Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells

In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis.     

The formation of the oncofetal J28 glycotope involves core-2 beta6-N-acetylglucosaminyltransferase and alpha3/4-fucosyltransferase activities.

The feto-acinar pancreatic protein or FAPP, the oncofetal glycoisoform of bile salt-dependent lipase (BSDL), is characterized by the presence of the J28 glycotope recognized by mAbJ28. This fucosylated epitope is carried out by the O-linked glycans of the C-terminal mucin-like region of BSDL. This glycotope is expressed by human tumoral pancreatic tissues and by human pancreatic tumoral cell lines such as SOJ-6 and BxPC-3 cells. However, it is not expressed by the normal human pancreatic tissues and by MiaPaCa-2 and Panc-1 cells. Due to the presence of many putative sites for O-glycosylation on FAPP and BSDL, the structure of the J28 glycotope cannot be attained by classical physical methods. In the first part of the present study, we have determined which glycosyltransferases were differently expressed in pancreatic tumoral cell lines compared to normal tissues, focusing in part on fucosyltransferases (Fuc-T) and core-2 beta6-N-acetylglucosaminyltransferase (Core2GlcNAc-T). Our data suggested that alpha2-Fuc-T activity was decreased in the four cell lines tested (SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1). The alpha(1-3) and alpha(1-4) fucosylations were decreased in tumor cells that do not express the J28 glycotope whereas alpha4-Fuc-T and Core2GlcNAc-T activities were significantly increased in SOJ-6 cells which best expressed the J28 glycotope. Therefore, we wished to gain information about glycosyltransferases involved in the building of this structure by transfecting the cDNA encoding the mucin-like region of BSDL in CHO-K1 also expressing Core2GlcNAc-T and/or FUT3 and/or FUT7 activities. These CHO-K1 cells have been previously transfected with the cDNA encoding Core2GlcNAc-T and/or FUT3 and/or FUT7. Data indicated that the C-terminal peptide of BSDL (Cter) produced by those cells did not carry out the J28 glycotope unless Core2GlcNAc-T activity is present. Further transfection with FUT3 cDNA, increased the antibody recognition. Nevertheless, transfection with FUT3 or FUT7 alone did not generate the formation of the J28 glycotope on the C-terminal peptide. Furthermore, the Cter peptide produced by CHO-K1 cells expressing Core2GlcNAc-T was more reactive to the mAbJ28 after in vitro fucosylation with the recombinant soluble form of FUT3. These data suggested that the J28 glycotope encompasses structures initiated by Core2GlcNAc-T and further fucosylated by alpha3/4-Fuc-T such as FUT3, likely on GlcNAc residues.     

The combinatorial extension method reveals a sphingolipid binding domain on pancreatic bile salt-dependent lipase: role in secretion

Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented.     

Impairment of bile salt-dependent lipase secretion in AR4-2J rat pancreatic cells induces its degradation by the proteasome

Bile salt-dependent lipase (BSDL, EC 3.1.1.13) is a lipolytic enzyme normally secreted by the pancreatic acinar cell. Co- and post-translational modifications, such as N- and O-linked glycosylation, regulate the secretion of this enzyme; therefore it was of first importance to determine the behaviour of BSDL under conditions that impaired its secretion. Using AR4-2J pancreatic cells as model, we showed, particularly when BSDL secretion is impaired, that proteasome inhibitors increased the amount of intracellular BSDL, suggesting that the proteasome is involved in the degradation of this protein. This was strengthened by the detection of ubiquitinated BSDL and of degradation product. Our results suggested that both ubiquitination and degradation of the enzyme occurred at the level of the cytosolic side of microsome membranes. ATP hydrolysis appears essential in ubiquitinated BSDL association with membranes and degradation. Furthermore, under normal secretory conditions, we have shown that a fraction of ubiquitinated BSDL is neither O-glycosylated nor N-glycosylated, suggesting that the N-glycosylation-deficient proteasome substrate does not reach the Golgi and could be degraded by the ER-associated degradation machinery. However, another fraction of ubiquitinated BSDL that is deficient in O-glycosylation, carries out endoglycosidase H-insensitive N-linked glycans, meaning that a second system, that detects abnormal BSDL molecules, could also operate at the level of the Golgi compartment. Consequently, it appears that impairment of BSDL secretion consecutive to secretion inhibition or to a deficient glycosylation leads to the proteasome-ubiquitin-dependent degradation of the protein. Therefore, this pathway is part of the quality control involved in BSDL secretion.     

Subcellular distribution and function of Rab3A-D in pancreatic acinar AR42J cells

Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J 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.     

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.     

Coupled induction of exocrine proteins and intracellular compartments involved in the secretory pathway in AR4-2J cells by glucocorticoids

Treatment of AR4-2J cells with dexamethasone at 10 nM for 96 h inhibited cell replication by 75% and increased cell size (30%), protein content (1.6-fold) and protein synthesis (2-fold). The increase in protein synthesis was largely due to a 5 to 10-fold increase in the synthesis of secretory proteins. Amylase activity increased 20 to 30-fold in cellular homogenates and 10 to 20-fold in culture medium. Both in the presence and absence of dexamethasone AR4-2J cells release their secretory proteins by constitutive secretion. The proportion of newly synthesized amylase retained by the cells over the 14 h labeling period increased from 15 to 30% with hormone treatment. As judged by comigration on polyacrylamide gels and Western blots analyzed by immunospecific sera, AR4-2J cells synthesize and secrete the majority of known pancreatic secretory proteins. Dexamethasone increased the synthesis of trypsinogen 12 to 16-fold, chymotrypsinogen 4.5 to 6-fold, the group of procarboxypeptidases 6-fold, and amylase 7 to 10-fold. Messenger RNA levels for trypsinogen, amylase and lipase were each increased 4 to 5-fold. At the ultrastructural level dexamethasone led to significant increases in rough endoplasmic reticulum (RER) (30-fold) and Golgi elements (1.5-fold) and to the de novo appearance of electron-opaque granules (0.1-0.5 microns) which were shown to contain amylase by immunolocalization techniques employing protein A-gold. Dexamethasone also led to the formation of gap junctions between AR4-2J cells. These findings indicate that AR4-2J cells provide a model for differentiation of pancreatic acinar cells which should also be studied for the differentiation markers for the regulated secretory pathway.     

Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins

Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca²⁺‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca²⁺‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca²⁺‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.      

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.     

Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport

Rab6 GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two-hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, A' and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP-dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6-binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6A'.     

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.     

Transcytosis of pancreatic bile salt-dependent lipase through human Int407 intestinal cells.

In previous studies, we have shown that the bile-salt-dependent-lipase (BSDL), secreted by pancreatic acinar cells and secreted into the duodenal lumen, can be transcytosed through intestinal cells up to the lamina propria. In this study, we used an in vitro system to provide insights into the apical to basolateral transport of BSDL, across the intestinal barrier. The Int407 human epithelial cell line, grown under conditions that optimize polarity, was used as a tight epithelium model. We attempted to delineate uptake mechanisms and the transcytotic pathway followed by this pancreatic enzyme within the intestinal Int407 cells, which do not produce BSDL. When added to the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first interact with the apical membrane. Further, BSDL forms clusters that are internalized via clathrin-coated pits. Following endocytosis, BSDL is directed to a nocodazole- and colchicin-sensitive multivesicular compartment. Interestingly, this protein transits through the Golgi apparatus, where it was found to colocalize with the KDEL retrieval-receptor. Finally, enzymatically active intact BSDL was released at the basolateral membrane level. This is the first demonstration for an apical-to-basolateral transcytotic pathway of a secreted pancreatic digestive enzyme through polarized intestinal cells.     

Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids

Dietary fat type influences fatty acids in rat pancreatic membranes, in association with modulation of secretory activity and cell signalling in viable acini. We aimed to confirm whether AR42J cells are a valid model to study the interactions between lipids and pancreatic acinar cell function. For this purpose we have (i) compared the baseline fatty acid composition of AR42J cells with that of pancreatic membranes from rats fed a standard chow; (ii) investigated if fatty acids in AR42J membranes can be modified in culture; and (iii) studied if similar compositional variations that can be evoked in rats when dietary fat type is altered occur in AR42J cells. Weaning Wistar rats were fed for 8 weeks either a commercial chow (C) or semi-purified diets containing virgin olive oil (VOO) or sunflower oil (SO) as fat source. AR42J cells were incubated for 72 hrs in medium containing unmodified fetal calf serum (FCS, AR42J-C cells), FCS enriched with 18:1 n-9 (AR42J-O cells), or FCS enriched with 18:2 n-6 (AR42J-L cells). Fatty acids in crude membranes from rat pancreas and AR42J cells were determined by gas-liquid chromatography. Differences in membrane fatty acids between C rats and AR42J-C cells can be explained in part by variations in the amount of fatty acids in the extracellular environment. Supplementation of FCS with 18:1 n-9 or 18:2 n-6 changed the fatty acid spectrum of AR42J cells in a manner that resembles the pattern found, respectively, in VOO and SO rats, although AR42J-L cells were unable to accumulate 20:4 n-6. The AR42J cell line can be a useful tool to assess the effect of membrane compositional changes on acinar cell function. However, differences in baseline characteristics, and perhaps fatty acid metabolism, indicate that results obtained in AR42J cells should be confirmed with experiments in the whole animal.     

Evidence for a protein-trafficking gene that rescues the defective glucocorticoid-regulated transport and Golgi retention of mouse mammary tumor virus glycoproteins in a rat hepatoma cell-sorting variant.

Glucocorticoids regulate the trafficking of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the virus-infected rat hepatoma cell line M1.54. The CR4 rat hepatoma sorting variant, which is derived from M1.54 cells by immunoselection, is uniquely defective in the glucocorticoid-regulated transport of MMTV glycoproteins. Indirect immunofluorescence of fixed permeabilized cells and subcellular fractionation of isolated microsomes revealed that variant CR4 cells retain the MMTV glycoproteins in Golgi-like membranes after glucocorticoid treatment. The variant CR4 phenotype can be complemented by interspecies cell fusions with human HepG2 hepatoma cells and by DNA rescue with genomic fragments isolated from either human or rat hepatoma cells. Transfected wild-type genomic fragments rescue the sorting defect in CR4 at a frequency consistant with a single genetic locus, whereas homologous transfection with CR4 genomic DNA has no effect. Thus, complementation of a rat hepatoma cell-sorting variant supports the existence of a novel protein-trafficking activity encoded by the human or rat genomes that acts in trans in the Golgi to selectively mediate the sorting of cell surface MMTV glycoproteins in glucocorticoid-treated cells.     

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.