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.     

The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen

Biosynthesis of type IV collagen in the parietal endodermal cells of 12 day gestant Sherman rats was examined following intraconceptal injection of 3H-proline. The concepti were removed at times varying from 2 minutes to 24 hours after the injection. The parietal wall of the yolk sac, including endodermal cells and the associated basement membrane known as Reichert's membrane were processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Radioactivity was high in endodermal cells during the first 2 hr after 3H-proline injection and later dropped to some extent, while radioactivity rose in Reichert's membrane. Examination of endodermal cell organelles showed some early labeling over rER and Golgi apparatus without a clear-cut trend, except for a drop in Golgi label at late times after 3H-proline injection. The density of silver grains over secretory granules rose significantly by 40 min, reached a high peak by 4 hr and then declined at the time when radioactivity increased over Reichert's membrane. Furthermore, the radioactively-labeled secretory granules were localized mainly at the trans Golgi face soon after injection and near the cell surface adjacent to Reichert's membrane at later times. Biochemical reports indicate that a substantial amount of the proline taken up by the 12-14.5 day rat embryo endodermal cells is incorporated into type IV collagen. Since there is high labeling of the secretory granules from 40 min to 4 hr and the labeled granules are associated with the Golgi apparatus at early times, it is proposed that collagen precursors are processed through rER and Golgi apparatus, packaged into secretory granules and then transported to the cell surface where type IV collagen or its precursors are released and subsequently deposited into Reichert's membrane.     

Changes in the fine structure of the parietal yolk sac of the rat placenta with increasing gestational age

The fine structure of the portion of the parietal yolk sac which extends across the fetal surface of the rat placenta (which is the only portion of the parietal yolk sac which persists after the sixteenth day of pregnancy) was examined sequentially on the even numbered days during the second half of gestation. The placental parietal yolk sac was seen to consist of two cellular layers, trophoblast and endodermal epithelium, which were separated by a thick, amorphous membrane (of Reichert). The trophoblast attenuated with increasing gestational age and, on day 18, became perforated by fenestrations which were closed by diaphragmata. By day 22, the attenuated trophoblast was patently perforate, and maternal blood was exposed to Reichert's membrane. Since throughout gestation, the cells of the parietal endoderm formed a discontinuous layer, at all stages Reichert's membrane was exposed to the vitelline cavity. When ferritin and thorotrast were injected i.v., both separately and together, the former passed readily from maternal blood through Reichert's membrane and was phagocytosed by cells of the parietal endoderm. Thorotrast, however, did not cross Reichert's membrane. Since the rodent yolk-sac complex is known to serve as a placental route, selective permeability of Reichert's membrane may help regulate maternofetal exchange.     

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography

The secretory pathway of clentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injectionof ³H serine, and the results were compared with those using ³H-proline as a tracer. Five min after injection of ³H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of ³H-proline was essentially the, same The pathway of H-proline movedmrore slowly as that of ³H-serine, ³H-proline than ³H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of ³H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with ³H-serine is closely related to the localization of phosphoproteins, whereas the pattern with ³H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by Odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.     

Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat foot pad

To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of ³H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of “intermediate vesicles” including similarly structured tubules which may be over 300 nm long and are referred to as “intermediate tubules”. The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after ³H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4–10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled. The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows. Collagen precursors arise in the rER and, by way of intermediate tubules or vesicles, reach the spherical Golgi distentions. These seem to migrate in a trans direction, while they become cylindrical and their looping threads straighten out into rods which have been identified as procollagen. The cylindrical distention is then freed from its saccule to become a secretory granule. The procollagen content of the granule is released outside the cell and presumably transforms into collagen which then polymerizes into fibrils. Thus, except for a minor fraction of the labeled precursors deviated toward the lysosomal pathway, the bulk of them give rise to collagen, which is added to the extracellular fibrils and, therefore, provides for their growth. No collagen is incorporated into the intracellular collagen fibrils, which, therefore, do not grow, but often undergo degeneration. Thus, the assembly of collagen into fibrils does not occur within cells but is entirely extracellular.     

The permeability of the guinea pig parietal yolk sac placenta to peroxidase and ferritin

The permeability of the guinea pig parietal yolk sac placenta late in gestation was investigated by means of electron microscopy using the tracer proteins ferritin and peroxidase. The parietal yolk sac consists of a layer of trophoblast, a thick extracellular lamina (Reichert's membrane) and a layer of endoderm. After injection into the maternal vascular system, the proteins crossed the trophoblast by means of small pinocytotic vesicles. Both proteins readily permeated Reichert's membrane and then moved by an intercellular pathway between endoderm cells to reach the uterine lumen. After injection of ferritin into the uterine lumen, the protein was observed between endoderm cells and throughout Reichert's membrane. Presumably the marked permeability of the endodermal epithelium to the tracer molecules is due to the absence of zonulae occludentes around the endoderm cells. Parietal endoderm cells exhibited limited pinocytotic activity regardless of the site of injection. The results indicate that the parietal yolk sac placenta of the guinea pig is permeable to relatively large molecules and therefore it may be an important pathway in overall maternal-fetal exchange in this species.     

Effect of colchicine on the transport of precursor enamel protein in secretory ameloblasts studied by 3H-proline radioautography in vitro

The incorporation of 3H-proline into the secretory ameloblasts of rat molar tooth germs cultured with or without colchicine was studied by light and electron microscope radioautography to determine the function of microtubules in the transport of precursor enamel protein from the rough-surfaced endoplasmic reticulum (rER) to the Golgi cisternae. The grain counts over the transitional vesicles, which accumulated in various cellular regions with colchicine treatment, continued to increase with chase time, unlike in controls. At 30 and 90 min chase, these counts were significantly higher than in controls. Moreover, the total grain count over the organelles (rER, pale granules, and transitional vesicles), which are positioned before the Golgi cisternae in the synthetic pathway, maintained a significantly higher level at 90 min chase in colchicine-treated tooth germs than in controls. The transport of synthesized protein to the Golgi cisternae via transitional vesicles was suppressed in colchicine-treated tooth germs. Some grains appeared with time over pale granular materials that appeared in the intercellular spaces of secretory ameloblasts with colchicine treatment. However, at each chase period, the grain count over pale granular materials was not so high as the count over the enamel in control. The present results indicate that colchicine affects the transport of newly synthesized protein from the rER to the Golgi cisterna via transitional vesicles, probably by interfering with the oriented transport related to microtubular function. It is suggested that the microtubular system may be concerned with the movement of the transitional vesicles.     

Fine structure of the venom gland epithelium of the south american rattlesnake and radioautographic studies of protein formation by the secretory cells

The South American rattlesnake venom gland is made up of secretory tubules lined by a simple columnar epithelium containing horizontal cells, mitochondria-rich cells, and the principal cell type, the columnar secretory cells. This cell has a round basal nucleus and abundant rough endoplasmic reticulum, the cisternae of which are variably distended with flocculent material containing many dense intracisternal granules. The supranuclear Golgi apparatus is spherical, with stacks of flattened saccules at the periphery and large vacuoles containing masses of dense material, and other dense granules in the center. Similar but smaller granules are present at the apex where they fuse with the microvillus-covered apical membrane and release their content into the lumen. Protein synthesis was studied in snakes injected with ³H-tyrosine and sacrificed at several times after injection. Radioautographs showed reactions at one half and one hour over the ribosomes and membranes of the rough endoplasmic reticulum. At two hours the immature face of the Golgi apparatus was labeled. At four hours Golgi saccules and vacuoles with dense masses (secretory granules) were labeled, and at eight hours the dense masses within the secretory granules were heavily labeled both in the Golgi region and in the apex near the lumen. Labeled material was found in the lumen at two days. Intracisternal granules were first labeled at eight hours, and by two days reactions remained only over the flocculent content and intracisternal granules of the rough endoplasmic reticulum. Thus, venom protein was synthesized on the rough endoplasmic reticulum, migrated through the Golgi apparatus and accumulated in the dense masses of the secretory granules, which moved to the apex and were extruded. The labeling of intracisternal granules at eight hours and two days after injection indicated a storage nature for these granules.     

An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: II. Colchicine-treated fibroblasts

Colchicine administered intravenously depolymerized microtubules and disrupted the normal organization of the Golgi apparatus in periodontal ligament fibroblasts. Radioautography with ³H-proline indicated that collagen secretion was completely inhibited during a period of approximately 4 hours following the onset of the colchicine effect. During this period of secretory inhibition, labeled collagen precursors were present within a variety of dense bodies, primarily located in a juxtanuclear location replacing the normal Golgi complex. The time course of ³H-proline labeling from 2 to 8 hours suggested that small, newly formed dense bodies fused to form larger dense bodies and pleomorphic structures (zebra bodies), within which collagen precursors appeared to undergo partial polymerization. Autophagosomes, many labeled with ³H-proline, also increased in number after colchicine administration. A gradual decline in ³H-proline label occurred from 4 to 24 hours, presumably due to exocytosis of dense bodies or by the digestion of labeled collagen precursors within autophagosomes. These results support the concept that an intact microtubular network is essential for the organized transport of collagen precursors, from the rough endoplasmic reticulum to the Golgi apparatus, and the eventual transport and exocytosis of collagen secretory granules.     

Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: Fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: I. Normal fibroblasts

Analysis of electon microscopic radioautographs revealed a maximum labeling with ³H-proline of rough endoplasmic reticulum (RER) at 3 minutes, Golgi saccules 1 and 2 at 10 minutes. Golgi saccules type 3 at 20 minutes, and presecretory and secretory granules at 30 minutes. Labeling of the extra-cellular collagen matrix occurred at 30 minutes and increased with time. These observations suggest that pro-a-chains of collagen in periodontal ligament fibroblasts are synthesized in the RER and transported to the Golgi apparatus within 10 minutes. These chains then undergo parallel alignment in Golgi saccules type 2 and form segment-long-spacing-like crystallites in Golgi saccules type 3 between 10 and 20 minutes. The peak labeling of presecretory granules and mature secretory granules in small amounts at 30 minutes and the rapid increase in labeling of extracellular collagen matrix which begins at 30 minutes, indicates that the formation of secretory granules requires approximately 30 minutes and that a rapid system of secretory granule translocation exists in periodontal ligament fibroblasts. This evidence futher supports the previously published morphologic evidence for a microtubuledependent system of collagen secretion in periodontal ligament fibroblasts (Cho and Garant, 1981b).     

Synthesis and secretion of collagen by cells of connective tissue, bone, and dentin

The production of type I collagen by fibroblasts, odontoblasts, and osteoblasts is reviewed on the basis of results obtained by electron microscopy, 3H-proline radioautography, and immunostaining for type I procollagen. In the three cell types, the precursors of type I collagen are processed along the rough endoplasmic reticulum (rER)-Golgi-secretory granule pathway in the same manner as secretory proteins, but the available evidence suggests a few special features: 1) From the rER site of synthesis, the initial collagen precursors, known as pro-alpha chains, are transported to the Golgi apparatus within tubular structures, referred to as intermediate tubules, rather than within vesicles. 2) The pro-alpha chains coil into a triple helix within spherical distensions present along the saccules on the cis side of Golgi stacks. 3) The resulting procollagens are fairly rigid and form bundles that cause spherical distensions to lengthen into cylindrical ones, whereas by an unknown mechanism these distensions become part of the saccules on the trans-side of Golgi stacks. 4) The procollagen-containing cylindrical distensions are released from trans-saccules to become secretory granules, and some procollagen material finds its way into lysosomes. 5) The secretory granules release their procollagen content by exocytosis at the cell surface. 6) The released procollagen is transformed into collagen before or, more probably, after associating with the surface of a collagen fibril.     

免疫组化电镜示垂体LH细胞

应用免疫组化电镜显示垂体黄体生成素(LH)细胞的形态特徵、超微结构以及反应阳性颗粒的分布状况等,并按此将LH细胞分为四型。一、二型细胞内大小分泌粒均含有LH;三型细胞长(?)的150 nm及200 nm颗粒显免疫反应阳性,且部分胞膜和微绒毛顶部界膜内外附着致密细粒;四型细胞出现反应阴性颗粒,仅在rER膜囊外显有直径30～40 nm黑色细粒。LH细胞的分泌可能以外排、分子渗透和局部分泌三种方式排出LH。     

The fine structure of the chinchilla placenta

The structure of the placental labyrinth, interlobular or “coarse” syncytium, visceral (splanchnopleuric) yolk sac, giant cells and subplacenta of the chinchilla was studied with the electron microscope. The fine structure of the interhemal membrane of the placental labyrinth was found to be hemomonochorial, consisting of a single layer of syncytial trophoblast. In this respect, the placental labyrinth was similar to that of another caviomorph rodent, the guinea pig. The labyrinthine trophoblast had pinocytotic vesicles as well as larger vacuoles and multivesicular bodies. The interlobular syncytium contained granular endoplasmic reticulum, and in one case from early in gestation there were intracisternal granules in the ER. The visceral endodermal cells of the inverted yolk sac placenta had a well-developed system of apical vesicles and tubules as well as larger cytoplasmic vacuoles. Their appearance was similar to that of endodermal cells found in other rodents which are known to absorb proteins and other substances from the uterine lumen. Towards term the giant cells were often vacuolated and contained large deposits of glycogen as well as lipid droplets. The syncytial trophoblast of the subplacenta contained numerous moderately electron-dense granules which may be secretory in function; cytotrophoblastic cells lacked these granules. The subplacental syncytium often surrounded spaces or lacunae which contained an electron-dense granular material.     

Ultrastructural localization of calcium in the chick yolk sac membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis

The yolk sac membrane (YSM) of the chick embryo transports calcium from the yolk into the embryonic circulation during the first half of development, but the intracellular pathway of calcium transport is poorly understood. In the present study, the ultrastructural localization of calcium was investigated in cells of the YSM of 9-day chick embryos. X-ray microanalysis as well as cytochemical techniques performed on yolk sac membrane cells treated with potassium oxalate, potassium ferricyanide and potassium antimonate demonstrated accumulation of calcium in yolk granules, digested yolk products, electron-dense bodies (EDBs; 100–400 nm diameter) and electron-dense granules (EDGs; 30–50 nm diameter). When strontium ions were injected into the yolk, they were incorporated into the endodermal cells and sequestered specifically in EDGs. From these results, we propose that calcium enters the endodermal cells by endocytosis of calcium-containing yolk granules, as well as through calcium channels in the apical cell membrane. In the cytoplasm, digested yolk products, EDBs, and EDGs act as sites of sequestration and accumulation of calcium. Extrusion of intracellular calcium into the extracellular space and embryonic circulation is accomplished by exocytosis of calcium-containing material and via an ion pump in the basal cell membrane.     

Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells

The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and α-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane.     

Southern society of anatomists prgram and abstracts presented at the. XIX annual meeting

The permeabilities of the parietal yolk sac placenta and the preplacental region of the hamster conceptus during early postimplantation (day 8) were compared by means of electron microscopy and a macromolecular protein tracer, horseradish peroxidase (HRP). HRP was administered by injection into the maternal venous system; samples of the two placental tissues were obtained for examination at intervals between 4 minutes and 1 hour later. The three layers of the parietal yolk sac wall (from outer to inner: capsular trophoblast, Reichert's membrane, parietal endoderm) appeared to provide little impediment to the passage of HRP from perivitelline maternal blood spaces to the yolk sac cavity. HRP passed through the outer trophoblast layer, both by way of intracellular fenestrae (60-200 nm diameter) and narrower intercellular channels, and completely permeated the meshwork of Reichert's membrane within minutes after injection. The inner parietal endoderm cell layer was widely discontinuous and clearly presented no barrier to HRP movement. HRP reaching the yolk sac cavity was avidly endocytosed by the visceral yolk sac epithelium. In contrast to the parietal yolk sac, the preplacental region of the conceptus was impermeable to HRP. Zonular occluding junctions located between contiguous cells of the chorionic ectoderm layer of the preplacenta were the obvious barrier to the HRP molecules. These results suggest that in this rodent species, during the early postimplantation period of gestation, the parietal yolk sac placenta potentially plays a more important role in the maternal-embryonic transfer of macromolecular substances than does the preplacenta.     

Calcium and the secretory cycle of prolactin cells: A cytochemical and ultrastructural study of dopamine inhibition and monobutyryl cyclic AMP-Stimulation of prolactin secretion

To identify intracellular calcium pools that may be involved in the secretory process in prolactin (PRL) cells, hemi pituitaries were incubated in medium containing 10^?6 M dopamine, 5 mM cyclic cAMP (experimentals), or in medium alone (controls) and then processed for electron microscopy using potassium pyroantimonate to localize intracellular calcium. PRL in the medium was measured by radioimmunoassay. The concentration of antimonate associated with mitochondria, Golgi saccules, and secretory granules was estimated. Dopamine inhibition of PRL secretion (>80% at 1, 2, 3 h) resulted in accumulation of secretory granules in all stages of maturation and dilation of Golgi saccules at 2 and 3 h, accompanied by increased mitochondrial antimonate and increased Golgi-associated antimonate. Cyclic AMP stimulation of secretion (635% at 5 min., declining to 34% at 1 h) resulted in marked exocytosis at 5 and 15 min., declining after 30 min. Mitochondrial antimonate decreased after 30 min. Stimulated cells exhibited numerous coated membrane structures at or near exocytotic pits and an amassing of microvesicles at the margin of the Golgi apparatus. Although some secretory granules consistently exhibited reactivity to antimonate (unchanged by inhibition or stimulation), plasma membrane, and granule membrane translocated to the plasma membrane during exocytosis, were not reactive.     

Synthesis of secretory and plasma membrane glycoproteins by striated duct cellsof 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 μm) 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, suggesting 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.     

A comparison of the protein synthetic activity of presecretory and secretory ameloblasts in rat incisors

Presecretory ameloblasts were compared morphologically and functionally to secretory ameloblasts in the rat incisor. Structurally, the cell types are similar except for the absence of a Tomes' process at the presecretory stage. The developmental changes at the apical end of the ameloblast were described and correlated sequentially with the onset of extracellular events. First, a layer a amorphous dense material appeared at the dentinal surface adjacent to the ameloblasts. Second, the initial layer of enamel began to develop. Third, inner enamel secretion began with the appearance of interrod enamel. This occurred concomitantly with the appearance of the interdigitating portions of Tomes' processes. Functionally, the protein synthetic activity of presecretory ameloblasts was compared to secretory ameloblasts. Light microscopic radioautography of 1-m?m thick Epon sections was used to localize ³H-proline and ³H-tyrosine at various times after injection. At time intervals up to 20 minutes the two presursors were localized as a band of labeled protein in the supranuclear cytoplasm of both presecretory and secretory ameloblasts. At 30 minutes an additional band of radioactivity was localized within the apices of both types of ameloblasts. In presecretory cells the apical reaction band was over the proximal portion of Tomes' processes which border on the dentin. In the secretory cells, the apical reaction band was over both proximal and interdigitating portions of Tomes' processes and over the enamel. Grain counts over the secretory ameloblasts showed that the incorporation of tyrosine increased by 7.5% as opposed to a 63% increase with proline when compared to the values in presecretory cells. The different increases with the two precursors were in keeping with the different amounts of the two amino acids reported to be present in enamel protein. It was concluded that while the secretory ameloblasts synthesize and secrete enamel protein, both presecretory and secretory cell types produce another category of protein which is involved in the apical reaction band. It was proposed that this material is structural protein being contributed to the continuously lengthening Tomes' process which is speculated to occur during formation of enamel.