Ultrastructural studies of endocrine-like cells in the fundic gastric mucosa of the bullfrog,Rana catesbeiana

Summary This study is concerned with electron-microscopic observations on endocrine or paracrine cells in the fundic gastric mucosa of the bullfrog. Also, an attempt was made to identify the histamine-releasing cells involved in the secretagogue response. At least three distinct endocrine-like cell types were found. The classification is based on the appearance of secretory granules and other organelles, and the relationship of endocrine-like cells with other cells in the tissue. The amphibian endocrine-like cells resemble the ECL, D and EC cells of mammals. Type-I (ECL) cells showed degranulation after repeated stimulation with tetragastrin (TG), acetylcholine (ACh) and K⁺ depolarizing solution, all of which release histamine.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

Summary The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasocative peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8–13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Immunohistochemical study on the morphology of enterochromaffin cells in the human fundic mucosa

Summary The morphology of enterochromaffin (EC) cells in the human fundic mucosa was investigated at the lightmicroscopic level by means of the unlabeled peroxidase anti-peroxidase method, with the use of a highly specific anti-serotonin serum.EC-cells in the human fundic mucosa were sparsely distributed below the neck portion of the gland, but were found to be rather numerous in its lower half. Immunohistochemistry revealed marked pleomorphic and seemingly polynuclear EC-cells or cells with long, sometimes multipolar cytoplasmic processes. In addition, luminal contacts and contiguity between EC-cells, or interglandular connections were also encountered.The present immunohistochemical procedure permits, for the first time, a clear-cut morphological visualization of the entire population of EC-cells, and reveals the distinctive morphological features of these cells in the human fundic mucosa. These morphological findings imply that EC-cells in the fundic mucosa may be crucial in gastric function.     

Vimentin-positive cells in the epithelium of rabbit ileal villi represent cup cells but not M-cells.

Membranous (M)-cells are specialized epithelial cells of the Peyer's patch domes that transport antigens from the intestinal lumen to the lymphoid tissue. Vimentin is a reliable marker for M-cells in rabbits. Using immunohistochemistry (IHC), a subpopulation of epithelial cells has recently been identified in ordinary rabbit ileal villi, which are vimentin-positive and share morphological characteristics with the M-cells of the domes. To test the hypothesis that these cells represent M-cells outside the organized lymphoid tissue, lectin labeling and tracer uptake experiments were performed. Lectins specific for N-acetyl-glucosamine oligomers selectively bound to the vimentin-positive villous cells but not to M-cells in the domes. Microbeads instilled into the ileal lumen were taken up by M-cells within 45 min but not by the vimentin-positive cells in the villi. Lectin-gold labeling on ultrathin sections revealed that the lectin binding sites were located in the brush border and in vesicles in the apical cytoplasm. The vimentin/lectin-positive cells shared ultrastructural characteristics with the so-called "cup cells." We conclude (a) that the vimentin-positive cells in ordinary villi represent cup cells but not M-cells, (b) that they are readily detectable by (GlucNAc)(N)-specific lectins, and (c) that they do not transcytose experimental tracers. Although the specific function of cup cells is still obscure, they most probably represent a cell type distinct from M-cells of the domes with respect to both function and expression of the two new markers.     

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of ceils. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal ceils. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal ceils whereas the high-affinity sites occur principally in the non-parietal cells.     

Glycoconjugate distribution in gastric fundic mucosa of Umbrina cirrosa L. revealed by lectin histochemistry

The stomach of adult shi drum Umbrina cirrosa was investigated using a battery of nine horseradish peroxidase‐conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N‐acetylglucosamine, mannose, N‐acetylgalactosamine, N‐acetylglucosamine, fucose and sialic acid‐galactosyl(β1→3)N‐acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N‐acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N‐acetylgalactosamine, N‐acetylglucosamine, galactosyl(β1→4) N‐acetylglucosamine, N‐acetylgalactosamine and a small amount of sialic acid linked to N‐acetylgalactosamine.     

Ultrastructural identification and immunocytochemical study of somatostatin cells in antral mucosa of the rabbit and the mouse (author's transl)]

In normal and L-Dopa treated rabbits and mice, combined immunochemical methods, photonic histological methods for endocrine cells and ultrastructural methods were used to elucidate ultrastructure and properties of somatostatin cells of the antral mucosa. In normal rabbits, immunoreactive cells giving no fluorescence with Falck's technic, they corresponded neither to serotonin cells nor gastrin cells; they were unreactive with Fontana, Hellerstr?m-Hellmann, Sevier-Munger and Mac Conaill methods but very slightly stained with Grimelius methods. In L-Dopa treated animals somatostatin cells gave formaldehyde induced fluorescence (they were included in GIC cells, thus in Apud group), exhibited a good reaction with Grimelius and Sevier-Munger methods. In order to carry out the alternate semi-thin/thin section procedure (semi-thin sections for immunofluorescence or immunoenzymatic detection and serial thin sections counter-stained for conventional ultrastructure studies), immunological treatment were performed on M.F.F.--glutaraldehyde fixed small fragments of mucosa before inclusion in Epon 812 or, after inclusion, on semi-thin sections. We succeeded in identifying ultrastructurally somatostatin cells. They displayed round or ovo?d shaped secretory granules, and three constant typical structures: numerous microfilaments--light and homogenous granules, often seeming like lipids---granules made up by coarsely filamentous cores surrounded by a large empty halo. Somatostatin cells seemed different of X cells because of their predominant localisation in the antral mucosa (in the rabbit X cells were predominantly in the fundus) and because of the lack of nuclear microfilaments; they also seemed ultrastructuraly different of D1 cells.     

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.     

Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection

The periodic acid-thiocarbohydrazide (SO2)--OsO4 method was used to examine the distribution of glycoproteins in rabbit fibroblast cells infected with Herpes simplex virus type 1. In non-infected cells, a low level of staining was seen over the plasma membrane and the membranes of the Golgi apparatus. At 17 hr post-infection, the intensity of reaction was increased to include not only a relatively heavy staining of the plasma membrane, including the numerous microvilli characteristic of infected cells, and of the newly proliferated Golgi membranes, but also the envelopes of intracytoplasmic and extracellular virions. A very faint but only occasional staining also was associated with the virus-induced reduplications of the inner nuclear membrane and the envelopes of associated enveloping nucleocapsids. We suggest that such differences in the intensity of staining may be related either to the amount of glycoproteins or to the sequential maturation of the viral glycoproteins. We also observed that the structurally modified portions of the Golgi membranes at the position where intracytoplasmic naked nucleocapsids bud into the Golgi cisternae usually exhibit a more intense reaction for glycoproteins than do the adjacent portions of the Golgi membranes. This supports the evidence for an envelopment of nucleocapsids in the cytoplasm, but it does not indicate whether this event obligatorily follows or only occasionally takes the place of the envelopment of nucleocapsids at the inner nuclear membrane. In either event, the envelopes of all mature virions exhibit a prominent reaction to glycoproteins.     

Isolation and identification of clara cells from rabbit lung

Summary A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion. Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy. This research was supported in part by the U.S. Environmental Protection Agency under an interagency agreement relating to the Federal Interagency Energy/Environmental Research and Development Program.     

Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa

The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies. Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.     

Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes

Summary— Ultrastructural changes of the germinal vesicle during the growth of rabbit oocytes were studied by means of light and electron microscopy, ³H-uridine autoradiography, Ag-NOR staining and E-PTA staining. Particular interest was paid to the nucleologenesis and condensation of chromatin. In contrast to other mammalian species, chromosome condensation in rabbit oocytes occurred concomitantly with rRNA synthesis-dependent nucleolar compaction and preceded nuclear envelope breakdown and resumption of meiosis.     

Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa

Summary The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies.Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.Abbreviations HGL Human gastric lipase - SDS PAGE Sodium dodecyl sulfate-polyAcrylamid gel electrophoresis - PBS Phosphate buffer saline     

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α-antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines. © 1996 Wiley-Liss, Inc.     

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr¹¹-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific, somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.