Effects of colchicine on osteoblast in rat: an ultrastructural study.

The present study was an attempt to investigate the effects of colchicine on the ultrastructure of rat osteoblasts with special reference to the microtubular function in vivo. Rats were killed at intervals of 2, 4, 8, 12 and 24 hours after the colchicine injection (0.1 mg per 100 g of body weight, subcutaneously). At 2 and 4 hours after the injection, the secretory granules and small vesicles accumulated in the Golgi area of the osteoblasts. The dilated spherical cisterna of the rough endoplasmic reticulum and the large vacuoles appeared, being located in the periphery of the cell. Microtubules were rarely found in the cytoplasm. Small masses of microfilaments of 80 to 110 A in diameter were found also in the cytoplasm. According to the special staining for collagen, it was indicated that the contents of the secretory granules might be collagen-like materials. At 8 hours after the injection, the shape of the osteoblasts transformed to a round profile and the autophagic vacuoles increased in the cytoplasm. At 12 and 24 hours after the injection, the osteoblasts seemed to be destroyed by the breakdown of the plasma membrane and by the increase of the autophagic vacuoles. It is suggested that colchicine affects the secretory process of the bone matrix and the cytoskeletal system of the osteoblasts by interfering with the structure and the function of the microtubules and colchicine also interferes with the function of the plasma membrane followed by the destruction of the osteoblasts.     

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo-l-thyronine (T₃) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T₃ (1mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T₃-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.     

Radioautographic analysis of [3H]-fucose utilization by mouse odontoblasts with emphasis on intracytoplasmic and plasma membrane glycoproteins

[3H]-fucose utilization by odontoblasts was studied by light and electron microscopic radioautography. At 10 min after injection, fucose label was concentrated in the Golgi area. By 20-30 min, there was a progressive decline in Golgi labelling with label present at the plasma membrane, terminal web, odontoblast process and predentine matrix. At 4 h, the predentine and the predentine-dentine junction were heavily labelled. At the ultrastructural level, Golgi labelling at 10 min was mostly localized to cisternal elements and at 20 and 30 min secretory granules and dense bodies were also labelled. Most of the silver grains observed in the terminal web were associated with microfilaments near the plasma membrane. In the predentine, the matrix itself accounted for 23.0 per cent of the label at 4 h and the plasma membrane of the odontoblast process accounted for 19 per cent. The results indicate that odontoblasts, in addition to secreting glycoproteins into the dentinal matrix, also continuously manufacture glycoproteins for incorporation into the cell surface, the lysosomal system and the terminal web.     

Salivary histatins in human deep posterior lingual glands (of von Ebner)

OBJECTIVE: Human saliva contains a family of low molecular weight histidine-rich proteins, named histatins, characterised by bactericidal and fungicidal activities in vitro against several microbial pathogens, such as Streptococcus mutans and Candida albicans. They represent a major component of an innate host non-immune defense system. In an earlier study we described the distribution of histatins in the glandular parenchyma of human major salivary glands, confirming that all human major salivary glands are involved in the secretion of histatins into saliva. In the present study we determined the expression and localisation of histatins in human posterior deep lingual glands (von Ebner's glands) by means of immunoelectron microscopy. DESIGN: Thin sections of normal human salivary glands, embedded in Epon resin, were incubated with rabbit polyclonal antibodies specific for human histatins and successively with a gold conjugated goat anti-rabbit IgG used as secondary antibody. Sections incubated with medium devoid of primary antibody or containing non-immune serum were used as controls. RESULTS: The serous secreting cells represented the main source of histatins in the glandular parenchyma of von Ebner's glands. At the electron microscopic level, labeling was associated with rough endoplasmic reticulum, Golgi complex and secretory granules that represented the main cytoplasmic site of histatin localisation. However, variability in the intensity of labeling was observed among adjacent cells. CONCLUSIONS: The present results show for the first time that human von Ebner's glands produce and represent a significant source of histatins, supporting the hypothesis of their important role in preventing microbial assaults on the tissues in the posterior region of the tongue and in the circumvallate papillae.     

Morphological and biochemical changes in mucous cells of the murine sublingual salivary gland during the carbamylcholine-induced secretory cycle

Changes in these cells have been evaluated over 6 h following cholinergic stimulation. Carbamylcholine administration resulted in the release of almost 50 per cent of secretory material within 15 min, which caused a reduction of 33 per cent in cell size. After 2 h the cells were depleted of secretory material. However, in the second hour the release of secretory material was accompanied by an enlargement of the nucleus, Golgi complexes and rough endoplasmic reticulum (RER), which suggests an elevation of biosynthetic activity. The enlargement of the RER was not the result of an increase in RNA, i.e. in the number of ribosomes, but of dilatation of its cisternal spaces. Before release took place, there was a continuous coalescence of secretory granules. After this extensive fusion, which is probably the result of an altered physiological state of the granule membrane and subsequent water uptake caused by cholinergic stimulation, the viscous mucins could be squeezed out, water transport is likely to assist in this ejection. Refilling of the mucous cells was almost complete within 6 h after stimulation.     

Radioprotection of the mice parotid gland by isoproterenol: study on morphometry of secretory granules and on autoradiography

Objectives The aim of the present study was to investigate the effect of radiosensitivity on acinar cells in the parotid gland when the secretory granules were released. Methods The parotid glands of mice were exposed to 10 Gy of X-radiation when the acinar cells were degranulated with isoproterenol (IPR). Three days later, morphological images and number and area of secretory granules within the acinar cells in the parotid glands were obtained and light microscope autoradiography (LMARG) was performed using ³H-leucine. Results The light microscopy images showed a disorderly arrangement and pycnosis of acinar cells and cellular atrophy in irradiated groups. The changes were milder in IPR-administered groups than in non-IPR-administered groups. The number of secretory granules in irradiated groups, which included both IPR-administered and non-IPR-administered sets, was significantly less than that in nonirradiated groups. The number of silver grains within acinar cells obtained by LMARG in the non-IPR-administered set of irradiated groups was significantly lower than that in the nonirradiated group or the IPR-administered set after 30 min of radioisotope administration, and it was significantly higher than that of the nonirradiated group after 240 min. Conclusions When the secretory granules of acinar cells in mouse parotid gland were degranulated by isoproterenol, alleviation of the effects of radiation exposure on morphological change as well as the ingestion and egestion of secretory substances were indicated.     

Morphologic changes in the granular convoluted tubule cells of the mouse submandibular gland following hypophysectomy and hormonal replacement

 Morphological changes in the granular convoluted tubule (GCT) cells of the male mouse submandibular gland (SMG) were examined following hypophysectomy and hormonal replacement. Semithin sections stained with Heidenhain's iron hematoxylin showed that hypophysectomy severely regressed the GCT phenotype. Although only a few dispersed cells containing secretory granules were observed in the GCT segments under a light microscope, electron microscopy revealed that many regressed cells continued to constitutively elaborate apical secretory granules (although they were very small) and contained a euchromatic nucleus at the center of the cell, poor rough endoplasmic reticulum (RER) and Golgi apparatus in the perinuclear region, and well-developed basal infoldings. These findings suggest that hypophysectomy resulted in atrophy of GCT cells, but that they retained evidence of being secretory cells. 5α-Dihydrotestosterone (DHT); 3,5,3′-triiodo-l-thyronine (T₃); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T₃ in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T₃ alone induced a normal female-like GCT phenotype, with considerably abundant secretory granules and the usual short basal infoldings, in hypophysectomized male glands. Furthermore, Dex was found to synergistically enlarge secretory granules when administered with T₃ and/or DHT, although it was only weakly effective in enhancing the GCT phenotype when used alone. Taken together, the above findings confirmed that the GCT phenotype of the mouse SMG is regulated by the synergistic action of pituitary-dependent hormones. Received: October 1, 2001 / Accepted: May 13, 2002     

Ultrastructure and cytochemistry of the Golgi apparatus and related organelles of the secretory ameloblasts of the rat incisor

Glutaraldehyde-fixed rat incisors were either post-fixed in ferrocyanide-reduced osmium impregnated with the Ur-Pb-Cu technique or incubated in the medium for acid-phosphatase (AcPase) reaction. The Golgi apparatus of the secretory ameloblast was composed of 4-7 cisternae, small vesicles and condensing vacuoles. It formed an elongated, continuous membrane system over from one- to two-thirds of the supranuclear cytoplasm. Condensing vacuoles seemed to be produced from the dilated margins of both Golgi cisternae and GERL. AcPase activity was demonstrated in the inner 2 or 3 Golgi cisternae, in GERL and in some condensing vacuoles and secretory granules within the Tomes process. It thus seemed that primary lysosomes originate from both the Golgi apparatus and GERL. Whereas many lysosomal bodies appeared in the supranuclear cytoplasm, autophagic vacuoles were rare. A well-developed Golgi apparatus and GERL were, therefore, considered to be involved in the digestion of exogeneous materials as well as in the formation of the precursor of enamel matrix. The simple Golgi apparatus consisting of only compactly stacked cisternae and small vesicles, were sometimes observed in the supranuclear cytoplasm and having no clear relationship with rough-surfaced endoplasmic reticula or GERL, may serve as a source of the plasma membranes necessary for continuous renewal of the cisternae of the well-developed Golgi apparatus.     

Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, alpha-amylase and B1-immunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, oamylase and Brimmunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

Cytochemical study of the Golgi apparatus and related organelles of the secretory ameloblasts of rat molar tooth germs cultured with and without colchicine

The Golgi apparatus and Golgi-associated endoplasmic reticulum lysosome (GERL) were examined in the ameloblasts with a cytochemical marker, osmium impregnation, and two enzyme markers, thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). In control cultured germs, osmium deposit appeared in one to two immature side cisternae of Golgi stacks; TPPase activity was restricted in a few mature side cisternae and condensing vacuoles. ACPase activity existed in the GERL and, sometimes, in the mature side-cisternae and condensing vacuoles. These findings show that Golgi stacks of ameloblasts consist of several distinct compartments. In colchicine-treated tooth germs, there were morphological and cytochemical changes in both Golgi stacks and GERL. The Golgi apparatus was fragmented and its stacks were scattered throughout the supranuclear region. In some stacks, the number of osmium-positive cisternae was greater than normal; in others they were absent. TPPase and ACPase activity was absent or diminished. These findings suggest the importance of microtubules in the organization of Golgi complex and GERL in the secretory ameloblast.     

Liquid-diet-induced alterations of rat parotid acinar cells studied by electron microscopy and enzyme cytochemistry

After 1 day on liquid diet, the acinar cells were filled with secretory granules, and gland amylase content was approx. 50 per cent greater than chow-fed controls. After 3 days, the number of secretory granules was reduced, and amylase levels had fallen to 50 per cent of controls. Altered or degenerating secretory granules and autophagic vacuoles containing rough endoplasmic reticulum, secretory granules and mitochondria were observed with increasing frequency during the 1st week. These structures were cytochemically reactive for trimetaphosphatase, a lysosomal hydrolase. Concomitantly, macrophages invaded the acinar parenchyma and phagocytosed the degenerating acinar cell components. Lipid droplets and large aggregates of glycogen particles were present in the acinar cells after 7–10 days. After 3 weeks, the acinar cells were considerably reduced in size and contained few secretory granules, but their structure appeared otherwise normal. These results indicate that the acinar-cell lysosomal system and invading macrophages, as in other experimental conditions, play an important role in the parotid-gland atrophy induced by liquid diet.     

Comparison of elemental concentrations in the acinar cells of the human labial salivary gland.

Two types of acinar cells were observed in human labial glands by conventional and analytical electron microscopic and light microscopic techniques. The predominant type contained large and prominent secretory granules that were strongly mucicarmine and PAS (with and without diastase) positive. The second type contained small, lacy, secretory granules, and these cells were faintly positive with these stains. The elemental contents of the two types of granules were measured by analytical electron microscopy using digital mapping and spot analysis applied to freeze-dried cryosections prepared from gland slices incubated in vitro under non-stimulated conditions. The large secretory granules had significantly higher Ca, S and Mg concentrations and significantly lower Cl and K concentrations than the small granules. The difference in elemental contents probably reflects differences in the content of secretory macromolecules. Specifically, the S content is thought to reflect the anionic properties of the secretory macromolecules, while the levels of divalent cations are thought to be determined by electroneutrality requirements for macromolecular folding and storage. No differences were found in nuclear or cytoplasmic elemental concentrations between the two cell types.     

The effects of ductal obstruction on the acinar cells of the parotid of cat

Twenty-nine parotids ligated for between 1 and 365 days were examined by light and electron microscopy. Major changes in the acini were seen at 4 days and included vacuolation, disintegration, extravasation, apoptosis, phagy and a reduction in number and size of secretory granules. There was a further reduction in secretory granules from 7 to 12 days, but acinar cells persisted even up to 365 days, some contained a luminal concentration of small secretory granules and occasionally acinar cells of a similar appearance to normal were found. These findings contrast with a reported absence of acinar cells from the obstructed parotid of rat and show that parotid acinar cells are able to persist and retain an appearance indicative of secretory activity.     

Immunocytochemical localization of enamelin proteins in developing bovine teeth

Enamelins were localized at both the light and electron microscopic level using an antienamelin monoclonal antibody and indirect immunogold methods. Bovine fetal incisors (crown-rump length 17-30 cm) were preserved in Karnovsky's fixative and embedded in Epon. For light microscopy, 2 microM thick sections were immunostained by the indirect method using the monoclonal antibody and goat anti-mouse IgG linked to 5 nM gold particles, followed by silver enhancement to increase the sensitivity of the method. For electron microscopy, thin sections were immunostained (indirect) with the antienamelin monoclonal antibody and goat anti-mouse IgG linked to 5 or 15 nM gold. Control samples were treated with an unrelated monoclonal antibody. The localization of enamelins was confined in the light microscopic sections to the extracellular enamel matrix. No gold staining was observed in the ameloblasts or other enamel organ cells even though the gold-silver technique is extremely sensitive. Ultrastructurally, enamelin was localized in the enamel extracellular matrix and associated ameloblasts. Both the crystal-containing and granular matrix were positively stained, with most gold particles being closely associated with the crystals. Counting of gold particles indicated more than 4 times as many amelogeninas enamelin-reactive antigenic sites in similar regions. Decalcification did not increase immunostaining with the anti-enamelin antibody in the extracellular matrix. Within ameloblasts, the gold particles were associated with secretory granules and Golgi complexes. Thus it appears that enamelins are synthesized in ameloblasts and secreted into the extracellular matrix in a similar manner to amelogenins and are preferentially associated with matrix hydroxyapatite crystals. Transient levels of enamelins within the ameloblasts are apparently too low to be detected by light microscopy.     

Ultrastructural phosphatase cytochemistry of the intercalary ducts of the parotid and submandibular salivary glands of man

Cells of the intercalary ducts showed concentrations of secretory granules adjacent to the luminal plasma membrane. Evidence of thiamine-pyrophosphatase activity was seen in the Golgi apparatus and of acid-phosphatase activity in GERL-like structures, lysosomes and immature secretory granules. Adenosine-triphosphatase reaction-product was present along surfaces of myoepithelial cells and to a lesser extent along contiguous surfaces of duct cells. The findings indicate secretory activity in the intercalary duct cells.     

Mast cells in oral lichen planus

Biopsies from lichen planus affected oral mucosa were compared with biopsies from healthy oral mucosa, in terms of the number of mast cells, their location and their morphological alteration at the light microscopic and electron microscopic level. In comparison with the normal oral mucosa an increased number of mast cells was found below the subepithelial infiltrate. This difference was statistically highly significant (p < 0.001). In the deeper part of the infiltrate mast cells were found to contain granules which presented an altered morphology upon electron microscopic examination. These cells had many of the ultrastructural changes that have been reported for mast cells undergoing degranulation. The present morphological observations suggest that mast cells participate in the recruitment of lymphocytes to the subepithelial infiltrate.     

Mast cells in oral lichen planus

Biopsies from lichen planus affected oral mucosa were compared with biopsies from healthy oral mucosa, in terms of the number of mast cells, their location and their morphological alteration at the light microscopic and electron microscopic level. In comparison with the normal oral mucosa an increased number of mast cells was found below the subepithelial infiltrate. This difference was statistically highly significant (p less than 0.001). In the deeper part of the infiltrate mast cells were found to contain granules which presented an altered morphology upon electron microscopic examination. These cells had many of the ultrastructural changes that have been reported for mast cells undergoing degranulation. The present morphological observations suggest that mast cells participate in the recruitment of lymphocytes to the subepithelial infiltrate.     

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after β-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B–GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.     

Age-related changes of the granular intercalated duct cells of male rat submandibular gland

The granular intercalated duct (GID) cells showed a progressive increase in number from two to four months of age (p less than 0.01). Their secretory granules also increased in number. Among all the age groups (2-22 months), the number of the GID cells in submandibular gland was highest of six months; they were then also most conspicuous with many electron-dense secretory granules in the cytoplasm. From 12 to 22 months, they showed regressive changes such as a decrease in cell number and in the number of secretory granules. The significance of these age-related changes of the GID cells is unknown.