular granules; (4) oval granules with a highly osmiophilic core; (5) oval, haloed granules. Five others are characterized by a heterogeneous population of granules: (6) small, round to oval, variably electron-dense granules; (7) oval medium-sized granules of variable electron density; (8) large irregular granules of variable electron density; (9) small dense granules and large vesicles with filamentous material; (10) small dense granules and very large pale vesicles.In addition, near the regenerative cells, large cells characterized by very large, irregular, dense granules (up to 4 m), lack contact with the lumen, and reach the basal lamina only by slender cytoplasmic processes.Several antisera raised against mammalian peptides and amine were used to reveal axonal fibers and endocrine cells. Serotonin-like immunoreactivity is localized in a profuse innervation of the muscle layers that surround the epithelium, whereas cholecystokinin and methionine-enkephalin antisera stain a more moderate number of axonal fibers. Cholecystokinin-, methionine-enkephalin-, substance P-, vasoactive intestinal peptide-, somatoliberin-, and gonadoliberin-like immunoreactivities were detected in endocrine cells of the epithelium. While most of the cells appear pyramidal, oval, fusiform or bowl-shaped, and seem to lack contact with the lumen, cells reaching it have been detected reacting with antisera to cholecystokinin, substance P, vasoactive intestinal peptide, somatoliberin and gonadoliberin.     

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.     

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.