Synthesis of secretory and plasma membrane glycoproteins by striated duct cells of rat salivary glands as visualized by radioautography after 3H-fucose injection

The ability of the striated ducts of rat salivary glands to incorporate 3H-fucose into glycoprotein was studied by light and electron microscope radioautography. At 3.5 to 20 minutes after intravenous injection, the majority of the radioautographic grains in the ducts of the parotid gland were localized to the Golgi apparatus. By 40 minutes, the percentage of grains over the Golgi apparatus had decreased; a corresponding increase in grains occurred over small (0.1-0.4 micrometer) apical granules and the highly infolded basal and lateral plasma membranes. By two hours, less than 10% of the label was associated with the Golgi apparatus, while 26% and 28% were attributed to the apical granules and plasma membrane, respectively. By 8 to 12 hours after injection, the number of grains over the apical cytoplasm had decreased, suggesint luminal discharge of the apical granules. In contrast, the basal and lateral plasma membranes remained labeled up to 30 hours after injection as judged by the distribution of grains in light microscope radioautographs. Mitochondria appeared capable of independent incorporation of fucose, accounting for about 20% of the grains from ten minutes to two hours after injection. Comparable results were obtained in the striated ducts of the submandibular and sublingual glands. These results indicate that the striated duct cells readily incorporate 3H-fucose into newly-synthesized glycoproteins. A portion of these are secretory glycoproteins which are packaged and stored in the apical granules, and a portion are membrane glycoproteins which are incorporated into the extensive plasma membrane of these cells.     

Synthesis and migration of glycoproteins in cells of the rat thymus,as shown by radioautography after 3H-fucose injection

Young male rats received a single intravenous injection of ³H-fucose and were killed after various time-intervals. Light- and electron-microscopic radioautographic studies of the thymus in animals killed shortly after injection showed that all of the different cell types present incorporated ³H-fucose label. The heaviest uptake occurred in macrophages and in hypertrophic epithelial cells located near the cortico-medullary border. Somewhat lighter incorporation was observed in medullary and cortical stellate epithelial cells and in cells designated as special cells, while the lightest reaction appeared over lymphocytes. In all cells the label was localized initially to the Golgi apparatus, where, presumably, it was incorporated into glycoproteins. With time, some of the labeled putative glycoproteins in all cell types migrated to the plasma membrane. In macrophages, much of the label migrated to lysosomal bodies, while in the special cells the label migrated to dense bodies which may also be of lysosomal nature. In stellate and hypertrophic epithelial cells much of the label migrated to characteristic vacuoles. The possible relationship between the observed glycoprotein synthesis in these cells and hormone production is discussed.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with ³H-fucose. Control rats received ³H-fucose only. All rats were sacrificed 90 min after ³H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

Formation and turnover of plasma membrane glycoproteins in kidney tubules of young rats and adult mice,as shown by radioautography after an injection of 3H-fucose

The formation and turnover of the glycoproteins of the plasma membrane have been investigated by quantitative radioautography in the kidney tubules of young rats and adult mice killed at various time intervals after an intravenous injection of ³H-fucose. In young (40 g) rats killed five to ten minutes after the injection, radioautographs of distal tubule cells show that the Golgi apparatus contained about 85% of the cell label. By 30 hours, only 8% of the label remained in this organelle, whereas 67% was in the plasma membrane, indicating that most of the label had migrated from Golgi apparatus to this membrane. Similarly, in proximal tubule cells, about 82% of the label was initially in the Golgi apparatus, but less than 2% remained at 30 hours, at which time 78% was in the plasma membrane. In the latter cells, the apical tubules and vacuoles became heavily labeled before the apical microvilli did and, therefore, may be involved in the transit of label from the Golgi apparatus to the microvillous membrane. The results are interpreted to mean that, in kidney tubule cells, the Golgi apparatus is the site of a continuous incorporation of fucose into glycoproteins and that these migrate to the plasma membrane. In fully formed cells, such a conclusion would imply a continuous turnover of plasma membrane glycoproteins. However, in the rapidly growing kidney of young rats many new cells are added daily, the growth of which might involve net addition as well as turnover of glycoproteins. Accordingly, the experiment has been repeated in adult mice, in which the cells are assumed to be fully formed. Furthermore, since turnover implies eventual decrease of incorporated label, some of the animals have been killed at longer intervals, up to 27 days after injection. In these adult mice, as in young rats, prompt Golgi uptake and subsequent migration of label to the plasma membrane were observed in distal and proximal tubule cells. With time the label content of the plasma membrane decreased gradually, and by 27 days had virtually disappeared. From grain counts, it is concluded that the mean half-life of glycoproteins in the apical membrane of distal tubule cells is about two days, whereas in both the apical and basal membranes of proximal tubule cells, it is slightly over three days.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with ³H-fucose. Control rats received ³H-fucose only. All rats were sacrificed 90 min after ³H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.     

Osteodentin formation in rat incisor as visualized by radioautography after 3H-proline administration

Osteodentin formation was studied in rat incisor pulp after adriamycin administration. Male Sprague Dawley rats (100 +/- 5 gm) were injected intravenously with adriamycin (5 mg/kg body weight), and after 7 days they were again injected intravenously with 3H-proline (3 microCi/gm). These animals were killed in groups of three from 5 minutes to 4 hours after proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by 2.5% phosphate-buffered glutaraldehyde. Control animals injected with only physiological saline, and 7 days later with 3H-proline (3 microCi/gm), and were killed at the same time intervals. Radioautography on sections showing osteodentin formation revealed that at 5 minutes after 3H-proline injection the labeling was located over the cells associated with the osteodentin matrix. At 1 hour after injection the labeling was located over the cells and the matrix, while at 4 hours the labeling was seen only over the matrix. It therefore appears that at least a proline-containing component of the osteodentin matrix is synthesized and secreted by the cells associated with it.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats,as visualized by radioautography

³H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated ³H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after ³H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.     

The effect of colcemid on the structure and secretory activity of ameloblasts in the rat incisor as shown by radioautography after injection of 3H-proline

Enamel secretion by ameloblasts was investigated in the incisors of 100 gm normal and colcemid-injected male rats. Morphological studies were done on rats given a single intraperitoneal injection of 0.1 mg (1.25 mM) of colcemid and sacrified 1 to 4 hours after injection. Protein synthesis and secretion were investigated with radioautography in normal and colcemid-treated rats injected with 3H-proline and sacrificed at intervals between 0.5 and 3.5 hours after injection. Colcemid was injected 0.5 hours prior to 3H-proline in each experimental rat. Electron microscopic examination revealed several morphological alterations between 1 and 4 hours after injection of colcemid. These changes included fragmentation of the normally elongated rough endoplasmic reticulum into shorter profiles; a disorganization of the normally tubular configuration of the Golgi apparatus into a number of seples and profiles of smooth endoplasmic reticulum from Tomes' processes; and the accumulation of secretion granules at the mature face of the Golgi stacks, as well as in the infranuclear cytoplasm where thye are normally not found. Radioautography revealed that protein synthesis by the rough endoplasmic reticulum had continued in colcemid-altered ameloblasts. Labeled secretion granules were found at the mature surface of the Golgi stacks and in the infranuclear cytoplasm, however they did not migrate into Tomes' processes. Consequently, labeled enamel matrix did not appear extracellularly at the same time as in normal controls. Quantitative radioautography in the light microscope revealed that the effect of colcemid, although reversed within 4 hours, had temporarily inhibited normal migration, and exocytosis of secretion granules.     

The effect of streptozotocin on the secretory activity of ameloblasts in rat incisor as revealed by radioautography after 3H-proline administration

The effect of a diabetogenic dose of streptozotocin on the secretory activity of ameloblasts was investigated in the rat incisor by radioautography. One group of male Sprague-Dawley rats was injected intravenously with streptozotocin in citrate buffer (pH 4.5). One hour later, this group was again injected intravenously with 3H-proline (2 mCi/kg). A control group of animals was injected with 3H-proline only. All the animals were sacrificed in groups of three at 5 min, 1 h, 2 h, 4 h and 8 h after 3H-proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by an additional perfusion with 2.5% phosphate-buffered glutaraldehyde. The incisors were extracted with the jaws, demineralized, and prepared for radioautographic observations and analysis. The principal effects of streptozotocin were as follows: There was an inhibition of 3H-proline incorporation into the secretory ameloblasts at 5 min after injection. This was followed by a larger uptake and a slower passage of the label out of the cells into the enamel matrix than that seen in the control sample. Finally, there was a slower secretion of labeled proteins out of Tomes' processes between 1 and 4 h after injection. Therefore, streptozotocin had a temporary inhibitory effect on the incorporation and secretion of 3H-proline by the secretory ameloblasts of the rat incisor. This effect was present for about 4 h and was completely reversed 9 h after streptozotocin injection.     

Low-grade mucoepidermoid carcinoma of salivary glands: characteristic immunohistochemical profile and evidence of striated duct differentiation

The purpose of the present study is to determine the presence and distribution of epithelial and myoepithelial cells in mucoepidermoid carcinoma (MEC) of salivary glands and to compare them with normal salivary gland tissue and other primary carcinomas. This is in order to establish novel diagnostic criteria and to better understand MEC histogenesis. Formalin-fixed paraffin-embedded tissues from ten well-differentiated MECs, three adenoid cystic carcinomas (ACC), four acinic cell carcinomas (AC), and three epithelial-myoepithelial carcinomas (EMCC) of salivary glands were studied with immunohistochemistry using antibodies that recognise antigens indicative of epithelial and myoepithelial cell differentiation. An anti-mitochondrial antibody was also employed. Normal salivary tissue was present for comparative study in non-tumorous areas of the same section from 12 cases. MEC contained numerous keratin-positive cells. Anti mitochondrial antibody was diffusely positive in all ten of these tumours. Smooth muscle actin, h-caldesmon, and smooth muscle heavy chain myosin, which are indicative of myoepithelial cell differentiation, were negative. Rare cells in only one case were stained by calponin. Cytokeratin 14 (CK14) and anti mitochondrial antibody stained cells located mainly at the periphery of neoplastic nests and cystic spaces, while CK7 was mainly present in cells bordering gland lumina (zoning pattern). The immunohistochemical cell profile was similar to that seen in striated normal ducts. All others tumours studied showed a different immunohistochemical pattern, mostly consisting of a lack of mitochondrion-rich cells and the presence of myoepithelial cells in ACC and EMCC. Immunoreactivity in MEC for CK7, CK14 and mitochondrial antibodies appears as a peculiar pattern of staining, different from that of other salivary gland tumors; this seems helpful for diagnostic purposes. In addition, a differentiation of the "striated duct phenotype" is suggested.     

Synthesis of lens capsule and plasma membrane glycoproteins by lens epithelial cells and fibers in the rat

The lens of the eye possesses a capsule which is a greatly hypertrophied basement membrane. To investigate the synthesis of glycoproteins destined for this capsule, ³H-fucose was iryected into the vitreous body of intact rats weighing approximately 200 gm. The animals were killed from 10 min to 14.5 months later, and their lenses were processed for electron microscope radioautography. At 10 min after injection, more than 58% of the silver grains were localized to the Golgi apparatus of the lens epithelial cells. By day 1, the heaviest sites of reaction were the plasma membrane (more than 50% of total label), the basal cytoplasm, and the adjacent lens capsule, where a heavy band of reaction was seen. The remainder of the capsule exhibited a lighter diffuse reaction. In the lens fibers, the label was at first localized to clusters of vesicles but then migrated to the plasma membrane and to the region of the capsule adjacent to the basal surface of these fibers. Light microscope radioautographs of the lens capsule at later time intervals revealed that by 1 month after injection the diffuse reaction had disappeared, and only the strongly labeled band remained. By 14.5 months after injection, this band had migrated partially across the lens capsule, but the capsule itself had increased considerably in thickness. On the other hand, the distance between the labeled band and the free edge of the capsule had decreased from that seen at the time of injection.     

Sulfation and transport of basement membrane proteoglycans,as visualized by 35S-sulfate radioautography in the endodermal cells of the rat parietal yolk sac

In the hope of clarifying the biogenesis of basement membrane proteoglycans, an injection of ³⁵S-sulfate was given to concepti of 12-day gestant Sherman rats. The parietal wall of the yolk sac (including endodermal cells and the associated basement membrane known as Reichert's membrane) was removed at times varying from 7 min to 24 hr after injection and processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Between 7 min and 2 hr after ³⁵S-sulfate injection, radioactivity was observed in the endodermal cells, while from 4 to 24 hr it was mostly present in Reichert's membrane. Detailed distribution of the cellular radioactivity at 7 and 15 min showed about 20% in the rough endoplasmic reticulum (rER), 60% in the Golgi apparatus, and 8% in secretory granules. The radiactivity present in rER and Golgi apparatus decreased to low levels by 2--4 hr after injection. In secretory granules, radioactivity increased to reach a peak at 2 hr and then declined; moreover, only the granules associated with the trans Golgi face were radioactive at early time intervals, while those scattered through the cytoplasm and along the cell surface became radioactive at later times. Between 4 and 24 hr, radioactivity became negligible over all cell organelles, while it was collected in Reichert's membrane. Biochemical reports indicate that when ³⁵S-sulfate is added to organ cultures of Reichert's membrane and endodermal cells, about 90% of the incorporated which these proteoglycans acquire sulfate are likely to be those labeled at 7 min after ³⁵S-sulfate injection, that is, the Golgi apparatus and to a lesser extent the rER, whereas some labeling of the secretory granules located at the trans Golgi face is explained by rapid acquisition of sulfated proteoglycans from the Golgi apparatus. Label later appears in the secretory granules along the cell surface and, eventually, in Reichert's membrane. It is concluded that secretory granules transport newly formed proteoglycans from the Golgi apparatus to the outside for deposition into Reichert's membrane.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting ³H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Autonomic denervation effects on the kallikrein and striated duct cells of the rat and cat submandibular glands

Chronic preganglionic parasympathetic denervation in the rat submandibular gland increases kallikrein activity whereas post ganglionic sympathetic denervation has no effect. Ultrastructural morphology of the striated duct following either denervation remains unaltered. Chronic preganglionic parasympathetic denervation in the cat gland dramatically reduces kallikrein activity and causes disruption and disappearance of subcellular organelles. Chronic sympathetic denervation has no effect.     

Cytological analysis of the secretory epithelium of the rat submandibular salivary glands hypertrophied after repeated amputation of the lower incisors

Repeated amputation of the lower incisors in August rats causes hypertrophy of the submandibular and sublingual salivary glands. Hypertrophy of the submandibular salivary glands is true in character and accompanied by hypertrophy of the structural components (acini, cells, nuclei). Hypertrophy of the cells is due to hypertrophy of the cell organelles and the accumulation of secretory masses in the cell. Hypertrophy of acinar cell nuclei is connected with polyploidization. Polyploidization and ultrastructural changes in the cells of the submandibular salivary gland are evidence of the increased functional activity of that organ.Laboratory of Growth and Development, Institute of Human Morphology, Academy of Medical Sciences of the USSR. Department of Cytology and Histology, M. V. Lomonosov Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 377–378, March, 1976.     

Cell turnover in the odontogenic organ of the rat incisor as visualized by graphic reconstructions following a single injection of 3H-thymidine

Turnover of cells within the odontogenic organ was studied in three dimensions by preparing serial sections of incisors from young male rats killed at various times following a single intraperitoneal injection of 1 μCi/g body weight of ³H-thymidine. Radioautographs showed that at 1 hour after injection labeled cells were present in all cell layers throughout the entire depth of the odontogenic organ. They were encountered frequently within the inner dental epithelium and stratum intermedium but appeared less abundant within the stellate reticulum and outer dental epithelium. With time, the frequency of labeled cells in each layer declined progressively, and more rapidly at the anterior and labial side of the odontogenic organ than toward its posterior and lingual side. Hence labeled cells were observed over the longest time interval in regions where cell layers were in closest proximity to the opening of the apical foramen, that is, near the apical and cervical loops. By 32 days after injection, numerous labeled cells could still be identified within the outer dental epithelium and stellate reticulum near the apical loop (bulbous part of the odontogenic organ) and the outer dental epithelium near the cervical loops (“U”-shaped part of the odontogenic organ). These findings support the hypothesis that cells originate within the bulbous part of the odontogenic organ and migrate anteriorly through the “U”-shaped and root sheath parts of the odontogenic organ during renewal of the incisor. It appears that individual stem cell compartments may be maintained for surface (outer/inner dental epithelium) and intermediate layers (stellate reticulum/stratum intermedium) in the odontogenic epithelium.