Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.     

Dominant negative Rab3D inhibits amylase release from mouse pancreatic acini

Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar cell exocytosis, adenoviral constructs were prepared encoding hemagglutinin-tagged wild type Rab3D and three mutant forms, N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis), which also expressed enhanced green fluorescent protein driven by a separate promoter. When isolated mouse pancreatic acini were cultured with 5 x 10(6) pfu/ml adenovirus, nearly 100% of acini were infected as visualized by expression of green fluorescent protein. Cultured acini showed a biphasic dose-response to cholecystokinin (CCK); basal amylase secretion was 1.8 +/- 0.3%/30 min, peak release was 7.3 +/- 0.2%/30 min at 30 pm CCK and reduced secretion was observed at higher CCK concentrations. Control beta-galactosidase virus infection had no effect on either basal or CCK-induced secretion in the titer range from 0.5 to 10 x 10(6) pfu/ml. While the expression of Rab3D and Rab3D Q81L had no effect on amylase secretion, Rab3D N135I and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40-50% at all points on the CCK dose-response curve from 3 to 300 pm. Inhibition was stronger during the first 5 min (71 +/- 5%) than over 30 min (36%+/-5%). Similar inhibition was found using other agonists including bombesin, carbachol, and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. Since both dominant negative Rab3D mutants had no effect on intracellular calcium increase induced by CCK, it is unlikely that they acted at receptors or transmembrane signaling. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is greatest on the early phase of amylase release.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells.     

Rab3D regulates amylase levels,not agonist-induced amylase release,in AR42J cells

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.     

Expression of Rab27B-binding protein Slp1 in pancreatic acinar cells and its involvement in amylase secretion

Slp1 is a putative Rab27 effector protein and implicated in intracellular membrane transport; however, the precise tissue distribution and function of Slp1 protein remain largely unknown. In this study we investigated the tissue distribution of Slp1 in mice and found that Slp1 is abundantly expressed in the pancreas, especially in the apical region of pancreatic acinar cells. Slp1 interacted with Rab27B in vivo and both proteins were co-localized on zymogen granules. Morphological analysis of fasted Slp1 knockout mice showed an increased number of zymogen granules in the pancreatic acinar cells, indicating that Slp1 is part of the machinery of amylase secretion by the exocrine pancreas.     

IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca²⁺. These aberrant Ca²⁺ elevations are triggered by release of Ca²⁺ from apical Ca²⁺ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca²⁺ release channel (IP3R2^−/−). Using live acinar cell Ca²⁺ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca²⁺ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2^−/− and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca²⁺ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.     

EPI64 protein functions as a physiological GTPase-activating protein for Rab27 protein and regulates amylase release in rat parotid acinar cells

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.     

Pancreatic acinar cells express vesicle-associated membrane protein 2- and 8-specific populations of zymogen granules with distinct and overlapping roles in secretion

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca(2+)-regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca(2+)-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca(2+)-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca(2+)-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca(2+)-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca(2+)-regulated secretory pathways.     

Organellar proteomics: analysis of pancreatic zymogen granule membranes

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

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.     

Cyclooxygenase-2 transcription is stimulated and amylase secretion is inhibited in pancreatic acinar cells after induction of acute pancreatitis

Cyclooxygenases as the key enzymes of prostaglandin synthesis have an important role in regulation of inflammation. We describe that Cox-1 and Cox-2 are synthesized in rat pancreatic acinar cells. Upon induction of pancreatitis, Cox-2 mRNA increases while Cox-1 expression remains constant. However, the cyclooxygenase inhibitor indomethacin has no influence by a feed-back mechanism on the expression of the two isoforms. We have previously shown that prostaglandins of the E-type inhibit cholecytoskinin-stimulated amylase secretion. Consistent with this observation, we find here that pancreatitis inhibits CCK-stimulated amylase secretion from isolated acini. In agreement with this result, the effect is neutralized by indomethacin inhibition of prostaglandin synthesis. In summary, we have found that both cyclooxygenases are synthesized in pancreatic acinar cells and that their expression is differentially regulated which in turn influences amylase secretion.     

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin

Aberrant cytosolic Ca(2+) flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca(2+)-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca(2+) changes were recorded in acinar cells loaded with the intermediate Ca(2+)-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 microM FK506 or 10 microM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca(2+) after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca(2+) signal.     

The GTPase Rab37 Participates in the Control of Insulin Exocytosis

Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.     

Increase in pancreatic exocrine secretion during uncoupling: evidence for a protein kinase C-independent effect

It has been demonstrated that blockade of the normal communication between pancreatic acinar cells leads to an increase in amylase release. Although the physiological mechanisms that regulate the gating of gap junction channels are unknown, the involvement of protein kinase C (PKC) in the inhibition of cell coupling has been reported in various cell lines. Since the activation of PKC also stimulates amylase secretion of pancreatic acinar cells, we sought to determine whether blockers of gap junctions and activators of PKC modify basal secretion by a similar mechanism. Thus, we have studied the effects of heptanol and of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the subcellular distribution of PKC, dye coupling, and amylase release of dispersed pancreatic acini. The data show that TPA activates PKC and stimulates amylase secretion without affecting the extensive dye coupling of acinar cells. By contrast, heptanol inhibits cell-to-cell coupling and increases enzyme output without altering the subcellular distribution of PKC. Heptanol also enhances significantly the secretion evoked by TPA. These results indicate that the stimulation of amylase release caused by uncoupling of acinar cells occurs by a mechanism(s) that does not involve the activation of PKC.     

Influence of chronic ethanol consumption on the muscarinic cholinergic control of rat pancreatic acinar cells

There are a number of hypothetical explanations for the actions of ethanol on the exocrine pancreas; among them, the cholinergic hypothesis has received special attention. According to this hypothesis, chronic alcohol consumption induces alterations in the control of exocrine pancreatic function resulting in cholinergic hyperstimulation of pancreatic acinar cells and their muscarinic receptors. Our aim was to investigate the cholinergic control of pancreatic enzyme secretion and the number and affinity of muscarinic receptors in the pancreatic acinar cells of rats subjected to chronic ethanol ingestion. We also investigated whether a high-fibre diet modifies the actions of ethanol on these aspects of the exocrine pancreatic function. Four groups of rats received either a standard or a high fibre diet, and either water or 20% (v/v) ethanol. After 6 months of treatment, isolated pancreatic acini were used for the determination of carbachol-stimulated amylase secretion and for the analysis of muscarinic receptors, using 1-[N-methyl-3H]scopolamine as a radioligand. Neither chronic ethanol intake nor a high fibre diet caused any apparent alteration in pancreatic histology, neither did them modify plasmatic amylase levels. Chronic alcoholization resulted in a significant increase in the amylase released from pancreatic acini in response to carbachol stimulation, but it did not affect either the number or the affinity of pancreatic acinar muscarinic receptors. The actions of ethanol are not significantly modified by the simultaneous consumption of a high fibre diet.     

The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.     

Rab3D redistribution and function in rat parotid acini

The original article to which this Erratum refers was published in J. Cell. Physiol. (2003) 197(3) 400–408 . Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition. J. Cell. Physiol. 199: 316, 2004© 2004 Wiley‐Liss, Inc.     

Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.