Carboxyl-methylation of Rab3D in the rat pancreatic acinar tumor cell line AR42J.

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.

An evaluation of the expression, subcellular localization, and function of rab4 in the exocrine pancreas.

The small GTP-binding protein, rab4, is involved in recycling of transferrin receptors and translocation of GLUT4. Recent studies suggest that rab4 controls regulated exocytosis in the exocrine pancreas. We conducted the present study to further investigate the role of rab4 in the exocrine pancreas. We found that the exocrine pancreas expresses two rab4 immunoanalogs, one of approximately 28 kDa identified previously in neonatal glands, and one of approximately 24 kDa which is similar to rab4 characterized in other systems. The latter species was mostly membrane-anchored and localized to endosome-like structures in a supranuclear region that was immunopositive for the transferrin receptor. The approximately 24-kDa rab4 form also localized to the apical plasmamembrane, and this immunofluorescence increased greatly in tissue challenged with a secretagogue. We propose that the approximately 24-kDa rab4 species is involved in compensatory membrane retrieval following regulated exocytosis, and that rab4-positive endocytic vesicles move through a supranuclear recycling compartment.

Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D.

The present study describes a novel phenomenon in pancreatic acinar cells undergoing regulated exocytosis. When acinar cell preparations were challenged with the secretagogue carbamylcholine, a subpopulation of zymogen granules became coated with filamentous actin. These zymogen granules were always in proximity of the acinar cell apical membrane (the site of exocytosis) but did not appear to have fused yet. They were distinct from regular zymogen granules not only because of their association with filamentous actin, but also because the majority of them lacked the zymogen granule marker rab3D, a small GTPase implicated in regulated exocytosis. The apparent loss of rab3D, presumed to result from the release of rab3D from the granule membranes, could be prevented by agents that modulate the actomyosin system as well as by GTP[gammaS]. These data suggest that zymogen granules engaging in exocytosis become coated with actin before fusion and that this actin coating is tightly coupled to the release of rab3D. We propose that rab3D is involved in the regulation of actin polymerization around secretory granules and that actin coating might facilitate the movement of granules across the subapical actin network and toward their fusion site.

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance.

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.

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.

Carboxyl methylation of rab3D is developmentally regulated in the rat pancreas: correlation with exocrine function.

Several GTPases of the rab family, including rab3A, are methylesterifled on their carboxy-terminal prenylcysteine residue. The significance of this reversible posttranslational modification for the function of rab proteins is unknown, although it has been postulated that carboxyl methylation facilitates the membrane association of prenylated proteins through a hydrophobic mechanism. We here demonstrate, that pancreatic rab3D undergoes developmentally regulated carboxyl methylation concurrently with the maturation of the regulated secretory apparatus in pancreatic acinar cells: in fetal glands, which are refractive to hormone stimulation, the majority of the rab3D protein was methylated, whereas in neonatal and adult glands, which are secretory competent, only 50% was methylated. The methylated form of rab3D was also predominant in a transplantable acinar cell tumor which displays impaired secretory responsiveness and morphological characteristics reminiscent of the fetal pancreas. In addition, treatment of AR42J pancreatic acinar tumor cells with dexamethasone to induce a regulated secretory pathway, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. Strikingly, membrane preparations from adult pancreata and parotid glands contained both methylated and unmethylated forms of rab3D indiscriminately. These results suggest that the acquisition of stimulus-secretion coupling by the exocrine pancreas correlates with the methylation state of rab3D, and that carboxyl methylation plays no significant role in enhancing the membrane association or determining the subcellular distribution of rab3D.

The Golgi complex: perspectives and prospectives.

We now have considerable understanding of the role of the Golgi complex in the posttranslational modifications of membrane and secretory proteins and of lysosomal hydrolases. It is now also clear that the Golgi plays a key role in the intracellular packaging, addressing, and sorting of these classes of proteins to their final destinations on the secretory and endocytic pathways. While it has been proposed that vesicular budding and fusion underlie entry of proteins into the Golgi from the ER and subsequent movement among its cisternae and exit to their final stations, recent observations indicate that this model may need to be revised based on studies in living cells where vesicular-tubular structures appear to mediate membrane trafficking. This will be a major challenge for investigators in the coming years who will rely again on the use of morphologic techniques of the sort that started it all in 1898.

Developmental changes of fetal rat lung Na-K-ATPase after maternal treatment with dexamethasone.

Late in gestation, the prenatal fetal alveolar epithelium switches from fluid secretion to resorption of salt and water via apical sodium channels and basal Na-K-ATPase. The amounts of lung sodium pump activity protein and mRNA increase in the lung just before birth. Because maternal glucocorticoids (GC) may promote maturation of the alveolar epithelium and augment fetal surfactant apoprotein levels, we hypothesized that GC increase the fetal lung Na-K-ATPase alpha- and beta-subunit gene expression in development. Timed-pregnant Sprague-Dawley rats were injected daily with intraperitoneal dexamethasone (1 mg/kg) or saline for 1, 3, or 5 days before death at fetal day (FD) 17 or 19. Maternal GC treatment altered the fetal lung wet to dry weight, decreasing it at FD17 and increasing it at FD19. Northern analysis of total lung RNA for the alpha1- and beta1-pump subunits demonstrated differential regulation of the mRNA in response to GC. At FD17, beta1-mRNA increased after 1 (FD16) or 3 days (FD14-FD16) of GC treatment, whereas alpha1-mRNA was not altered. There were accompanying increases in beta1-, but not alpha1-, protein. At FD19, GC treatment for 5 days (FD14-FD18) increased beta1- and decreased alpha1-mRNA levels, but treatment for 1 (FD18) or 3 days (FD16-FD18) had no effect. In all groups, the alpha1-Na-K-ATPase protein was predominantly on the basolateral surface of airspace epithelium by immunofluorescence. In summary, maternal dexamethasone differentially affected the fetal lung mRNA levels of the two sodium pump subunits in a complex manner, with increased beta1-mRNA levels dependent on duration of treatment and fetal age.

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.

Developmental regulation of Na, K-ATPase in rat lung.

Late in gestation lung epithelium changes from net chloride and fluid secretion to sodium and fluid absorption. Fluid resorption is required for postnatal gas exchange and occurs by combined action of epithelial sodium channels and Na, K-ATPase. We hypothesized that alveolar epithelial Na, K-ATPase increases perinatally. Immunofluorescence (IF) and immunoelectron microscopy (IEM) with a monoclonal anti-alpha subunit antibody demonstrated that Na, K-ATPase was present on the basolateral surfaces of columnar epithelial cells at fetal day (FD) 17 and on type II cells throughout development. However, type I epithelial cells did not have detectable Na,K-ATPase. The steady-state levels of both the alpha 1 isoform and the beta-subunit mRNAs were maximal at FD20-neonatal day (ND) 1, with consistent increases from FD17 level. Na, K-ATPase alpha-subunit protein also increased from FD17 to FD20-22 and then decreased in the early postnatal period. The ouabain-inhibitable sodium pump activity per milligram membrane protein increased 2.6-fold from FD17 to FD22-ND1 (P < 0.05). The quantities of sodium pump mRNA, antigenic protein, and enzyme activity increase in late gestation in accord with a proposed role for Na, K-ATPase in resorption of alveolar sodium and fluid in preparation for birth.

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.

gamma-Glutamyl transpeptidase is a polarized alveolar epithelial membrane protein.

In many diseases the lung is injured by oxidants. gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme on the apical plasma membrane of many epithelial cells that protects against oxidants by replenishing intracellular glutathione. We sought to localize GGT within rat lungs in vivo and in cultured alveolar epithelial cells. In the adult rat lung, indirect immunofluorescence (IF) with a polyclonal antibody to triton-solubilized GGT revealed linear staining outlining the alveoli. Immunoelectron microscopy (IEM) localized the protein on the apical surface of the alveolar epithelial cells, but more densely on type I cells than type II cells, as well as on the apical surface of some ciliated bronchial cells. On Western blots of whole lung and isolated type II cell membrane proteins, the antibody predominantly recognized a broad protein band of 110-120 kDa, consistent with the uncleaved, glycosylated form of GGT. Over time in culture, isolated rat type II cells had increasing immunoreactivity on Western blots and indirect IF but decreasing enzyme activity. At 2 days in culture, confocal laser scanning microscopy demonstrated that GGT was polarized to the apical surface of nonconfluent type II cells. Thus GGT is a polarized apical membrane protein in type I and II cells, suggesting a role in the metabolic functions of these cells. The increased immunoreactive GGT of cultured type II cells is consistent with their acquisition of properties similar to type I cells, but the lack of correlation between immunoreactive protein and enzyme activity awaits explanation.

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.