Ultrastructural aspects of the effect of calcium ionophore A23187 on incubated anterior pituitary cells of rats

In order to study the fine structural effect of calcium influx on secretory activity of rat anterior pituitary cells, small pieces of anterior pituitary were incubated in Krebs' medium containing the calcium ionophore A23187 (0.15 mM) and were examined electron microscopically. Marked changes were present in all types of secretory cells incubated for 3, 12 and 20 min in the medium containing calcium and A23187. Secretory granules tended to accumulate in the peripheral cytoplasm of the secretory cells, and more numerous images of granule release by exocytosis were observed in somatotroph (STH cell), luteotroph (LTH cell), thyrotroph (TSH cell), corticotroph (ACTH cell), type 1 gonadotroph (Type 1 GTH cell), and type 2 gonadotroph (Type 2 GTH cell). In addition to the increase in the number of exocytosis of single granules, the simultaneous extrusion of multiple granules, "multigranular exocytosis", was often observed in all kinds of secretory cells, especially the ACTH-cells. Large numbers of granule cores were often located in large vacuole-like or channel-like structures, irregular in shape and size, which were open to the intercellular or pericapillary space. Some parts of the membrane of the vacuole-like or channel-like structures were coated. These observations are interpreted to suggest that the calcium influx stimulates the extrusion of the secretory granules by single or multigranular exocytosis.     

Ultrastructure of the conjugates of cytolytic T-lymphocytes and target cells]

Conjugates of target cells and of cytological T-lymphocytes obtained on the 11th day after alloimmunization were investigated. The conjugates formed small and medium lymphocytes; mature secretory granules, crystal-like structures and lipids were revealed in their cytoplasm. The lymphocyte is spherical, the area of contact with the target cell does not exceed 5 to 15%. Cytolysis of target cells is observed after 30 to 60 minutes of incubation. The lymphocyte becomes flattened, its nucleus acquires an oval form, and the area of contact with the target cell increases considerably. At the same time hypertrophy and change of orientation of Golgi's complex to the area of contact with the target cell, coalescence of the secretory granules with lipids and crystal-like structures, the appearance of immature secretory granules and vacuolar degeneration of mitochondria are demonstrated. The lymphocyte membrane becomes "desquamated"; structures connected with it, named "membranosomes" are described. It is suggested that the secretory processes in the cytoplasm of cytolytic T-lymphocytes are activated in their interaction with target cells.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

Studies on secretory activity in the pars intermedia of Xenopus laevis 1: Fine structural changes related to the onset of secretory activity in vivo

Changes in the fine structural organization of the pars intermedia related to the onset of secretory activity within the gland have been studied. It is shown that during the first seven days, following the onset of secretory activity, there is an extensive membraneogenesis within the cytoplasm of the parenchymatous cells of the pars intermedia which results in the formation of a well-organized array of endoplasmic reticulum and an increased development of the Golgi complex. Simultaneously the large population of secretory granules present in the cells in the inactive condition is reduced, the granules fusing with the plasma membrane and releasing their contents into the extracellular space. During the process of intracellular reorganization, in addition to the elaboration of those components of the Golgi complex which are believed to be concerned with the formation of the secretory granules, a second distinctive cisternal element develops within the Golgi area. This component which remains confined to the Golgi area for only a short time (days 2-4) appears to be responsible for the production of membrane-bound dense bodies with a finely granular content. The dense bodies, in turn, become transformed into the larger heterogeneous structures which are a prominent feature of actively secreting pars intermedia cells.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Structural and ultrastructural features of boar seminal vesicles

The morphological features of boar seminal vesicles were examined by light and transmission microscopy. Boar seminal vesicles consist of glandular tissue arranged in multiple lobules containing a system of ramified secretory tubules. The secretory tubules are composed of a mucosa formed by an epithelium and an underlying lamina propria and, are surrounded by a muscular layer. The epithelium is made up of columnar cells and occasional basal cells. Mast cells are frequently found among epithelial cells. Three types of columnar cells, considered different stages of the secretory cell cycle, are present: principal cells, clear cells and dense cells. Principal cells are functionally differentiated cells characterised by abundant mitochondria, great development of the rough endoplasmic reticulum and presence of secretory granules in their cytoplasm. The apical surface of many principal cells shows apical blebs filled with PAS-positive material. No acid mucosubstances are detected. Microvilli cover the apical surface except in the apical blebs. Dense cells, arranged between principal cells, are also functional differentiated cells but with signs of cellular degeneration. Clear cells are an initial differentiated stage of columnar cells and are characterised by the presence of a poorly developed rough endoplasmic reticulum and by the absence of secretory granules. Proliferating cells are present among columnar cells. Basal cells contain scarce cytoplasm, few organelles and no secretory granules. The lack of mitotic activity in these cells suggests that they do not act as precursors of columnar cells.     

THE CYTOLOGY OF THE NORMAL PARATHYROID GLANDS OF MAN AND VIRGINIA DEER : A Light and Electron Microscopic Study with Morphologic Evidence of Secretory Activity

The normal parathyroids of six humans and a Virginia deer were studied by light and electron microscopy. The parenchyma of the deer parathyroid is composed of uniform chief cells, which contained 100 to 400 mµ electron-opaque, membrane-limited granules, presumed to be secretory granules, in addition to the usual cytoplasmic organelles. Desmosomes are present between adjacent cells, and rare cilia are observed protruding from the chief cells into the intercellular space. The human parathyroids contain chief cells in two phases—active and inactive—as well as oxyphil cells. Active chief cells have a large Golgi apparatus, sparse glycogen, numerous secretory granules, and rare cilia. Inactive chief cells contain a small Golgi apparatus, abundant glycogen, and few secretory granules. Both forms have the usual cytoplasmic organelles and, between adjacent cells, desmosomes. Oxyphil cell cytoplasm is composed of tightly packed mitochondria and glycogen granules, with rare secretory granules. Cells with cytoplasmic characteristics intermediate between chief and oxyphil cells, possibly representing transitional cells, have been observed. Secretory granules of both man and deer are composed of 100 to 200 A particles and short rods, and the granules develop from prosecretory granules in the Golgi region of the cell. The human secretory granules are smaller and more variable in shape than those of the deer. The granules are iron and chrome alum hematoxylin-positive, argyrophilic, and aldehyde fuchsin-positive, permitting light microscopic identification. They are also found in the capillary endothelial cells of the parathyroid and in its surrounding connective tissue. The secretory granules of the parathyroid cells can thus be followed from their formation in the Golgi apparatus almost to their extrusion into the blood stream.     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

Tissue culture study on Mongolian gerbil's (Meriones unguiculatus) malignant melanoma

We tried to culture melanoma cells from a Mongolian gerbil's (Meriones unguiculatus) malignant tumor. In primary culture, most of cells have abundant melanin granules in their cytoplasm. Melanin granules decreased through 5 to 15 serial passages and disappeared after 15 th passage. The morphology of the cells varied from spindle to large polydendritic cells. Although typical melanin granules were not seen when the cells were stained by Masson-Fontana method, the cells were positive for DOPA reaction. Electron microscopically, most of the cells have well-developed Golgi apparatus and rough-surfaced endoplasmic reticulum with dilated cistern, and premelanosome-like granules were frequently observed in their cytoplasm.     

Vacuolated pinealocytes in the hedgehog (Erinaceus europaeus L.) and the mole (Talpa europaea L.)

Special cells in the hedgehog and mole pineal gland which are characterized by an enormous single vacuole were studied. The presence of secretory granules (in the hedgehog) and of paracrystalline structures (in the mole) in the cytoplasm of these cells indicates that they are pinealocytes.     

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: Evidence for the colocalization of different peptides in single secretory granules

Summary Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A₂-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60–100 nm diameter), electron-dense, membranebounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immunostaining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.     

Histochemical localization of calcium in the enamel organ of rat incisors in early-stage amelogenesis

The localization of calcium in the enamel organ of rapidly-frozen, freeze-substituted rat incisors in early-stage amelogenesis was examined by a histochemical calcium-staining method. In secretory ameloblasts, glyoxal bis(2-hydroxyanil) (GBHA) staining revealed intense red reactions in mitochondria and tubulovesicular structures located throughout the cytoplasm, while no reaction was seen in the nucleus and cytosol, nor along the plasma membranes of the respective cells. No significant GBHA reaction was observed in the intercellular compartment and other cells of the enamel organ. Some granular reactions were localized in the cells of the adjacent connective tissue. Control tests confirmed the specificity of GBHA reactions for calcium. Thus, the present observations provide histochemical evidence indicating an exclusive localization of calcium in mitochondria and tubulovesicular structures of the secretory ameloblast, and support their contributions to the translocation of calcium from the proximal to the distal pole of the cytoplasm.     

Quantitative analyses of atrial myoendocrine cells and plasma atrial natriuretic peptides (ANP) of the rat with special reference to the twenty-four-hour variations in secretory granules and plasma ANP concentrations

Summary Subcellular structures of atrial myoendocrine cells in the rat heart and plasma concentrations of atrial natriuretic peptides (ANP) were examined at six evenlyspaced time points over 24 h, using morphometric techniques and radioimmunoassay.Myofibrils and mitochondria of the cells occupied 73.3% of the cytoplasm; 2% of the cytoplasm was occupied by secretory granules, rough endoplasmic reticulum and Golgi complexes, structures characteristic of endocrine cells. Plasma ANP concentration was maximal at 08.00 h, when the individual volume of secretory granules was minimal. The numerical density of secretory granules was increased at 12.00 h. The plasma ANP concentration was minimal at 20.00 h, when the numerical density was minimal and the individual volume was maximal. The fluctuation in plasma ANP concentrations over 24 h was thus parallel to that in the numerical densities of secretory granules and inverse to that in individual volumes.These results suggest that in rats the secretory activity of atrial myoendocrine cells increases at the beginning of the resting period, whereas it decreases at the beginning of the active phase.     

Prostaglandin E2 localization in the rat ileum.

Summary The application of anti-prostaglandin E₂ immunoglobulin to plastic-embedded thin sections of the rat ileum has permitted the localization of prostaglandin E₂ in this tissue. In agreement with the published data (Chock & Schmauder-Chock (1988), Schmauder-Chock & Chock (1989)), the results also suggest the presence of an arachidonic acid cascade in the granules of various secretory cells of the gut. Since antibody labelling was found within the secretory granules of connective tissue mast cells, goblet cells, and Paneth cells, the presence of the arachidonic acid cascade in these granules is implied. The appearance of prostaglandin E₂ over the non-cellular internal elastic lamina of arterioles suggests that it may have been secreted along with the elastin. The even distribution of prostaglandin throughout the cytoplasm of the erythrocyte is consistent with the concept that this cell scavenges the eicosanoid from the circulation. These data further link the secretorh granule to the production of eicosanoids and therefore illustrate the potential sources of prostaglandins in the rat ileum.     

The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae)

The ventriculus and the midgut caeca of the fed females of Anystis baccarum (L.) were investigated by using light and electron microscopy. In addition to the main type of polyfunctional digestive cells, special secretory cells were detected in the anterior region of the ventriculus. The shape and the ultrastructure of the digestive cells vary depending on their physiological state. Intracellular digestion, absorption or excretion processes prevail at different stages of the cell cycle. The secretory cells are characterized by the presence of extensive rough endoplasmic reticulum, filling whole space of the cell. These cells do not contain the apical network of pinocytotic canals, which are typical for the digestive cells. Three types of secretory granules were found in the cytoplasm of the secretory cells that probably correspond to three sequential stages of granulogenesis. The primary secretory granules are formed by the fusion of Golgi vesicles. The primary granules fuse to form complex vesicles with heterogeneous contents. These secondary granules aggregate to form very large inclusions of high electron density (tertiary secretory granules), which probably represent the storage of the secretory product. All types of secretory granules were observed close to the apical plasmalemma.     

Seasonal ultrastructural changes of the hypophyseal pars tuberalis in the hedgehog (Erinaceus europaeus L.)

The comparison of the hypophyseal pars tuberalis in active and in hibernating hedgehogs reveals different cytological characteristics in the specific secretory cells. In active animals, these cells show oval nuclei and the cytoplasm contains numerous secretory granules near or attached to the cell membrane, suggesting exocytotic release. In hibernating animals, the specific secretory cells are characterized by irregularly shaped and often invaginated nuclei. Clusters of secretory granules lying within the cytoplasm are often joined by lysosomes. These areas are observed to be encircled by cisterns of the endoplasmic reticulum. Degenerative processes in the structures sequestered in this way are interpreted as signs of crinophagy which has no equivalent in the cell types of the pars distalis.     

Chemical inducers of differentiation,dimethylsulfoxide, butyric acid,and dimethylthiourea,induce selective ultrastructural patterns in B16 melanoma cells

The morphology and ultrastructure of B16 melanoma cells was examined after treatment of the cells with the chemical inducers of differentiation dimethylsulfoxide (DMSO), butyric acid, and dimethylthiourea (DMTU). The treated B16 melanoma cells seemed to be enlarged and more flattened, and to possess dendrite-like structures as revealed by scanning electron microscopy. The main ultrastructural features, depicted by transmission electron microscopy in DMSO-treated B16 cells were: a marked increase in melanin granules, migration of the melanin granules to the dendrites, and appearance of melanosome aggregates. Butyric acid did not induce melanin biosynthesis; however, it stimulated rough endoplasmic reticulum (RER) formation all over the cytoplasm. The DMTU-treated cells also showed a well developed RER accompanied by early stages of melanosomes and melanin granules. The increase in the endoplasmic reticulum was also reflected by enhancement of NADPH cytochrome c reductase activity, an enzymatic marker of the endoplasmic reticulum. The mitochondria in the DMTU-treated cells were swollen with disrupted cristae. The results indicate that DMSO, butyric acid, and DMTU induce different ultrastructural patterns in B16 melanoma cells. These findings correlate with the biochemical alterations induced in melanoma cells by these agents.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.