Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.     

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Tridimensional architecture of the Golgi apparatus and its components in mucous cells of Brunner's glands of the mouse

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in thin and thick sections of mucous cells of mouse Brunner's glands by using low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of glands impregnated with osmium or treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase) or thiamine pyrophosphatase (TPPase) activity, the Golgi apparatus appeared, at low magnification, as a continuous network located in the supranuclear region. At higher magnifications and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following components were observed: on the cis-face of the Golgi stacks, an osmiophilic tubular network referred to as the cis-element; a cis-saccular-compartment composed of a distended porous saccule slightly reactive for NADPase and three or four underlying NADPase-positive, flattened, poorly fenestrated saccules; a trans-saccular-compartment consisting of four to six TPPase-positive saccules or sacculo-tubular elements, prosecretory granules, and "peeling off" trans-tubular networks. The saccules of the cis-compartment were often perforated by large pores in register. The cavities thus formed in the stacks were called wells and were pan-shaped with a mouth directed toward the cis-face of the stacks and a bottom closed by TPPase-positive saccules. The wells always contained 80-nm vesicles. The saccules of the trans-compartment were involved in the formation of secretory granules according to the following proposed sequence of transformation. The secretion product appeared initially as a granular material evenly distributed throughout a slightly distended, poorly fenestrated saccule. These saccules appeared to transform into fenestrated elements with irregular pores and with parts of them taking on the appearance of a tubular network; they were thus referred to as sacculotubular elements. The secretory material initially distributed throughout these elements accumulated in nodular dilatations randomly distributed along the tubular portions of the elements. The dilatations, considered as prosecretory granules, increased in size as they drained the secretory material from the rest of the sacculotubular elements. Such prosecretory granules, large and irregular in shape, "peeled off" from the stacks of saccules with residual saccular or tubular structures still attached to them, some of the latter forming trans-tubular networks. The prosecretory granules detached from such membranous residues, condensed, and finally transformed into spherical secretion granules.     

Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. III. Immunostaining for laminin and its precursors

Reichert's membrane and the endodermal cells of the parietal yolk sac were examined for the presence of laminin antigenicity using anti-laminin antibodies and the peroxidase-antiperoxidase sequence. Immunostaining was observed through the full width of Reichert's membrane and within endodermal cells. In these cells immunostaining was observed in rough endoplasmic reticulum (rER) cisternae and Golgi apparatus. The Golgi staining could occur in any saccule, but predominated in components interpreted as the last saccule of the stack, the GERL element, and associated prosecretory granules. The secretory granules found in the ectoplasm were also immunostained. Finally, multivesicular bodies showed some staining. The immunostaining of Reichert's membrane indicates the presence of laminin itself, while that of rER cisternae and the Golgi apparatus is attributed to laminin precursors. Presumably the biosynthesis of laminin occurs along the usual protein pathway, that is, from rER through Golgi saccules and the GERL element to secretory granules, which release their content into Reichert's membrane. The laminin immunostaining of Reichert's membrane and endodermal cells is similar to that of type IV collagen. It is, therefore, likely that the two substances are processed and secreted simultaneously.     

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity within columnar, goblet, and Paneth cells of rat small intestine

The distribution of nicotinamide adenine dinucleotide phosphatase (NADPase) activity was examined in epithelial cells of rat small intestine. Segments of ileum were fixed with glutaraldehyde and tissue chopper sections were incubated for up to 4 hr at pH 5.0 in cytochemical media prepared with NADP as substrate. NADPase activity was found primarily within the Golgi saccules of columnar, goblet, and Paneth cells. Columnar and goblet cells showed most of the NADPase activity within the saccules which were intermediate between the cis and trans faces of the Golgi stack. Paneth cells, however, showed the heaviest staining within saccules between the intermediate and innermost saccule at the trans aspect of the Golgi stack. Both columnar cells and Paneth cells also contained spotty, and sometimes heavy, deposits of reaction product within an occasional focal area of the GERL system and within an occasional lysosome. Control experiments indicated that the Golgi-associated NADPase activity was enhanced if cells were pretreated for about 12 hr with EGTA prior to incubation. No similar enhancement was apparent if the tissues were pretreated with DMSO. Furthermore, NADPase activity within the Golgi saccules could be inhibited completely by incubating intestinal epithelial cells with NADP in the presence of sodium fluoride or L(+)-tartrate.     

Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.     

Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.     

Ultrastructural localization of CMPase, TPPase, and NADPase activity in neurons, satellite cells, and Schwann cells in frog dorsal root ganglia

Sections of bullfrog dorsal root ganglia were analyzed for cytidine monophosphatase (CMPase), thiamine pyrophosphatase (TPPase), and nicotinamide adenine dinucleotide phosphatase (NADPase) activity, and the distributions of these enzymatic activities were compared with those traditionally found in other cell types (e.g., CMPase: Golgi trans-sacculotubular network; TPPase: trans-Golgi saccule(s); NADPase: intermediate Golgi saccules). In the present study, CMPase activity in neurons was localized mainly to the Golgi trans-sacculotubular network and lysosomes, but sometimes also occurred at the ends of the trans and most distal intermediate Golgi saccules. A similar distribution was found in satellite and Schwann cells. TPPase activity in neurons occurred not only in the trans-Golgi saccule but also in the trans-sacculotubular network, lysosomes, and scattered tubular elements. In satellite and Schwann cells, activity was found in both the trans saccule and trans-sacculotubular network, and substantial activity often appeared in the more distal of the intermediate saccules. NADPase activity in neurons was usually absent from the intermediate Golgi saccules and was confined to the trans-sacculotubular network and lysosomes; however, activity was sometimes also found in the intermediate and/or trans-Golgi saccules. In satellite and Schwann cells, activity appeared consistently in both the trans-sacculotubular network and intermediate saccules, as well as in lysosomes. These distributions, especially in the case of TPPase and NADPase, differ substantially from the most frequently reported localizations of the above enzymes, indicating that the Golgi complex may exhibit considerable plasticity of structure and function in different cell types.     

Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. I. Ultrastructure and phosphatase activity of endodermal cells

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.     

Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma-aminobutyric acid (GABA)

Summary The GABAergic innervation of the mouse pituitary, including the median eminence, was studied at light microscopic and ultrastructural levels by use of a pre-embedding immunocytochemical technique with antibodies directed against GABA. In the median eminence, a high density of GABA-immunoreactive fibers was found in the external layer where the GABAergic varicosities were frequently observed surrounding the blood vessels of the primary capillary plexus. In the internal and subependymal layers, only few fibers were immunoreactive. The intense labeling of the external layer was observed in the entire rostro-caudal extent of the median eminence. In the pituitary proper, a dense network of GABA-immunoreactive fibers was revealed throughout the neural and intermediate lobes, entering via the hypophyseal stalk. The anterior and tuberal lobes were devoid of any immunoreactivity. The GABA-immunoreactive terminals were characterized in the median eminence, and in the intermediate and posterior lobes at the electron-microscopic level. They contained small clear vesicles, occasionally associated with dense-core vesicles or neurosecretory granules. In the intermediate lobe they were seen to be in contact with the glandular cells. In the posterior lobe and in the median eminence, GABA-immunoreactive terminals were frequently located in the vicinity of blood vessels. These results further support the concept of a role of GABA in the regulation of hypophyseal functions, via the portal blood for the anterior lobe, directly on the cells in the intermediate lobe, and via axo-axonic mechanisms in the median eminence and posterior lobe.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbonlike Golgi apparatus. 1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. 2) A first, poorly fenestrated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. 3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. 4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. 5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

DETECTION OF COMPLEX CARBOHYDRATES IN THE GOLGI APPARATUS OF RAT CELLS

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.     

Fine structural localization of peroxidase activity in the epithelium and the gland of the rat larynx

Summary Endogenous peroxidase activity was demonstrated in ciliated cells and secretory cells of the laryngeal epithelium and gland of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The intensity of peroxidase activity was greatly varied from cell to cell, but the fine structural localization of the activity was similar in various cell types. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum including nuclear envelope, some vesicles and saccules of the Golgi complex, large membrane-limited granules, multivesicular bodies and probable lysosomes. In secretory cells, the activity was also found in secretory granules.The significance of peroxidase activity is not unclear, while the activity, at least a part of it, seems to be secreted into the cavity of the larynx. The possibility that peroxidase participates bactericidal mechanism, deserves further investigation.     

Endocytotic pathways in the melanotroph of the rat pituitary

Summary The internalization of the extracellular markers horseradish peroxidase (HRP) and cationized ferritin (CF) by the melanotrophs of the intermediate lobe of the rat pituitary was studied during short-time incubation of mechanically dissociated cells or in cell culture after 5 days. After a 30 min exposure, the tracers were found in electron-lucent granules or vacuoles of approximately the same size as the secretory granules, situated 200–500 nm from the cell membrane. In the cultured cells, which showed a higher rate of tracer uptake, internalization was followed for 1, 2 and 5 min after labelling and during 2 h of exposure. Initially, the label was seen only in coated pits and coated vesicles at the cell membrane. Larger vacuoles were first seen after 2–5 min of incubation. After 2 h of exposure the labelling pattern was distinctly different for the two tracers. CF was found in larger vacuoles of varying morphology, in dilatations at the base of cilia, within Golgi saccules and at the edge of the electron-dense core of forming secretory granules. HRP was found in an extensive array of tubulovesicular structures extending throughout the cytoplasm. The Golgi complex and forming granules were, however, not labelled with HRP. The study identifies part of the electron-lucent granules or vacuoles in the melanotroph as endosomes, and shows that the melanotrophs sort CF and HRP via diverting pathways after internalization, suggesting that granule membrane, and possibly its functional components, can be recycled in these cells.     

Delivery of lectin-labeled membrane to the trans-Golgi network and secretory granules in cultured atrial myocytes

To examine whether and how internalized plasma membrane components are routed to the compartment of the biosynthetic-exocytic pathway in cultured atrial myocytes, the plasma membrane labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was traced electron microscopically by cytochemical detection of HRP. The WGA-HRP label was internalized via a coated pit-small vesicle pathway and reached vacuoles and endosomes by 3 min. Labeled endosomes comprised vacuoles and tubular elements containing reaction product. By 15 min, similar tubular structures containing reaction product accumulated in the area of the trans-Golgi network (TGN). The labeled TGN consisted of interconnected tubular elements, which often connected to atrial granules containing reaction product. In contrast, neither native HRP nor Lucifer Yellow reached Golgi elements or atrial granules. These results suggest that a proportion of the plasma membrane labeled with WGA-HRP is delivered to endosomes, from which tubules might bud off to transfer the tracer molecules to the TGN, where the lectin conjugate and associated membranes are packaged into atrial granules.     

SYNTHESIS OF THE CARBOHYDRATE OF MUCUS IN THE GOLGI COMPLEX AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY OF GOBLET CELLS FROM RATS INJECTED WITH GLUCOSE-H3

It is known that colonic goblet cells utilize glucose to synthesize the carbohydrate portion of mucus glycoprotein. To determine the intracellular site of this synthesis, glucose-H³ was injected into 10-g rats. At 5, 20, 40 min, 1, 1½, and 4 hr after injection, segments of colon were fixed and prepared for electron microscope radioautography. By 5 min after injection, label had been incorporated into substances present in the flattened saccules of the Golgi complex. At 20 min, both Golgi saccules and nearby mucigen granules were labeled. By 40 min, mucigen granules carried almost all detectable radioactivity. Between 1 and 4 hr, these labeled granules migrated from the supranuclear region to the apical membrane; here, they were extruded singly, retaining their limiting membrane. The evidence indicates that the Golgi saccule is the site where complex carbohydrate is synthesized and is added to immigrant protein to form the complete glycoprotein of mucus. The Golgi saccule, distended by this material, becomes mucigen granules. It is roughly estimated that one saccule is released by each Golgi stack every 2 to 4 min: a conclusion implying continuous renewal of Golgi stacks. It appears that the Golgi synthesis, intracellular migration, and release of mucus glycoprotein occur continually throughout the life of the goblet cell.     

Secretion of renin-angiotensin system (RAS) components by normal and tumoral lactotropes. A comparative study using reverse hemolytic plaque assay (RHPA) and immunoelectron microscopy.

Immunodetection of renin-angiotensin system (RAS) components indicates that there is a local RAS in anterior pituitary cells, particularly in lactotropes. We have attempted to determine if RAS molecules are secreted by lactotropes and the secretory pathways and intracellular sites of maturation. We investigated the secretory activity of individual lactotropes, using the reverse hemolytic plaque assay (RHPA), with GH3B6 tumor cells and normal male rat pituitary cells. We also determined the subcellular distributions of RAS components in these cells. Both tumor and normal cells secreted angiotensinogen, prorenin, renin, angiotensin I, angiotensin-converting enzyme, and angiotensin II, although at different levels. The percentage of secretory cells was generally higher in tumor lactotropes than in normal cells. The subcellular distribution of RAS components obtained by immunoperoxidase was very similar in both cell types, although the intensities of immunoreactivity differed. Cleaved and uncleaved components were found in rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules, all compartments of the secretory pathway. The cleaved components in the RER suggest the existence of early maturation, whereas the presence of uncleaved products in the secretory granules of normal lactotropes might indicate late maturation sites.     

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.