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Originally published In Press as doi:10.1074/jbc.M201248200 on March 1, 2002

J. Biol. Chem., Vol. 277, Issue 20, 18002-18009, May 17, 2002
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Dominant Negative Rab3D Inhibits Amylase Release from Mouse Pancreatic Acini*

Xuequn ChenDagger §, Julie A. S. Edwards, Craig D. LogsdonDagger , Stephen A. Ernst, and John A. WilliamsDagger ||

From the Departments of Dagger  Physiology,  Cell and Developmental Biology, and || Internal Medicine, The University of Michigan, Ann Arbor, Michigan 48109-0622

Received for publication, February 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 × 106 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 × 106 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, A23187, 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pancreatic acinar cells are the functional unit of digestive enzyme secretion and have long been used as a model to study the packaging and secretion of secretory proteins and its control by neurotransmitters and hormones (1-3). The major intracellular signaling pathway leading to acinar secretion of stored proteins in most species is an increase in intracellular Ca2+ (4, 5). Whereas the details of transmembrane and intracellular signaling pathways leading to an increase in cytosolic Ca2+ have been well investigated, much less is known of the downstream events leading to the release of digestive enzymes by zymogen granule exocytosis. This process is believed to share basic mechanism with other vesicular fusion systems from yeast to neurons (6). Current models of membrane fusion have been dominated by the consideration of two types of proteins: small G proteins of the Rab family and SNARE proteins. Small GTPases of the Rab/Ypt family form the largest branch of the Ras-related small G-protein superfamily and are recognized as key protein components involved in vesicular trafficking and membrane fusion in eukaryotic cells (6, 7). Rab proteins act as molecular switches which cycle between the GDP-bound inactive and GTP-bound active forms. As with other small G proteins, the conversion from the GDP-bound form to the GTP-bound form is stimulated by a Rab GEF1 (guanine nucleotide exchange factor) and the conversion of the GTP-bound form to the GDP-bound form is catalyzed by a Rab GTPase-activating protein (8).

The Rab3 proteins are the Rab species associated with synaptic or secretory vesicles in neurons, neuroendocrine, endocrine, and exocrine cells, and are thought to play an important role in regulated exocytosis (9). Four isoforms of Rab3 exist, denoted A through D, with different members present in different tissues. Rab3A and Rab3C are both associated with synaptic vesicles and secretory granules in brain and neuroendocrine cells (7). Rab3B is expressed in epithelial cells (10) and anterior pituitary (11). Rab3D was recently found in pancreatic acinar cells and other exocrine cells (12, 13). In recent years, the functions of Rab3 proteins have been intensively investigated in neurons (14), chromaffin cells, PC12 cells (15, 16), and mast cells (17, 18), mainly by using GTP-binding and GTP hydrolysis-deficient Rab3 mutants. Whereas several different approaches indicate that Rab3A is a negative modulator of exocytosis, there is also evidence that Rab3B and Rab3D are positive regulators of secretion (11, 19, 20).

Recent work in our laboratory and others (12, 13, 21) has demonstrated that Rab3D is the only detectable Rab3 isoform in pancreatic acini and is localized on zymogen granules. The secretory granule localization of Rab3D in various exocrine cells implies that it may be involved in regulated exocytosis. Recently, direct evidence has suggested that Rab3D may play a positive role in regulated exocytosis. Baldini et al. (22) reported that expression of Rab3D N135I, a dominant negative Rab3D mutant, inhibited positioning of dense core granule near the plasma membrane and blocked regulated secretion of mature ACTH in AtT-20 cells. However, they also found that the expression of this mutated Rab3D impaired the membrane association of endogenous Rab3A in AtT-20 cells, and hypothesized that this alteration in Rab3A may be at least one of the defects that inhibits exocytosis of dense core granules in AtT-20 cells. In pancreatic acini, Onishi et al. (20) reported that overexpression of Rab3D enhanced the initial phase of regulated amylase secretion from pancreatic acini of transgenic mice. The authors suggested that further overexpression of mutant Rab molecules affecting the GTPase cycle was necessary to understand the regulation of exocytosis by Rab3D. We have now overexpressed in vitro Rab3D mutants that interfere with the Rab3D GTP/GDP cycle in pancreatic acinar cells and investigated their effect on acinar exocytosis. We utilized adenoviral constructs encoding HA-tagged wild type Rab3D and three mutant forms: N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis) (23, 24) and compared the effect of their expression on amylase secretion. We found that both Rab3D mutants, N135I and T36N, functioned as dominant negative mutants and inhibited regulated exocytosis in pancreatic acinar cells, preferentially in the early phase of secretion. In contrast to wild type Rab3D which was localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Collagenase (CLSPA grade) was purchased from Worthington Biochemicals (Freehold, NJ); protein G-agarose beads from Pierce (Rockford, IL); [alpha -32P]GTP (3,000 Ci/mmol) from Amersham Biosciences (Piscataway, NJ); CCK octapeptide (CCK-8) from Research Plus (Bayonne, NJ); CPT-cAMP from Sigma; A23187 from Calbiochem (La Jolla, CA); Fura-2/AM from Molecular Probes (Eugene, OR). Anti-Rab3D antisera was a gift from Dr. Mark McNiven (Mayo Clinic, Rochester, MN). Mouse monoclonal anti-HA antibody 12CA5 and rat monoclonal anti-HA antibody 3F10 were purchased from Roche Molecular Biochemicals (Indianapolis, IN) and rabbit polyclonal anti-HA antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-GFP antibody was from CLONTECH (Palo Alto, CA). All other chemical reagents were obtained from Sigma.

Construction of Recombinant Adenoviruses Encoding Wild Type and Mutant Rab3D-- The recombinant adenoviruses encoding the wild type and mutant Rab3D were produced according to the method of He et al. (25). Briefly, the HA-tagged wild type and mutant mouse Rab3D cDNAs were cloned into the shuttle vector pAdTrack-CMV, linearized, and co-transformed into Escherichia coli BJ5183 cells along with the adenoviral backbone vector pAdEasy-1. Recombinants were selected for kanamycin resistance and confirmed by restriction endonuclease analyses. Finally, linearized recombinant plasmid was transfected into an adenovirus packaging cell line, HEK293 cells. Recombinant adenoviruses were collected 7 to 12 days after infection and were concentrated using a CsCl gradient. The shuttle vector pAdTrack-CMV also encodes EGFP driven by a separate CMV promoter and thus the titers of the viral stocks were estimated by counting EGFP-expressing cells. An adenovirus (AdLacZ) expressing bacterial beta -galactosidase and EGFP, each under the control of a separate CMV promoter, was a gift from Dr. He (John Hopkins Oncology Center, Baltimore, MD) and used as a control. Rab3D T36N, N135I, and Q81L mutants were created using QuikChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA) and confirmed by DNA sequencing.

Isolation, Short-term Culture, and Viral Infection of Pancreatic Acini-- Pancreatic acini were isolated from male ICR mice by collagenase digestion as previously described (26, 27). Isolated acini from two pancreases were resuspended in Dulbecco's modified Eagle's medium and divided into four or five 150-mm Petri dishes each containing 30 ml of Dulbecco's modified Eagle's medium enriched with 0.5% fetal bovine serum, 0.02% soybean trypsin inhibitor, and antibiotics, and incubated at 37 °C for 4-20 h. In the viral infection experiments, either control beta -galactosidase or various Rab3D adenoviruses were added at a specified titer, usually 5 × 106 pfu/ml, to the culture medium at the beginning of the incubation.

Analysis of CCK-stimulated Amylase Secretion-- After incubation for 9 h, cultured acini were allowed to settle by gravity, resuspended in Hepes-Ringer buffer containing essential amino acids, and soybean trypsin inhibitor, and then incubated with various concentration of CCK. After the specified time the acinar suspension was centrifuged for 30 s in a microcentrifuge and supernatant assayed for amylase activity using Phadebas reagent (Amersham Biosciences and Upjohn) as described previously (26). Results were expressed as a percentage of initial acinar amylase content. The data presented are the mean ± S.E. Statistical significance was calculated by the Student's t test with p < 0.05 representing significance.

Analysis of CCK-induced Increase in Intracellular Ca2+ Concentration-- As previously described (27), isolated acini were incubated with 1 µM fura-2/AM at 37 °C for 30 min and then washed and resuspended in fresh HR buffer. For measurement of intracellular Ca2+, fura-2-loaded acini were allowed to stick to a coverslip, transferred to a low volume closed chamber mounted on the stage of a Zeiss Axiovert inverted microscope, and continuously superfused at 1 ml/min with 37 °C HR buffer alone or HR buffer containing 300 pM CCK. Solution changes were accomplished rapidly by means of a valve attached to an 8-chambered superfusion reservoir. Measurement of intracellular Ca2+ was performed using an Attofluor digital imaging system and software (Rockville, MD). Under the conditions used, the presence of EGFP in acini did not interfere with the fura-2 signal or calculated intracellular Ca2+ concentration.

SDS-PAGE and Western Blotting-- Lysates of cultured acini were prepared in buffer containing 25 mM Tris, 150 mM NaCl, 0.2% Triton X-100, 5 mM EDTA, and supplemented with the proteinase inhibitors: 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride. Protein concentration was determined using protein assay reagent (Bio-Rad). An aliquot of sample was mixed with 4 × SDS stop solution (final concentrations 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromphenol blue, and 2% 2-mercaptoethanol) and boiled for 5 min. Acinar samples (20 µg of protein/lane) were then loaded onto 12% SDS-polyacrylamide electrophoresis ready gels (Bio-Rad). After gel electrophoresis, proteins were transferred to nitrocellulose membranes at 55 V for 1 h on ice. Western blotting was then carried out as previously described (20) using anti-HA antibody 12CA5 (0.4 µg/ml) overnight at 4 °C. ECL chemiluminescence reagents were used to visualize the secondary antibody.

Immunoprecipitation and GTP Overlay-- HA-tagged wild type and mutant Rab3D proteins were immunoprecipitated from the corresponding acinar lysate (1 mg of protein) by incubation with 4 µg of anti-HA monoclonal antibody at 4 °C overnight. Protein G-agarose beads (20 µl) were added to each sample and incubated for an additional hour at 4 °C. The beads were collected by centrifugation, and after three washes, SDS sample buffer was added. Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Following the procedure of Bhullar et al. (28), the transfer blot was soaked for 20 min in GTP binding buffer (50 mM Tris, pH 7.5, 12 µM MgSO4, 1 mM 2-mercaptoethanol, 10 µM MgATP, 0.3% Tween 20) and then incubated with 1 µCi/ml [alpha -32P]GTP in fresh GTP binding buffer for 2 h at room temperature. After five 10-min washes with fresh buffer, the membrane was autoradiographed for 6 h at -70 °C.

Immunocytochemistry and Confocal Fluorescence Microscopy-- Control and virus-treated pancreatic mouse acini were allowed to settle in test tubes and then were fixed for 2 h at room temperature with 4% formaldehyde (prepared from paraformaldehyde) in phosphate-buffered saline, pH 7.4. Acinar preparations were rinsed in phosphate-buffered saline, cryoprotected, and frozen with isopentane cooled with liquid nitrogen as previously described (12). Cryostat sections (6-µm thick) were mounted on SuperFrost Plus slides (Fischer Scientific) and processed as previously described in detail for immunofluorscence localization (12). Primary antibodies were rabbit anti-HA polyclonal antibody (diluted 1:1000 to 1:2000) and rat anti-HA monoclonal antibody (diluted 1:100). HA localization patterns were identical with these antibodies; in later experiments, the monoclonal antibody was generally used as it gave a slightly better signal. Secondary antibodies were Cy3-conjugated donkey anti-rabbit IgG (1:200) and anti-rat IgG (diluted 1:200 to 1:600). Slides were viewed with a Zeiss LSM 510 confocal microscope. Digitized images were generally collected as a Z-series (0.5 µm steps). A single image from the Z-stack was then chosen (based on the most informative and sharpest fluorescence) and processed using Photoshop 5.5 software (Adobe Systems Inc., Mountain View, CA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adenovirus-mediated Expression of HA-Rab3D Proteins in Pancreatic Acinar Cells-- Adenovirus-mediated HA-Rab3D expression was examined by Western blotting using anti-HA antibody and found to be viral titer- and incubation time-dependent. When the incubation time was fixed at 9 h, the amount of expression increased with virus titer (Fig. 1 top panel). When the viral titer was fixed at 5 × 106 pfu/ml, expression was detected between 4 and 6 h, was significantly increased at 9 h, and gradually reached a plateau after 12 h (Fig. 1, bottom panel). To obtain adequate overexpression but also preserve acinar polarity and responsiveness, 9 h was used as a standard incubation time period. Since the recombinant adenoviral vector also encodes EGFP under the control of a separate CMV promoter, the efficiency of viral infection can be assessed by monitoring EGFP fluorescence in acinar cells. For the viral titer used in most experiments (5 × 106 pfu/ml), nearly 100% of acini displayed EGFP fluorescence (Fig. 2), although not all individual cells were fluorescent as shown latter by confocal microscopy. These data demonstrate the ability to express exogenous protein in acinar cells using recombinant adenovirus. Next we tested whether adenoviral infection itself affected acinar secretion using adenovirus expressing beta -galactosidase as control (Fig. 3). Control adenoviral infection had no effect on either basal or CCK-induced amylase release within the titer range examined from 5 × 105 to 107 pfu/ml.


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  		Fig. 1.   Adenoviral-mediated HA-Rab3D expression was viral titer- and incubation time-dependent. Isolated mouse pancreatic acini were incubated with various titers of HA-Rab3D adenovirus for 9 h and then acinar lysates were analyzed for Rab3D overexpression by Western blotting (WB) using anti-HA antibody (top panel). In the bottom panel isolated acini were incubated with 5 × 106 pfu/ml HA-Rab3D adenovirus for the indicated period of time and then acinar lysates were analyzed for Rab3D overexpression. Results shown are representative of three independent experiments.


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  		Fig. 2.   Adenovirus efficiently infects isolated pancreatic acini. After a 9-h incubation with 5 × 106 pfu/ml HA-Rab3D adenovirus, acini were placed in a culture dish with a glass coverslip bottom. Fluorescence and bright field images were captured using a ×10 objective with a Spot digital camera. The efficiency of adenoviral infection was estimated by comparing the fluorescent and bright field images within the same field.


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  		Fig. 3.   Control adenovirus infection had no effect on either basal or CCK-induced amylase release. Isolated acini were incubated without or with various titers of beta -galactosidase adenovirus. After a 9-h incubation, acini were resuspended in fresh HR buffer and basal and CCK (30 pM)-induced amylase release was measured over a 30-min period. Results shown are means and S.E. for three to five independent experiments.

Dominant Negative Rab3D Mutants Inhibit CCK-induced Amylase Release-- Three Rab3D mutants were prepared within the conserved sequence motifs that are necessary for guanine nucleotide binding and GTP hydrolysis among all Rab proteins. The mutants T36N (threonine at position 36 substituted by asparagine) and N135I (asparagine at position 135 substituted by isoleucine) were both expected to have reduced affinity for guanine nucleotides (23). The mutant Q81L (glutamine at position 81 substituted by leucine) is deficient in GTP hydrolysis and should persist in the GTP bound conformation for longer time than the wild type protein (23). The expression and GTP binding activity of the wild type and three mutants Rab3D are shown in Fig. 4. When 5 × 106 pfu/ml and 9 h were used as the standard viral titer and incubation time, all four HA-Rab3D proteins were expressed in acinar cells in equivalent amounts (Fig. 4), and were properly isoprenylated as indicated by partitioning into the detergent phase upon Triton X-114 phase separation (data not shown). Consistent with the observations of others (16, 19), Rab3D N135I seemed to have higher mobility on SDS-PAGE than the other three constructs. The expression of the four Rab3D constructs and beta -galactosidase control adenovirus led to equivalent EGFP expression by Western blotting (data not shown). While the wild type and Q81L mutant Rab3D showed strong GTP binding, the other two Rab3D mutants, N135I and T36N, had undetectably low GTP binding activities. The effect of expressing the four Rab3D proteins on amylase release was then compared (Fig. 5). Amylase release from each group, stimulated by 30 pM CCK for the 30-min incubation period, is shown after the basal release had been subtracted. While the expression of wild type Rab3D and the Q81L mutant had no effect, the N135I and T36N mutants inhibited CCK-induced amylase release by 45 ± 1 and 47 ± 3%, respectively. Given their deficiency in GTP binding, the Rab3D mutants, N135I and T36N, thus behave as dominant negative mutants.


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  		Fig. 4.   Expression and GTP binding of wild type and mutant HA-Rab3D. Isolated acini were incubated with 5 × 106 pfu/ml control beta -galactosidase, wild type, and mutant Rab3D adenoviruses for 9 h. Acini were then lysed and wild type and mutant HA-Rab3D immunoprecipitated. After washing, SDS sample buffer was added to each sample which was then heated to 95 °C, divided into two aliquots and loaded on 12% SDS-polyacrylamide gels. One aliquot of each immunoprecipitant was used for Western blotting using anti-HA antibody and the other for [alpha -32P]GTP overlay. Results shown are representative of three independent experiments.


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  		Fig. 5.   Dominant negative Rab3D mutants inhibit CCK-induced amylase release. Isolated acini were incubated with 5 × 106 pfu/ml of either control beta -galactosidase, wild type, or mutant Rab3D adenovirus for 9 h. Amylase release during a subsequent 30 min incubation with or without 30 pM CCK was measured. Basal secretion was subtracted and 30 pM CCK induced secretion was compared among different groups. Results are means and S.E. for three to five independent experiments. **, p < 0.01.

Dominant Negative Rab3D Mutants Inhibit Amylase Release across the Dose-Response Curve and Preferentially during the Early Phase-- Since in normal acini the CCK dose-response curve is biphasic and characterized by supermaximal inhibition of secretion, we determined if the inhibition by dominant negative Rab3D mutants affected the biphasic shape of the CCK dose-response curve. In the cultured acini with control viral infection, amylase release was stimulated by 3 pM CCK, reached a maximum at 30 pM, and showed reduced release (supermaximal inhibition) at 300 pM (Fig. 6). Adenoviral expression of wild type Rab3D had no effect at any CCK concentration, but Rab3D T36N inhibited secretion across the CCK dose-response curve with preservation of the biphasic shape. Dominant negative Rab3D mutant N135I showed identical results as Rab3D T36N (data not shown). It is well known that the time course of stimulated amylase release has two phases with the highest rate of secretion in the early phase (during the first 5-10 min) and thereafter a slower or late phase. We therefore examined whether the inhibition by the dominant negative Rab3D mutants was greater during the early or late phase. Fig. 7 shows that the inhibition was stronger when measured at 5 min (71 ± 5%) than at 30 min (36 ± 5%). In contrast to the early phase of stimulated amylase release which is almost completely blocked, the late phase was unaffected (Fig. 7, inset). A similar result was seen from the Rab3D N135I virus treated group but not from either wild type or the Q81L virus-treated group (data not shown). Thus dominant negative Rab3D mutants preferentially inhibit the early phase of stimulated amylase secretion.


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  		Fig. 6.   Dominant negative Rab3D mutants inhibit amylase release across the CCK dose-response curve. Isolated mouse acini were incubated with 5 × 106 pfu/ml of either beta -galactosidase (open circle ), Rab3D (), or Rab3D T36N (black-down-triangle ) adenovirus for 9 h. Acini were then resuspended in fresh buffer and incubated with various concentrations of CCK for 30 min. Results are mean ± S.E. for three independent experiments. *, p < 0.05.


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  		Fig. 7.   Dominant negative Rab3D mutants inhibit amylase release preferentially during the early phase. Isolated acini were incubated with 5 × 106 pfu/ml of either beta -galactosidase (filled symbols) or Rab3D T36N (open symbols) adenovirus for 9 h. Acini were then resuspended in fresh buffer and incubated with (circles) or without (triangles) 30 pM CCK for the indicated period of time. Results are mean ± S.E. for three independent experiments. When error bars are not shown they were smaller than the size of the symbol. The inset shows 30 pM CCK induced secretion (with basal secretion subtracted) during the first 5 min and during the period from 20 to 30 min comparing the effect of beta -galactosidase (filled bars) and Rab3D T36N (hatched bars) groups. *, p < 0.05.

Dominant Negative Rab3D Mutants Inhibit Amylase Release Induced by Various Secretagogues-- To determine whether the dominant negative inhibition was general or stimulus specific, we studied the inhibitory effect of dominant negative Rab3D mutants on amylase release induced by different agonists including CPT-cAMP (100 µM), carbachol (1 µM), bombesin (300 pM), and calcium ionophore A23187 (2 µM). The amylase release induced by these secretagogues were all inhibited by Rab3D T36N to a similar extent to that induced by CCK (Fig. 8); similar results were seen with Rab3D N135I (data not shown). These results suggest that the inhibition occurs at a late step of stimulus-secretion coupling downstream of intracellular calcium mobilization. To directly rule out the possibility that dominant negative Rab3D inhibited regulated secretion by affecting intracellular calcium signaling, we examined the effect of the dominant negative Rab3D on the intracellular calcium increase in response to CCK stimulation (Fig. 9). Compared with the acini cultured 9 h without virus or infected with beta -galactosidase control virus, neither Rab3D N135I nor T36N virus treatment had an effect on the increase of intracellular calcium in response to maximal CCK stimulation, although cultured acini showed an impaired stimulated calcium response compared with fresh isolated acini where CCK increased intracellular calcium to 577 ± 32 nM in response to 100 pM CCK.


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  		Fig. 8.   Dominant negative Rab3D mutants inhibit amylase release induced by various secretagogues. Acini were incubated with 5 × 106 pfu/ml adenovirus expressing beta -galactosidase (filled bars) or Rab3D T36N (hatched bars) for 9 h. Amylase release induced by various agonists during 30 min was then determined. CCK (30 pM), bombesin (300 pM), carbachol (1 µM), CPT-cAMP (100 µM), or A23187 (2 µM) were used as secretagogues. Results are mean ± S.E. for four independent experiments. *, p < 0.05.


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  		Fig. 9.   Dominant negative Rab3D mutants had no effect on intracellular Ca2+ signaling in pancreatic acinar cells. Isolated acini were incubated without adenovirus or with 5 × 106 pfu/ml of either beta -galactosidase or dominant negative Rab3D mutant adenovirus for 9 h. Acini were then resuspended in HR buffer and incubated with fura-2/AM for 30 min and intracellular calcium concentration was measured in an Attofluor dual wavelength imaging work station. A, representative calcium traces from an acini incubated with beta -galactosidase adenovirus. B, representative calcium traces from an acini incubated with Rab3D T36N adenovirus. C, average basal and peak values of intracellular calcium concentration stimulated with 300 pM CCK. Each result is the mean and S.E. of 6-7 acini from three to four independent experiments. In each acinus intracellular calcium concentration was recorded from 6-10 cells.

Localization of Overexpressed Wild Type and Mutant Rab3D in Pancreatic Acinar Cells-- The cellular distribution of HA-tagged wild type and mutant Rab3D in pancreatic acinar cells was determined by immunofluorescence confocal localization of the HA tag in cryostat sections of acinar cell preparations infected with adenovirus. The adenoviral vectors also encoded EGFP to allow Rab-independent identification of acinar cells infected with virus. In control preparations expressing untagged beta -galactosidase (Fig. 10A), HA immunofluorscence staining was at background levels, requiring high gain to resolve, and was distributed primarily to the basal cytoplasm of acinar cells with little or no signal in apical regions. EGFP fluorescence was diffusely distributed throughout the acinar cell cytoplasm, and ranged from intense, often with even stronger nuclear localization, to relatively weak or absent (insets to Fig. 10A). In cells infected with the wild type HA-Rab3D construct, HA immunofluorescence was present in the apical cytoplasm (Fig. 10, B and D) where it strongly outlined the periphery of individual zymogen granules (Fig. 10, B and B, inset). This pattern of localization is similar to that seen when Rab3D distribution was examined using antibody to Rab3D protein (data not shown), and corresponds to the localization of endogenous Rab3D previously described in acinar cells (12). HA staining was only present in EGFP positive cells, and the intensity of the HA signal generally reflected that of EGFP, such that apical HA staining was proportionately low in cells that poorly expressed EGFP (Fig. 10B). In addition to the localization of HA-Rab3D to granules, a variable amount of diffuse HA staining was present in the basal cytoplasm of acinar cells, the intensity again generally correlating with that of EGFP.


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  		Fig. 10.   Immunofluorescence localization of wild type HA-Rab3D. Immunofluorescence localization of HA (red) in pancreatic acini infected with adenovirus encoding GFP together with untagged beta -galactosidase (A), or with wild type HA-tagged Rab3D (B and D). There was no specific staining for HA in the beta -galactosidase control (A); the insets show the corresponding Nomarski image and EGFP expression, respectively. EGFP was diffusely distributed throughout the cytoplasm and often concentrated in nuclei. Several cells in the interior of this acinus were EGFP negative. Wild type HA-Rab3D was targeted primarily to the secretory granules in the apical cytoplasm of acinar cells (B, overlay of HA and EGFP fluorescence; C, corresponding Nomarski image; D, overlay of B and C). The inset in B shows a higher magnification of the granule localization of HA-Rab3D. HA staining outlines the periphery of granules in the apical region (B and B, inset), consistent with targeting of HA-Rab3D to granule membranes. Several acinar cells in this acinus express EGFP poorly and have a correspondingly low signal for HA granule staining (B and D). Scale bar for: A-D, 25 µm; inset in A, 25 µm; inset in B, 2 µm.

Expression of either the N135I or T36N mutant of Rab3D (Fig. 11, A and B, respectively) gave strikingly different patterns of HA immunofluorescence distribution compared with the predominant zymogen granule localization of the wild type construct. Apical, granule-associated staining was not present with either N135I or T36N mutant. In both constructs, diffuse basal cytoplasmic fluorescence was present to varying degrees of intensity, and was generally more abundant with Rab3D N135I. In a few cells with particularly strong expression of the N135I mutant, appreciable HA staining was present in apical cytoplasm as well, but an unambiguous localization to granule membranes could not be resolved. Although occasional cells in some T36N mutant expressing acini showed a strong EGFP signal accompanied by strong basal cytoplasmic HA staining and an even more intense nuclear localization (data not shown), there was little HA fluorescence in the apical cytoplasm. N135I mutant expressing acinar cells often exhibited a punctate form of HA staining, which was scattered throughout the cytoplasm (Fig. 11A). As shown in Fig. 11A, there was considerable variability in the number of these HA-reactive deposits within an acinus, and some acini lacked this type of expression entirely. Punctate staining was seen only occasionally with T36N mutant. The nature of the cytoplasmic structure corresponding to the punctate staining seen with Rab3D N135I was not explored. However, this punctate staining differed significantly from wild type Rab3D localization in that the latter outlined only the peripheries of the granules and was abundantly distributed to the granules comprising the bulk of apical cytoplasm (see Fig. 10, B and D).


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  		Fig. 11.   Immunofluorescence localization of dominant negative Rab3D mutants, N1235I and T36N. Immunofluorescence localization of HA (red) in pancreatic acini infected with adenovirus encoding EGFP together with Rab3D N135I (A) or Rab3D T36N (B). The insets in A and B show corresponding Nomarski images and EGFP expression. HA-tagged Rab3D N135I was diffusely distributed in the acinar cytoplasm (A) with little evidence of the specific type of granule staining seen with wild type Rab3D. The basal cytoplasm of the acinar cells was generally more strongly stained than the apical region which also contained scattered punctate HA staining in some cells. HA-tagged Rab3D T36N was diffusely distributed in the acinar cytoplasm (B). The granule region exhibited a low signal for HA, whereas the basal cytoplasm was more intensely fluorescent. EGFP negative cells were devoid of HA staining. Scale bar for A and B, and for insets in A and B, 25 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study utilized adenoviral-mediated gene transfer to express wild type and three mutant forms of Rab3D in pancreatic acini and examined the effects of their expression on acinar amylase release. Adenoviral infection has been shown previously (29-31), and confirmed in this study, to be a highly efficient means of gene transfer to pancreatic acinar cells with nearly 100% efficiency. In the current study, isolated mouse pancreatic acini were cultured in vitro for a short period of time and various culture media were tested to obtain optimal conditions for amylase secretion. In most experiments, the 9-h time point was used to obtain adequate expression of Rab3D proteins and also to preserve as much as possible the acinar polarity and secretory responsiveness. Despite the adverse effect of in vitro incubation on acinar secretion, adenoviral infection within the titer range used in this study did not further perturb either the responsiveness or the sensitivity of acinar secretion. Moreover, the intracellular calcium response and endogenous Rab3D localization were also not affected by adenoviral infection. Altogether, adenoviral-mediated gene transfer is an efficient way to overexpress protein in pancreatic acinar cells and thus allows the study of regulated exocytosis using acinar cells as a model.

Four isoforms of Rab3 denoted A through D have been found in different tissues. Rab3D was recently found in pancreatic acinar cells and other exocrine cells (3, 4). Compared with Rab3A which has been intensively studied in recent years, the role of Rab3D in regulated exocytosis is not well understood. Since Rab3D coexpresses with Rab3A in some of the widely used models for studying exocytosis, such as bovine chromaffin cells, PC12 cells, and insulin secreting cells (32), using these models may complicate the study of the role of Rab3D in exocytosis. Pancreatic acini have been utilized extensively as a classical model for regulated exocytosis by nonexcitable cells (1, 2). Previous work has demonstrated that Rab3D is the only detectable Rab3 isoform in pancreatic acinar cells. Therefore pancreatic acini are a good model to study the role of Rab3D in regulated exocytosis.

In pancreatic acini, Onishi et al. (20) reported that overexpression of Rab3D enhanced the initial phase of regulated amylase secretion from pancreatic acini of transgenic mice. In the current study, to further understand the function of Rab3D in regulated exocytosis in acinar cells, we overexpressed in vitro Rab3D mutants that interfere with the Rab3D GTP/GDP cycle in pancreatic acinar cells. The three mutants used were Rab3D N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis) (23, 24) and the effect of their expression on amylase secretion were compared. We found that both Rab3D mutants, N135I and T36N, functioned as dominant negative mutants and inhibited regulated exocytosis in pancreatic acinar cells, while the wild type and Q81L did not have significant effects. This pattern is consistent with the findings previously reported using equivalent mutants in Rab1 (33), Rab7 (34), Rab27a (35), and yeast Rab homologue SEC4p (36), but it differs from the findings of equivalent mutants in Rab3A (15, 37) and Rab2 (33) in which the mutants equivalent to N135I and Q81L were strong inhibitors while the T36N equivalent mutants had no effect. These observations led to the concept that the GTP hydrolysis of Rab3A is the regulatory step and Rab3A acts as a negative regulator in regulated exocytosis. If this is true, the different effects between equivalent Rab3A and Rab3D mutants may reflect the fundamental differences in the regulation and/or function of these individual Rab proteins. In the case of Ras, a consensus has been reached that the dominant-inhibitory mutants work in cells by competing with normal Ras for binding to RasGEFs in their nucleotide-free conformations and form "dead-end" complexes. This prevents the activation of endogenous Ras by RasGEFs (38). The dominant negative Rab mutants are believed to act similarly (34, 35, 39-41). In the case of Sec4p, Dss4, a GDP dissociation stimulator, formed a complex with dominant negative alleles of SEC4, T34N, and N133I, and Dss4 expressed at high copy suppressed dominant negative alleles of SEC4 (40). Moreover, nucleotide-free Ypt mutant proteins inhibited the Ypt GEF activity of TRAPP (41). Since only a few mammalian Rab GEFs have been identified and characterized, there is yet little direct evidence to prove the notion that dominant negative Rab mutants disrupt the function of endogenous Rab by sequestering the corresponding GEF(s). If this hypothesis is true, it implies that the activation of Rab3D, namely GDP/GTP exchange, instead of inactivation, namely GTP hydrolysis, is a rate-limiting step in regulating Rab3D function. This might explain the lack of effect from wild type and Q81L mutant of Rab3D overexpression on amylase secretion. The stimulatory effect observed in Rab3D transgenic mice may be due to a long term secondary effect such as stabilizing Rab3D effector(s) other than a direct effect of Rab3D overexpression. The localization of the dominant negative Rab3D mutants away from the zymogen granules is also more consistent with blocking an upstream regulator (GEF) rather than an effector.

In pancreatic acini, two phases of exocytosis are proposed: an initial phase, which is completed within 5 min of stimulation, and a second phase which is sustained for the duration of agonist stimulation. In analogy to neurotransmission, it is postulated that the first phase represents fusion of docked granules in the vicinity of the plasma membrane, while the second phase is due to release from a reserve pool (42). According to this scheme, our finding that dominant negative Rab3D blocked the first phase of CCK-stimulated amylase secretion from pancreatic acini suggests that Rab3D participates in the first phase of pancreatic acinar exocytosis, which is consistent with the current model for the role of Rabs in tethering/docking (8, 43). The role of Rab3D in ZG tethering/docking near the plasma membrane is supported by the finding that the expression of Rab3D N135I inhibited positioning of dense core granule near the plasma membrane and blocked regulated secretion of mature ACTH in AtT-20 cells (22) and a more recent finding that Rab3D N135I decreased the total granule numbers and the fraction of granules docked to the plasma membrane in undifferentiated PC12 cells (44). An alternative explanation to the selective inhibition of dominant negative Rab3D mutants is that there are two components of amylase release which contribute to two phases of regulated exocytosis. One is Rab3D- dependent and the other is relatively Rab3D-independent. In fact, in acinar cells ample evidence has showed that the first phase of acinar exocytosis is mediated by an increase in [Ca2+]i released from intracellular calcium stores, whereas the second phase is associated with stimulation of PKC (42).

It is well known that the CCK dose-response curve of acinar amylase release has a biphasic shape characterized by supermaximal inhibition presumably due to the binding of CCK to its receptors in low affinity state (29). Our result showed that dominant negative Rab3D inhibited amylase secretion across the CCK dose-response curve, but did not alter the biphasic shape, in other words, dominant negative Rab3D did not affect the supermaximal inhibition by high dose CCK. This result is consistent with the finding in alpha -toxin-permeabilized acini that a high dose of CCK8 (1 µM) did not influence the first phase of Ca2+-dependent amylase secretion, but inhibited the second sustained phase of the response (45).

In conclusion, the current study demonstrates that Rab3D plays an important role in regulating acinar exocytosis. Upon activation, it may recruit its downstream effectors functioning as tethering/docking factors and position ZGs at the apical plasma membrane. Fusion of those ZGs with the apical plasma membrane may contribute to the initial phase of acinar secretion in response to stimuli. To elucidate the pathway(s) through which the Rab3D activity is regulated in acinar cells, it is necessary to identify the upstream regulators of Rab3D, e.g. GEFs. To date, two Rab3 GEFs have been identified: Rab3 GEP (46, 47) and GRAB (48). Whether either or both of these GEFs are expressed in acinar cells are currently being investigated. On the other hand, efforts are also needed to identify the downstream effectors of Rab3D in acinar cells to understand the mechanism by which Rab3D exerts its function and through these interactions to understand the relations between Rab3D and those proteins already known to mediate membrane fusion, such as NSF and SNARE proteins.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK41122 (to J. A. W.), Michigan Gastrointestinal Peptide Center Grant P30 DK34933, and Michigan Diabetes Research and Training Center Grant P60DK20572.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan, 7734 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622. Tel.: 734-764-9456; Fax: 734-936-8813; E-mail: xuequnc@umich.edu.

Published, JBC Papers in Press, March 1, 2002, DOI 10.1074/jbc.M201248200

    ABBREVIATIONS

The abbreviations used are: GEF, guanine nucleotide exchange factor; HA, hemagglutinin; CCK, cholecystokinin; EGFP, enhanced green fluorescent protein; CMV, cytomegalovirus; HR, Hepes-Ringer.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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