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     

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.     

Mechanisms in regulating the release of serotonin from the perfused rat stomach

The mechanisms regulating the release of serotonin into the portal circulation as well as into the gastric lumen were studied in the isolated vascularly and luminally perfused rat stomach. Immunohistochemical study of the rat stomach showed that serotonin-containing enterochromaffin (EC) cells were densely packed in the antral mucosa, sparsely scattered in the corpus, and not found in the fundus. Such morphological findings suggest that serotonin detected in this study may have originated from antral EC cells. Luminal acidification stimulated the vascular release of serotonin but did not affect the luminal release of serotonin. The basal release of serotonin into the vasculature was 10 times higher than that into the gastric lumen at intragastric pH 2. The vascular release of serotonin is regulated by stimulation from cholinergic nicotinic mechanisms, whereas inhibitory neurotransmitters such as vasoactive intestinal peptide and NO are probably not involved. Somatostatin and peptide YY originating from endocrine cells may exert direct inhibitory effects, possibly via somatostatin and peptide YY receptors on the EC cells, and a cholinergic muscarinic mechanism may exert indirect effects on the vascular release of serotonin via the muscarinic receptor on the endocrine cells.     

Ultrastructural and Fluorescence Microscopical Characterization of the Intestinal Endocrine Cells in a Cyclostome,Myxine glutinosa

Endocrine cells of so-called basal-granulated-open type in the intestinal epithelium of a cyclostome, the Atlantic hagfish (Myxine glutinosa), are characterized ultrastructurally and fluorescence microscopically. These cells regularly extend from the basal lamina to the gut lumen, ending in an apical process with microvilli and a filamentous surface coat. Fasting results in an accumulation of secretion granules in all cytoplasmic portions, except for the terminal web area. A similarity is recorded between the distribution of secretion granules and the finely granular fluorescamine-induced fluorescence, suggesting that the fluorescence is associated to some component(s) of the secretory granules. Granule release may take place at the base after an adequate stimulus (presence of food?) at the luminal portion of the cells. The formaldehyde condensation technique shows that insulin-containing hagfish islet parenchymal cells, but not intestinal endocrine cells, store dopamine after intestinal supply of the amine precursor. Acidification of formaldehyde vapour-fixed intestinal epithelium induces fluorescence in the granules of zymogen cells but not of endocrine cells, indicating a low concentration of tryptophyl-peptide(s) in the secretory granules of hagfish intestinal endocrine cells.     

Serotonin stimulates endotoxin translocation via 5-HT3 receptors in the rat ileum

Because few previous studies have investigated the mechanisms of endotoxin translocation induced by intestinal obstruction, we aimed to clarify whether or not serotonin [5-hydroxytryptamine (5-HT)], which is released from enterochromaffin (EC) cells, is responsible for alterations of the mucosal permeability to endotoxin and to identify the 5-HT receptor subtypes that mediate this action. FITC-labeled LPS (FITC-LPS) was injected into the ileum of rats, and the FITC-LPS level in the superior mesenteric vein was subsequently measured by using a fluorescence spectrophotometer. To measure the 5-HT release induced by high intraluminal pressure, ex vivo preparation of vascularly and luminally perfused rat ileum was used. Results demonstrated that elevated intraluminal pressure stimulates the translocation of FITC-LPS and the release of 5-HT from the EC cells into the intestinal lumen but not into the portal circulation. This FITC-LPS translocation, which was stimulated by exogenously applied 5-HT in the lumen and the jugular vein, was inhibited by 5-HT(3) receptor antagonist administration both intaluminally and intravenously. The stimulatory effect of elevated intraluminal pressure on the translocation of FITC-LPS was inhibited by the intraluminal and intravenous administration of 5-HT(3) receptor antagonist. These results suggest that 5-HT released from EC cells may be involved in the translocation of FITC-LPS induced by elevated intraluminal pressure and that this effect is mediated by 5-HT(3) receptors that may be located in the intestinal epithelium.     

Expression of fucose residues in entero-endocrine cells

 The binding of the fucose-specific lectin, Ulex europaeus agglutinin (UEA-I), to entero-endocrine cells was studied in the ileum and caecum of humans, rabbits, rats, and mice. In all species investigated, numerous cells scattered in the crypt and villus epithelia intensely bound the UEA-I lectin. These cells proved to be argyrophilic and were identified as enterochromaffin cells and peptide tyrosine tyrosine cells by immunohistochemistry. They mostly reached the gut lumen (”open type”) by slender cellular processes. At the ultrastructural level, fucose binding sites were located in the matrix of the electron-dense secretory granules of these cells and in the glycocalyx covering their apical membrane. The results show that in various mammalian species entero-endocrine cells of defined types express fucose-bearing glycoconjugates. The presence of fucose residues in the apical membrane of entero-endocrine cells indicates that this membrane domain has a specialized composition of intramembranous glycoconjugates which could be involved in receptive and/or secretory functions. Accepted: 12 June 1997     

An ultrastructural study of the cysts in chicken ultimobranchial glands,with special reference to C-cells

Summary The ultimobranchial glands of the chicken were examined by electron microscopy and immunocytochemistry using a calcitonin antiserum. Electron microscopy confirmed the presence of C-cells, containing numerous secretory granules storing calcitonin, in the luminal lining of cyst-like structures found in these glands. These cells were furnished with prominent microvillar projections at their luminal surface, and the cytoplasm of the apical region was filled with fibril material. Furthermore, the cells contained prominent junctional complexes and desmosomes at their apico-lateral surfaces. In these C-cells, secretory granules were concentrated near the lumen and some were attached to the apical cell membrane. The luminal content of the cysts had a colloid-like and flocculent appearance, and was frequently seen attached to the cytoplasmic projections or apical cell membrane of the C-cells. Since the cysts progressively increase in volume and number with age, it is suggested that they may partly play a role in the storage of excess or unneeded hormonal products.     

Studies on the histomorphology and histochemistry of the saccus vasculosus of Cyprinion macrostomus.

The saccus vasculosus of Cyprinion macrostomus is made up of coronet and supporting cells. Neurones are not observed. The luminal surface of the coronet cells exhibits hair-like protrusions. The saccus vasculosus is mainly concerned with a secretory function. Plenty of secretory material is seen in the lumen. PAS and alcian blue tests are positive for the secretory material in the lumen and the apical protrusions of the coronet cells. Some cells also exhibit small PAS- and alcian blue-positive granules. The possibility of the elaboration of glycogen by the coronet cells is ruled out. The toluidine blue test is also positive for the secretory material in the lumen and the apical protrusions. Small metachromatic granules are also seen in some of the coronet cells. The acetone-Sudan black B and Sudan black B tests point to lipid moiety of the coronet cells, the apical protrusions and the secretory material in the lumen. The acrolein-Schiff and ninhydrin-Schiff tests point to a protein moiety of the coronet cells and the secretory material in the lumen. The performic acid-alcian blue test showed cystine to be present in small amount.     

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.     

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit

The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.     

Electron microscopic study of the anterior midgut in Cenocorixa bifida Hung. (Hemiptera: Corixidae) with reference to its secretory function

The epithelium of anterior midgut of adult Cenocorixa bifida was examined with light and electron microscopy. The folded epithelium is composed of tall columnar cells extending to the lumen, differentiating dark and light cells with interdigitating apices and regenerative basal cells in the nidi surrounded by villiform ridges that penetrate deeply into the epithelium. The columnar cells display microvilli at their luminal surface. Microvilli lined intercellular spaces and basal plasma membrane infoldings are associated with mitochondria. These ultrastructural features suggest their role in absorption of electrolytes and nutrients from the midgut lumen. The columnar cells contain large oval nuclei with prominent nucleoli. Their cytoplasm is rich in rough endoplasmic reticulum, Golgi complexes and electron-dense secretory granules indicating that they are also engaged in synthesis of digestive enzymes. The presence of secretory granules in close proximity of the apical plasma membrane suggests the release of secretion is by exocytosis. The presence of degenerating cells containing secretory granules at the luminal surface and the occurance of empty vesicles and cell fragments in the lumen are consistent with the holocrine secretion of digestive enzymes. Apical extrusions of columnar cells filled with fine granular material are most likely formed in response to the lack of food in the midgut. The presence of laminated concretions in the cytoplasm is indicative of storageexcretion of surplus minerals. The peritrophic membrane is absent from the midgut of C. bifida.     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

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.     

The immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

Luminally released serotonin stimulates colonic motility and accelerates colonic transit in rats

Enterochromaffin (EC) cells of the epithelial cells release 5-HT into the lumen, as well as basolateral border. However, the physiological role of released 5-HT into the lumen is poorly understood. Concentrations of 5-HT in the colonic mucosa, colonic lumen, and feces were measured by HPLC in rats. To investigate whether intraluminal 5-HT accelerates colonic transit, 5-HT and (51)Cr were administered into the lumen of the proximal colon, and colonic transit was measured. To investigate whether 5-HT is released into the lumen, we used an ex vivo model of isolated vascularly and luminally perfused rat proximal colon. To investigate whether luminal 5-HT is involved in regulating stress-induced colonic motility, the distal colonic motility was recorded under the stress loading, and a 5-HT(3) receptor antagonist (ondansetron, 10(-6) M, 0.5 ml) was administered intraluminally of the distal colon. Tissue content of 5-HT in the proximal colon (15.2 +/- 4.3 ng/mg wet tissue) was significantly higher than that in the distal colon (3.3 +/- 0.7 ng/mg wet tissue), while fecal content and luminal concentration of 5-HT was almost the same between the proximal and distal colon. Luminal administration of 5-HT (10(-6)-10(-5) M) significantly accelerated colonic transit. Elevation of intraluminal pressure by 10 cmH(2)O significantly increased the luminal concentration of 5-HT but not the vascular concentration of 5-HT. Stress-induced stimulation of the distal colonic motility was significantly attenuated by the luminal administration of ondansetron. These results suggest that luminally released 5-HT from EC cells plays an important role in regulating colonic motility in rats.     

Effect of α-fluoromethylhistidine-evoked histamine depletion on ultrastructure of endocrine cells in acid-producing mucosa of stomach in mouse,rat and hamster

The oxyntic mucosa of the mammalian stomach is rich in endocrine cells, such as ECL cells, A-like cells, somatostatin cells, D₁/P cells and, in some species, enterochromaffin cells. The various endocrine cell types can be distinguished on the basis of their characteristic cytoplasmic granules and vesicles. The ECL cells contain numerous large secretory vesicles and relatively few, small electron-dense granules and small clear microvesicles. We have suggested that in the rat the ECL cells contain most of the gastric histamine with the secretory vesicles as the major histamine storage site in these cells. α-Fluoromethylhistidine is an irreversible inhibitor of histidine decarboxylase, the histamine-forming enzyme. We have previously shown that this enzyme inhibitor depletes histamine from the ECL cells in the rat and reduces the number of secretory vesicles in the cytoplasm. In the present study, we have examined whether α-fluoromethylhistidine affects the ECL cells in other species and whether it affects other types of endocrine cells in the oxyntic mucosa of the rat. Mice, rats and hamsters were treated with the inhibitor (3 mg/kg per h) via minipumps subcutaneously for 24 h. This treatment lowered the oxyntic mucosal histamine concentration by 65–90% and the number and volume density of the secretory vesicles by 85–95% in the ECL cells of the three species examined. In contrast, the number and volume density of granules and microvesicles were not greatly affected. No evidence was found for an effect of α-fluoromethylhistidine on A-like cells, somatostatin cells or D₁/P cells of the rat stomach, suggesting that, unlike the ECL cells, they do not contain histamine. Received: 18 January 1996 / Accepted: 23 May 1996     

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells

Summary 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.Supported by grants from the Swedish Medical Research Council (537, 2207, 5220). Göteborgs Läkaresällskap, and The Medical Faculty of Göteborg