The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells.

We examined the effects of disruption of the actin cytoskeleton by cytochalasin D (cytoD) on basal and carbamylcholine-stimulated exocytosis and on compensatory membrane retrieval in pancreatic acinar cells. Although the involvement of actin in exocytosis is reasonably well established, its role in these coupled processes is not understood. Our findings suggested that cytoD inhibited stimulated secretion of amylase. However, morphometry revealed that exocytosis had occurred: the number of zymogen granules decreased, the size of the lumen increased, and large vacuolar structures continuous with the lumen formed into which amylase accumulated. Large amounts of amylase were released to the medium on removal of secretagogue and cytoD, suggesting that the subapical actin network provides contractile forces that expel the lumenal contents. Strikingly, we observed that at the apical pole of the cells where exocytosis occurred, cytoD induced an accumulation of membrane invaginations into a vastly enlarged apical membrane. These pits were often surrounded by a clathrin-like coat. Concomitantly, AP-2-, clathrin-, dynamin- and caveolin-like immunoreactivity concentrated around the enlarged lumina, suggesting that incorporation of zymogen granule membrane into the apical plasma membrane triggered the recruitment of these proteins. After wash out of cytoD and carbamylcholine and reformation of the subapical actin cytoskeleton, the coated invaginations largely disappeared in association with a reduction in lumenal size, and relocation of clathrin, AP-2, dynamin and caveolin into the cell. We suggest that the actin terminal web also controls compensatory membrane retrieval following exocytosis.

Rab4 associates with the actin terminal web in developing rat pancreatic acinar cells.

Rab4 is a small GTP-binding protein that has been implicated in the regulation of membrane traffic and recycling of transferrin receptors and GLUT4 transporters along the endocytic pathway. Here we present data that suggest a novel and very different role for rab4 during development in the rat exocrine pancreas. On immunoblots of pancreatic homogenates, a dramatic increase in rab4 expression occurred over the first 40 h after birth, concomitant with the time of acquisition of stimulus-secretion coupling. Following high-speed centrifugation of postnuclear supernatants prepared from 1-day neonatal pancreatic homogenates, rab4 partitioned into a Triton X-100 insoluble particulate fraction and was partially solubilized upon extraction with 0.1 M Na2CO3, pH 11.5, or 1 M KCl, suggesting that rab4 was not an integral membrane protein. This was confirmed by Triton X-114 extractions of post-nuclear supernatants showing that rab4 partitioned into the aqueous phase of Triton X-114, which is indicative of a lack of isoprenylation. Confocal and electron microscopic immunocytochemistry revealed that rab4 colocalized with the actin terminal web and microvilli in the apical region of the exocrine acinar cells. In view of these findings, we propose that rab4 is involved in the maturation of the regulated secretory pathway in pancreatic acinar cells through an interaction with the apical actin cytoskeleton.

The expression pattern of rab3D in the developing rat exocrine pancreas coincides with the acquisition of regulated exocytosis.

Although the secretory apparatus of rat pancreatic acinar cells already has a mature appearance in late stage fetuses, the regulated exocytotic pathway becomes functional only after birth. In this study we tested the hypothesis that the acquisition of stimulus-secretion coupling in the acinar cells depends on the developmental expression of rab3D, a small GTP-binding protein which we have previously shown to be associated with zymogen granule membranes in the adult pancreatic acinar cell. On immunoblots of pancreatic homogenates, rab3D became detectable on gestational day 18. This developmental pattern was consistent with that observed by confocal immunocytochemistry. In 20- and 21-day-old fetal pancreata, rab3D was mostly associated with the cytosolic fraction, whereas a redistribution to the membrane fraction occurred after birth, concomitant with a decrease in rab-GDI expression. Overlays of Western blots with 32P-alpha-GTP revealed that rab3D immunoprecipitated from fetal pancreatic homogenates possessed very little GTP-binding capacity as compared to adult homogenates. Triton X-114 extractions showed that the fetal rab3D partitioned into the detergent phase, suggesting that it was posttranslationally isoprenylated. Taken together, the present data indicate that the expression and localization of rab3D is developmentally regulated and strongly suggest that the maturation of the regulated exocytotic pathway in the exocrine pancreas depends on the membrane association of rab3D.

Localization of cellubrevin to the Golgi complex in pancreatic acinar cells.

Cellubrevin is the smallest (14 kDa) isoform of the synaptobrevin (VAMP) protein family and is found in a wide variety of tissues. Western blot analysis with a polyclonal antibody against the unique N-terminus of cellubrevin identified a protein of 14 kDa in rat pancreas. This protein distributed predominantly to the particulate fractions from the rat exocrine pancreas and was totally resistant to NaHCO3 washes, indicating that it is an integral membrane protein. Subcellular fractionation of pancreatic homogenates showed enrichment of this protein in the smooth microsomal fraction while negligible amounts were present in the zymogen granule membrane or the rough microsomal membrane fractions. As seen in other tissues, the 14 kDa immunoreactive form was proteolyzed by tetanus toxin. Light and electron microscopic immunocytochemistry localized cellubrevin immunoreactivity primarily to small vesicles and condensing vacuoles originating from the Golgi region, with significantly lower labeling on zymogen granules. Based on the intracellular localization of cellubrevin detected in acinar cells by immunocytochemistry and cell fractionation, we suggest that cellubrevin may be involved in the maturation of secretory granules.

Rab3D localizes to secretory granules in rat pancreatic acinar cells.

This study reports of presence of rab3D, a low M(r) GTP-binding protein, in rat pancreatic acinar cells and islets using a combination of Western blot analysis, two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis/isoelectric focusing, and light and electron microscopic immunocytochemistry. For these purposes, we used an affinity-purified rabbit polyclonal antibody generated against the exclusive amino terminus of rab3D. Failure to detect rab3A, B or C in pancreatic acinar cells with their respective antisera indicated that the rab3D immunoreactivity was not due to cross-reaction with rab3A, B or C. A monoclonal antiserum which recognized rab3A, B, C and D (clone 42.1) detected a second lower M, band in gradient gels. This protein may be an additional member of the rab family. Double label electron microscopic immunogold localizations for rab3D, and the monoclonal antibody that recognizes all members of the rab3 family, showed a preferential localization of rab3D to zymogen granules. In contrast, clone 42.1 detected both zymogen granules and elements of the Golgi complex. Rab3D also localized to the secretory granule field in pancreatic islet cells which additionally expressed rab3A. The majority of rab3D in acinar cells was tightly associated with membrane fractions as indicated by its resistance to alkaline pH extraction. It is likely associated with membranes via isoprenyl groups as suggested by its partitioning into the detergent phase in Triton X-114 extractions. In contrast, bacterially expressed rab3D partitioned solely into the aqueous phase in Triton X-114 extractions. Because of its exclusive location on zymogen granules, rab3D may play a role in regulated exocytosis from pancreatic acinar cells.

rab3D protein is a specific marker for zymogen granules in gastric chief cells of rats and rabbits.

BACKGROUND & AIMS: Pepsinogen secretion from gastric chief cells requires a series of intracellular vesicle interactions. The final step of exocytosis involves fusion of secretory granules with the plasmalemma, culminating in the release of the digestive enzyme pepsinogen into the lumina of gastric glands. A group of specialized proteins tht confer membrane recognition, targeting, and fusion events are involved in regulated exocytosis. The aim of this study was to examine the distribution of rab3D, a low-molecular-weight guanosine 5'-triphosphate-binding protein, in gastric chief cells. METHODS: rab3D immunoreactivity was evaluated by Western blot analysis and by immunocytochemical studies at both confocal and electron-microscopic levels. RESULTS: rab3D immunoreactivity was specifically localized to the secretory granules in chief cells. Double labeling of gastric glands with rab3D and pepsinogen antibodies confirmed rab3D immunoreactivity localization to chief cells but not acid-secreting parietal cells. Immunoelectron microscopy revealed a near exclusive localization of the rab3D-like protein to the cytoplasmic face of the zymogen granules. After short-term stimulation consequent to feeding, rab3D immunoreactivity appeared to relocate from an apical region of duct cells to an unidentified cytoplasmic compartment. CONCLUSIONS: The localization and redistribution of the rab3D-like protein in the chief cell suggests that it may play an important role in regulated exocytosis.

Hyperoxia enhances expression of gamma-glutamyl transpeptidase and increases protein S-glutathiolation in rat lung.

By participating in glutathione (GSH) synthesis, gamma-glutamyl transpeptidase (GGT) influences the GSH redox cycle, which is a major contributor in protecting against reactive oxygen metabolites. This study determined the effect of prolonged exposure of neonatal rats to > 98% oxygen on expression of GGT and on GSH metabolism. Lungs of neonatal rats chronically exposed to hyperoxia had increased expression of GGT mRNA, resulting in significantly higher GGT protein levels and enzyme activity than in lungs of animals raised in room air. Hyperoxia also upregulated glucose-6-phosphate dehydrogenase, but Na-K-ATPase activity was not changed. GGT mRNA, protein level, and enzyme activity returned to control levels after recovery in room air for 3 days. Levels of GSH, glutathione disulfide, and protein-bound GSH (S-glutathiolated protein) rose with hyperoxia and fell during recovery. S-glutathiolation is likely a mechanism for protection and a regulatory modification of protein sulfhydryl groups. Hyperoxia-induced upregulation of GGT and the concomitant increase in protein S-glutathiolation appear to be additional components fundamental in protecting the lung against oxidative injury.

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis.

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy.

Tenascin/hexabrachion is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that tenascin is associated with epithelial-mesenchymal interfaces during embryogenesis and is prominent in the matrix of many tumors. However, the distribution of tenascin is more restricted in adult tissues. We have found tenascin to be present in normal human skin in a distribution distinct from other matrix proteins. Immunohistochemical studies showed staining of the papillary dermis immediately beneath the basal lamina. Examination of skin that had been split within the lamina lucida of the basement membrane suggested a localization of tenascin beneath the lamina lucida. In addition, there was finely localized staining within the walls of blood vessels and in the smooth muscle bundles of the arrectori pilorem. Very prominent staining was seen around the cuboidal cells that formed the basal layer of sweat gland ducts. The sweat glands themselves did not stain. The distribution of tenascin in the papillary dermis was studied at high resolution by immunoelectron microscopy. Staining was concentrated in small amorphous patches scattered amongst the collagen fibers beneath the basal lamina. These patches were not associated with cell structures, collagen, or elastic fibers. Tenascin could be partially extracted from the papillary dermis by urea, guanidine hydrochloride, or high pH solution. The extracted protein showed a 320-kD subunit similar to that purified from fibroblast or glioma cell cultures. We have developed a sensitive ELISA assay that can quantitate tenascin at concentrations as low as 5 ng/ml. Tests on extracts of the papillary dermis showed tenascin constituted about 0.02-0.05% of the protein extracted.

Specificity of adhesion between murine tumor cells and capillary endothelium: an in vitro correlate of preferential metastasis in vivo.

We have compared the rate and extent of adhesion of various types of mouse tumor cells to endothelial cells derived from different organ sources. Our panel of tumors has included sarcoma, bladder carcinoma, glioma, teratoma, hepatoma, endothelioma, mammary adenocarcinoma, and lymphoma cells. Endothelial cell monolayers have included murine microvascular endothelial cells from ovary, brain, lung, and liver as well as large vessel endothelium from thoracic duct and dorsal aorta. Tumor cells differ both in the adhesive propensity and adhesive preference for different endothelial cells. Some, but not all, of the adhesive preferences correlate with the known in vivo metastatic behavior of these tumors. Our results support the hypothesis that endothelial cell surface-associated specificities may play a significant role in determining the pattern of metastasis.

Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells.

Microvascular endothelial cell cultures have been established from mouse lung, liver, brain, heart, placenta, kidney, urinary bladder, mammary gland, ovary and epididymal fat pad. In addition, large vessel endothelial cells have been obtained from the mouse aorta and thoracic duct. The heterogeneity of these cells has been shown by flow cytometric determination of angiotensin-converting enzyme, by differential presence of the acetyl low density lipoprotein receptor, by the variable expression of cell surface antigens, and by differential binding of various plant lectins. The endothelial cell lines we have developed provide the means to examine in the mouse, long a key species for biomedical research, a wide range of biological functions and properties of the vascular endothelium.

Immunocytochemical localization of protein antigens in large sections of tissues embedded in water-soluble embedding media.

JB4 and Immunobed are water-soluble embedding media used for embedding large blocks of tissue. Immunobed was specifically designed for immunocytochemistry because ethanol extraction of an additive in the monomer of the resin is reported to render tissue sections permeable to immunoglobulins. We have modified the manufacturer's protocol to accomplish localization of two protein antigens in tissues embedded in either JB4 or Immunobed. Luteinizing hormone-beta (LH beta) was localized in sections of rat and bovine pituitary tissues and bovine placental lactogen (bPL) was localized in sections of placentomes from bovine placentas. Sections received one of the following pre-treatments: absolute EtOH; NaHCO3 buffer, pH 6-10; EtOH followed by NaHCO3 buffer; one of several enzymes; EtOH followed by enzyme; NaHCO3 buffer followed by enzyme. Anti-LH beta stained only pituitary gonadotrophs and anti-bPL stained only placental binucleate cells, as assessed by absorption controls. Pre-treatment with enzyme was required for staining of sections, but an alkaline pH change (NaHCO3) had little or no effect. Ethanol pretreatment had little or no effect alone or in conjunction with NaHCO3 or enzyme. Sections were sufficiently thin (1.5 micron) to afford resolution of structure, but suitably large (approximately 2 cm2) to minimize problems of sampling.

Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex.

Normal, unimmunized mouse serum from several strains (BALB/c, C57/b, DBA/2, NZB, SJL, CD/1) contains an endogenous IgG antibody that localizes to the Golgi complex of rat pancreatic acinar cells. Treatment of pancreatic acini with 5 microM monensin resulted in the swelling and vacuolization of the Golgi cisternae, and in a corresponding annular staining by the mouse serum as observed by immunofluorescence, suggesting that the antigen recognized is on the Golgi complex cisternal membrane. The antiserum did not react with pancreatic secretory proteins, and its binding to smooth microsomal membranes was retained following sodium carbonate washing, supporting a Golgi membrane localization. Advantage was taken of the existence of the endogenous murine antibody for the isolation of monoclonal antibodies directed to the Golgi complex of the rat pancreas. Two antibodies, antiGolgi 1 and antiGolgi 2, are described. Both antibodies are IgMs that recognize integral membrane proteins of the trans-Golgi cisternae, with lighter and patchy staining of the pancreatic lumen membrane, as observed both by light and electron microscopy. AntiGolgi 1 recognizes predominately a protein of molecular weight 103,000-108,000, whereas antiGolgi 2 shows a strong reaction to a 180-kd band as well as the 103-108-kd protein.