Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells

Benjamin C. Calhoun, Lynne A. Lapierre, Catherine S. Chew, and James R. Goldenring

Institute for Molecular Medicine and Genetics, Departments of Medicine, Surgery, and Cellular Biology and Anatomy, Medical College of Georgia, and Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous investigations in several systems have demonstrated that Rab3 family members redistribute to soluble fractions on fusion of secretory granules with target plasma membranes. Rab proteins are then recycled back onto mature secretory vesicles after reinternalization of the membrane. Although this cycle is well established for Rab3, far less is known about redistribution of other Rab proteins during vesicle fusion and recycling. In the gastric parietal cell, Rab11a is associated with H-K-ATPase-containing tubulovesicles, which fuse with the apical plasma membrane (secretory canaliculus) in response to agonists such as histamine. We have analyzed distribution of Rab11a and other tubulovesicle proteins in resting and histamine-stimulated rabbit parietal cells. Stimulation of isolated gastric glands in the presence of 100 µM histamine and 100 µM 3-isobutyl-1-methylxanthine did not cause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a, Rab25, syntaxin 3, and SCAMPs increased immunoreactivity in stimulus-associated vesicles prepared from rabbits treated with histamine compared with those from ranitidine-treated animals. The large GTPase dynamin was found in both vesicle preparations, but there was no change in amount of immunoreactivity. Immunofluorescence staining of resting and histamine-stimulated primary cultures of parietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate that Rab11a does not cycle off the membrane during the process of tubulovesicle fusion with the secretory canaliculus. Thus Rab11a may remain associated with recycling apical membrane vesicle populations.

tubulovesicle proteins; rabbit; dynamin; syntaxin; SNARE proteins

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PROCESS OF VESICLE trafficking within cells is critical to the orderly functioning of intracellular systems. This trafficking requires a series of regulated vesicle fusion and membrane budding and retrieval events at all points along the exocytotic and endocytotic pathways. Recent investigations have stressed the importance of highly conserved mechanisms for membrane vesicle docking at points of vesicle processing. These docking complexes are formed from the association of SNARE proteins on vesicles (v-SNARE) and their target fusion membrane surfaces (t-SNARE) (38). In addition, other proteins are required for the orderly assembly and recognition of the docking complex (25). In particular, members of the Rab small GTP-binding protein family appear to be required for assembly of SNARE complexes (40).

The Rab family of small GTP-binding proteins regulates various stages of vesicle trafficking along the endocytotic and exocytotic pathways (3, 34). Present evidence suggests that Rab proteins are necessary for the orderly fusion of specific vesicle populations with target acceptor membranes. This process has been studied most extensively for the Rab3 subfamily members that are present on small synaptic vesicles and packaged secretory granules from endocrine and exocrine cells. Studies in synaptosomes (14, 27), adrenal chromaffin cells (12), and pancreatic acinar cells (26) have demonstrated that Rab3 isoforms cycle off granule and vesicle membranes during the process of fusion. This process appears to be mediated by the guanine nucleotide dissociation inhibitor (GDI) protein (37, 49). After reinternalization of secretory membranes, Rab3 isoforms appear to recycle back to the mature secretory vesicle membranes (14, 26). This pathway of Rab3 recycling has led to the suggestion that the association of Rab3 isoforms with membranes indicates maturation to secretory-competent vesicles.

In contrast to packaged secretory processes, epithelial cells maintain and regulate the repertoire of apical and basolateral membrane ion channels and transporters through the control of their insertion into plasma membrane surfaces. This mechanism is well established for three critical cAMP-dependent processes: 1) insertion of water channels into the apical membranes of cortical collecting duct cells (30, 32, 39), 2) insertion of cystic fibrosis transmembrane conductance regulator into the apical membranes of colonic and other chloride-secreting epithelia (31, 44), and 3) delivery of the H-K-ATPase to the apical surface of gastric parietal cells (15). In the case of proton-pumping gastric parietal cells, H-K-ATPase molecules are sequestered within a class of intracellular tubulovesicles that lie deep to an extensive invagination of the apical membrane referred to as the intracellular canaliculus (6, 16, 41). On stimulation of an increase in intracellular cAMP by histamine, the tubulovesicle membranes fuse with the canaliculus to deliver the H-K-ATPase to the luminal surface (16, 41, 47). This massive fusion event elicits a fivefold increase in apical plasma membrane surface area, the largest reversible membrane fusion event observed in mammalian cells. Nevertheless, on cessation of the stimulus and return of intracellular cAMP to resting levels, the H-K-ATPase is rapidly reincorporated into tubulovesicles that are competent for another round of fusion (6, 16). This dynamic membrane fusion and retrieval process makes the parietal cell an important model for the study of apical membrane recycling.

Little is known concerning the proteins that regulate apical plasma membrane recycling. Recently, a number of investigations have indicated that Rab11a is involved in the process of recycling to the plasma membrane in nonpolarized and polarized cells. Ullrich et al. (46) showed that Rab11a was associated with the pericentrosomal recycling system responsible for recycling of transferrin to the membrane surface in Chinese hamster ovary cells and fibroblasts. Similar results were reported by Green et al. (23) in K-562 cells. We recently noted that Rab11a and Rab25, which shares 68% sequence identity with Rab11a, are associated with the recycling system in polarized Madin-Darbin canine kidney (MDCK) cells (19). We previously noted the enrichment of Rab11a in gastric parietal cells (21), and more recently we demonstrated that Rab11a and Rab25 are present on immunoisolated H-K-ATPase-containing membranes (7). These results have supported the hypothesis that the tubulovesicles represent an elaboration of the apical epithelial recycling system present in a number of epithelial cells.

We have not been able to document the presence of any Rab3 family member in gastric parietal cells. Thus the parietal cell represents an important system to compare the recycling of Rab11a with that observed in other systems for Rab3 family members. In our original investigations we observed that, in concert with stimulation, Rab11a redistributed into heavier membrane fractions in parallel with the H-K-ATPase (21). In these studies no redistribution of Rab11a into a cytosolic fraction was observed. We have now studied in detail the redistribution of Rab11a along with other tubulovesicle proteins in parietal cells during stimulation. These studies, with use of biochemical and immunocytochemical criteria, suggest that Rab11a does not cycle off the membranes into the cytosol during secretory stimulation.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Male New Zealand White rabbits were obtained from Shelton's Bunny Barn. The monoclonal antibody (MAb) specific for the alpha -subunit of the gastric H-K-ATPase (12.18) was a generous gift from Dr. Adam Smolka (Medical University of South Carolina, Charleston, SC). Affinity-purified anti-syntaxin 3 and anti-syntaxin 4 antibodies were a gift of Mark Knepper (National Institutes of Health) (29). The production of Rab11a- and Rab25-specific MAb has been described elsewhere (20). Mouse monoclonal anti-dynamin (clone 41) was obtained from Transduction Laboratories (Lexington, KY). Anti-mouse IgG-coated magnetic Dynabeads were obtained from Dynal (Great Neck, NY). Fc fragment-specific secondary antibodies conjugated with horseradish peroxidase and Cy5-conjugated secondary antibodies were from Jackson ImmunoResearch Labs (West Grove, PA). Enhanced chemiluminescence (ECL) substrate (SuperSignal) was obtained from Pierce (Rockford, IL). Nonimmune control IgG2b was purchased from Sigma Chemical (St. Louis, MO). Immobilon-P polyvinylidine difluoride membranes were purchased from Millipore (Bedford, MA). Bodipy-phallacidin was obtained from Molecular Probes (Portland, OR). All other reagents were from standard suppliers and were of the highest purity available.

Tubulovesicle preparation. Gastric tubulovesicles were prepared from resting rabbit gastric mucosa, as described by Crothers et al. (10). Briefly, male New Zealand White rabbits were anesthetized by intravenous administration of a mixture of ketamine and xylazine, and their stomachs were perfused under high pressure with oxygenated PBS and removed. The gastric mucosa was scraped off the serosa with a glass slide, minced with scissors, and homogenized in five volumes of homogenization buffer [in mM: 113 mannitol, 37 sucrose, 0.4 EDTA, 5 MES, pH 6.7, 5 benzamidine, and 0.1 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)] plus a protease inhibitor cocktail (in µg/ml: 1.75 aprotinin, 2.5 soybean trypsin inhibitor, 1 chymostatin, pepstatin A, and leupeptin). The homogenate was centrifuged sequentially at 50, 4,000, 14,000, and 100,000 g. The 100,000-g pellet was resuspended in 10% sucrose buffer (5 mM HEPES-NaOH at pH 7.4, 300 mM sucrose) and fractionated over discontinuous sucrose gradients consisting of layers of 20, 27, and 33% sucrose. The vesicles partitioning at the 10-20% sucrose interface were used for immunoadsorption experiments.

Immunoadsorption of tubulovesicles. For a single immunoadsorption experiment, 750 µg of Dynabeads were washed three times in PBS containing 1% BSA, blocked for 30 min at 4°C in PBS-1% BSA, and washed twice in PBS-0.1% BSA. The blocked beads were incubated overnight at 4°C with MAb specific for the alpha -subunit of the gastric H-K-ATPase (12.18) or nonimmune IgG2b. The beads were washed four times for 30 min at room temperature with PBS-0.1% BSA and incubated with tubulovesicles (20 µg of protein) for 2 h at room temperature in PBS-0.1% BSA plus a protease inhibitor cocktail (5 mM benzamidine, 0.1 mM AEBSF, 1.75 µg/ml aprotinin, 2.5 µg/ml soybean trypsin inhibitor, and 1 µg/ml chymostatin, pepstatin A, and leupeptin). After 2 h the unbound material was removed, centrifuged at 20 psi for 5 min at 4°C in an Airfuge (Beckman Instruments, Stanford, CA), and resuspended in SDS sample buffer. The bound material was eluted from the beads in SDS sample buffer, and all samples were heated at 65°C for 5 min. The proteins were separated by SDS-PAGE, electrophoretically transferred to Immobilon-P polyvinylidine difluoride membranes, and analyzed by immunoblotting. The proteins of interest were then detected by incubating the blots sequentially with an Fc fragment-specific secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Labs) and an ECL substrate (SuperSignal). For quantitation, autoradiographs were digitized using an Alpha Innotech image analyzer and Alpha Ease software (San Diego, CA), and integrated densities from three identical experiments were determined. The exposure of the images analyzed by densitometry was monitored using the Saturation Palette of the Alpha Ease software (Alpha Innotech, San Leandro,CA). All images quantitated by densitometry were within the optimal exposure range. Results (means ± SE) are expressed as percent recovery. The statistical significance of the densitometry data was analyzed using the nonparametric t-test.

Rab11a and Rab25 redistribution in gastric glands. Isolated gastric glands were prepared from the fundic mucosa of New Zealand White rabbits as previously described (4, 18). Gastric glands (200 µl of packed glands in 8 ml of media) were incubated in the presence of 100 µM ranitidine or 100 µM histamine with 100 µM 3-isobutyl-1-methylxanthine (IBMX) for 10 min at 37°C in a shaking water bath. At the end of the incubation, glands were rapidly separated from media by centrifugation, and the gland pellet was homogenized on ice in 800 µl of homogenization buffer with 30 strokes of a Dounce homogenizer. The membrane and supernate fractions were then separated rapidly through successive centrifugation at 100 g for 1 min, then the supernate was centrifuged for 5 min in an Airfuge at 150,000 g. The high-speed pellet was resuspended in 500 µl of homogenization buffer. For detection of Rab11a, 50 µg of high-speed supernate protein and 25 µg of membrane protein were resolved on SDS-PAGE gels and transferred to Immobilon-P. For detection of Rab25, 100 µg of high-speed supernate protein and 50 µg of membrane protein were resolved on SDS-PAGE gels and transferred to Immobilon-P. Blots were probed with the anti-Rab11a and anti-Rab25 MAb, and specific labeling was determined using ECL, as described above. Autoradiographic images were quantitated digitally as described above, and Rab11a or Rab25 immunoreactivity in membrane and cytosolic fractions was determined as a percentage of total immunoreactive material (n = 3).

Preparation of gastric stimulus-associated vesicles. Stimulus-associated (SA) vesicles were prepared from New Zealand White rabbits by the method of Crothers et al. (11). Briefly, rabbits were pretreated with 3 mg/kg sc chlorpheniramine maleate 10 min before injection of ranitidine or histamine (2 mg/kg iv). In the case of histamine, three injections were given at 10-min intervals. After treatment, under ketamine-xylazine anesthesia, the abdominal aorta was isolated and the celiac axis was retrograde perfused under high pressure with PBS. The fundic mucosa was scraped from the serosa and homogenized in a Teflon-on-glass homogenizer in five volumes of buffer A (in mM: 113 mannitol, 37 sucrose, 0.4 EDTA, 5 MES, pH 6.7, 5 benzamidine, 0.1 AEBSF) with four strokes at 300 rpm (Master Servodyne, Cole Parmer). The homogenate was centrifuged consecutively at 50 g for 5 min and at 1,000 g for 10 min. The 1,000-g pellet was resuspended in 18% Ficoll in buffer B (in mM: 300 sucrose, 0.2 EDTA, 5 HEPES, pH 7.4) and overlayed with buffer B. The gradient was then centrifuged at 135,000 g for 2 h in an SW28 rotor. SA vesicles were harvested from the interface of the Ficoll and sucrose buffer layers and, after fourfold dilution with buffer B, were pelleted at 135,000 g in a 50.2 Ti rotor for 60 min. Immunoblotting and quantitation were performed as described above.

Immunofluorescence studies of primary cultures of parietal cells. Parietal cells were isolated from New Zealand White rabbits and maintained on Matrigel-coated coverslips in primary culture, as previously described (8, 41). Parietal cells maintained in culture for 24 h were incubated in the presence of 100 µM ranitidine (resting) or 100 µM histamine-50 µM IBMX for 20 min. After stimulation, cells were rapidly washed in PBS and then fixed in 4% paraformaldehyde for 15 min at 4°C. Cells were permeabilized and blocked with 17% goat serum-0.3% Triton X-100 in PBS for 30 min and then incubated with monoclonal murine anti-H-K-ATPase (MAb 12.18 ascites, 1:2,000), monoclonal murine anti-Rab11a (MAb 8H10 ascites, 1:100), or monoclonal murine anti-dynamin (1:1,000; Transduction Laboratories) for 2 h at room temperature. After they were washed in PBS, all cells were incubated with Bodipy-phallacidin (Molecular Probes) for 60 min at room temperature. Cells were then incubated with Cy5-conjugated donkey anti-mouse IgG or donkey anti-rabbit IgG for 30 min. After a final wash, cells were mounted in Prolong Antifade solution (Molecular Probes) and examined with scanning confocal fluorescence microscopy (Molecular Dynamics, Sunnyvale, CA).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Syntaxins on immunoisolated tubulovesicles. We have utilized immunoisolation of rabbit tubulovesicles to identify definitively components of H-K-ATPase-containing vesicles (7). Although syntaxin 1 immunoreactivity was present in gradient-isolated vesicles, no syntaxin 1 was recovered in the immunoisolated vesicles (7). Thus, although we did identify a v-SNARE, VAMP-2, on tubulovesicles, no t-SNARE was definitively identified. Therefore, to obtain further integral membrane markers in tubulovesicles, we investigated the presence of other syntaxins in tubulovesicle fractions immunoisolated with antibodies against the H-K-ATPase alpha -subunit (Fig. 1). The gradient-isolated tubulovesicle fraction contained immunoreactivity for syntaxin 3 and syntaxin 4. Neither syntaxin isoform was recovered in association with beads coated with a nonspecific subclass-matched IgG2b monoclonal antibody. Similarly, although the majority of H-K-ATPase was recovered in association with anti-H-K-ATPase-coated beads, none of the syntaxin 4 was recovered on immune beads. In contrast, syntaxin 3 immunoreactivity was recovered in the immunoisolated vesicles to an extent similar to H-K-ATPase (Table 1). These results in rabbit tubulovesicle membranes are similar to results obtained recently by Peng and colleagues (36) in rat gastric vesicles.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1.   Immunoisolation of tubulovesicles. Enriched gastric tubulovesicles were immunoadsorbed with magnetic beads coated with a nonimmune murine monoclonal IgG2b or murine monoclonal antibodies specific for alpha -subunit of H-K-ATPase (alpha -H/K). Enriched tubulovesicles (TV), immunoadsorbed tubulovesicles (P, pellet), and nonadsorbed material (S, supernate) were analyzed by immunoblotting with monoclonal anti-H-K-ATPase and polyclonal antibodies against syntaxin 3 and syntaxin 4. Syntaxin 3 immunoreactivity was consistently recovered in immunoadsorbed membranes. Syntaxin 4 immunoreactivity was recovered in nonadsorbed fraction. Results are representative of 3 separate experiments and are quantitated in Table 1.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Syntaxins in gastric tubulovesicles

Distribution of Rab11a and Rab25 in resting and stimulated glands. Our earlier investigations suggested that stimulation of parietal cells elicited a redistribution of Rab11a into heavy membrane fractions in parallel with the redistribution of H-K-ATPase (21). In these earlier studies we did not observe an increase in soluble Rab11a during stimulation for 45 min. Because the cycling of Rab proteins on and off membranes may be very rapid, we first sought to investigate the possible movement of Rab11a into the cytosolic fraction during stimulation of tubulovesicle fusion with the canaliculus. We therefore incubated isolated gastric glands in the presence of 100 µM ranitidine (resting) or 100 µM histamine-100 µM IBMX (stimulated) for 10 min, which coincides with the exponential phase of the induction of secretion (1). After incubation, glands were rapidly homogenized and the soluble fraction was separated from whole membranes by centrifugation at 150,000 g. Supernates and membrane pellets were then analyzed for Rab11a content on Western blots to determine distribution. In resting glands only 1.06 ± 0.36% of total gland Rab11a was present in the cytosolic fraction. After 10 min of incubation with ranitidine, we observed 0.90 ± 0.50% of Rab11a in the cytosolic fraction compared with 0.95 ± 0.55% in the cytosol of glands stimulated with a combination of histamine and IBMX. No changes were observed with incubations with histamine and IBMX at any time point from 5 to 45 min (data not shown). When blots were probed for Rab25, no immunoreactivity was detectable in the high-speed supernate fractions from resting or stimulated glands. All the Rab25 immunoreactivity was recovered in the membrane fraction. These findings are similar to those previously published for Rab25 (7), and we have been unable to detect any cytosolic Rab25 in parietal cells. The results suggest that there was no redistribution of Rab11a or Rab25 to the cytosol with maximal stimulation of the parietal cells with a combination of histamine and IBMX.

Movement of tubulovesicle contents into SA vesicles after stimulation. Urushidani and Forte (47) characterized a fraction of SA vesicles isolated from rabbits treated with histamine that are enriched in the canalicular membranes of parietal cells. To examine further the possible distribution of Rab11a during stimulation of parietal cells, we studied the redistribution of Rab11a and other putative components of the tubulovesicle into SA vesicle fractions. Results from a representative experiment are shown in Fig. 2 with quantitation presented in Table 2. As noted previously (47), SA vesicle fractions from stimulated rabbits demonstrate a marked increase in H-K-ATPase compared with fractions from rabbits treated with the histamine-receptor antagonist ranitidine. Rab11a showed a similar increase in immunoreactivity in the SA vesicle fractions from stimulated animals. This increase in immunoreactivity was also noted for syntaxin 3 as well as SCAMPs and Rab25, all of which have been localized to immunoisolated tubulovesicle membranes (7). In contrast, although prominent immunoreactivity was observed for dynamin, a large GTPase associated with membrane endocytosis (13, 48), the concentration of this protein was not significantly altered in membranes from stimulated rabbits.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2.   Enrichment of tubulovesicle proteins into stimulus-associated (SA) vesicles. SA vesicle fractions were isolated from rabbits treated with ranitidine (R, resting) or histamine (S, stimulated). Protein (25 µg) from resting and stimulated SA vesicles was analyzed using immunoblotting with monoclonal antibodies against H-K-ATPase, Rab11a, VAMP-2, Rab25, and dynamin and polyclonal antibodies against syntaxin 3. Immunoreactivity for H-K-ATPase, Rab11a, VAMP-2, syntaxin 3, and Rab25 increased in SA vesicle fraction prepared from rabbits stimulated with histamine. Although a prominent immunoreactivity for dynamin was observed in both preparations, there was no significant change with stimulation. Results are representative of 3 separate preparations and are quantitated in Table 2.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Redistribution of tubulovesicular antigens in SA vesicle fractions

Immunocytochemical redistribution of Rab11a during parietal cell stimulation. We and others reported that the redistribution of H-K-ATPase to the canalicular membrane of stimulated parietal cells can be demonstrated immunocytochemically in preparations of isolated glands and cultured parietal cells (24, 41). We therefore sought to compare the redistribution of H-K-ATPase to the F-actin-containing canaliculus with that for Rab11a. We utilized primary cultures of rabbit parietal cells, because the canalicular architecture is simplified with internalization of the canalicular membrane as a large intracellular vesicle structure that is clearly separable from the tubulovesicle population (41). Figure 3 demonstrates in cultured parietal cells that stimulation with histamine elicits a redistribution of Rab11a immunoreactivity from an intracellular punctate distribution to a more linear staining pattern colocalizing with F-actin staining. As previously described (41), a similar pattern was observed for H-K-ATPase with redistribution of staining from an intracellular punctate pattern to one that coincided with F-actin.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 3.   Immunocytochemical localization of H-K-ATPase, Rab11a, and dynamin in resting and stimulated cultured parietal cells. Cultured parietal cells treated with ranitidine (a, b, e, f, i, and j) or histamine and 3-isobutyl-1-methylxanthine (c, d, g, h, k, and l) were dual stained with Bodipy-phallacidin (b, d, f, h, j, and l) along with specific antibodies against Rab11a (a and c), H-K-ATPase (e and g), or dynamin (i and k). Although immunostaining for H-K-ATPase and Rab11a showed a punctate intracellular distribution in resting cells, it colocalized with F-actin staining in stimulated cells. Dynamin staining was present on the canaliculus in resting and stimulated cells. Images are representative 0.29-µm confocal fluorescence sections. Scale bar, 2 µm.

Because of the prominent immunoreactivity observed for dynamin in SA vesicle fractions, we also investigated the distribution of dynamin by immunocytochemistry. In resting gastric glands, dynamin immunoreactivity was enriched in parietal cells and completely colocalized with the F-actin of the intracellular canaliculus (Fig. 3, i and j). The dynamin immunoreactivity remained associated with the canaliculus, colocalizing with F-actin staining, in histamine-stimulated parietal cells.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The coordination of vesicle trafficking is critical to the dynamic processes within all cells. Inherent in these pathways are the orderly vectorial fusion and retrieval of membrane vesicles. Although SNARE proteins may mediate the interaction of fusing membranes (25, 38), it appears likely that other factors are necessary to account for the orderly recycling of membrane domains. The results presented here and in previous investigations (7, 9, 36) establish the trafficking of the parietal cell H-K-ATPase as a critical model for regulated apical recycling with discrete localization of SNARE proteins, Rab proteins, and apical endocytotic machinery.

Forte et al. (16) first proposed the membrane recycling hypothesis for parietal cell secretion in 1977. Studies from Peng and colleagues (36) in rat and from our own laboratory in rabbit (7) demonstrated the presence of v- and t-SNARE proteins on tubulovesicle membranes. SNARE proteins have been implicated in the assembly of a critical docking complex necessary for vesicle-membrane fusion (38). We previously demonstrated the presence of VAMP-2, a v-SNARE protein, on immunoisolated tubulovesicles. The present data demonstrate the presence of syntaxin 3, but not syntaxin 4, on immunoisolated tubulovesicles. These results are similar to those observed in rat tubulovesicle membranes (36). Thus, although immunoreactivity for syntaxins 1, 2, 3, and 4 can be documented in parietal cells, only syntaxin 3 is present on tubulovesicles. It is not clear which of the syntaxins is present on the canalicular target membranes. However, we have noted localization of syntaxin 4 immunoreactivity to basolateral membranes in isolated gastric glands and cultured parietal cells, as assessed by immunocytochemistry (unpublished results). Unfortunately, none of the other antibodies presently available have allowed definitive immunocytochemical localization. Still, it is of interest to note that syntaxin 3 has been localized to the basolateral membrane in collecting duct cells (29), whereas it is present in the apical membranes of MDCK cells (28) and on zymogen granules in pancreatic acinar cells (17). Thus syntaxin localization may be more complex than previously understood. Nevertheless, the presence of SNARE proteins on tubulovesicles lends further credence to the notion that vesicle fusion mediates the regulated insertion of the H-K-ATPase into the apical membranes of parietal cells.

Previous investigations have hypothesized that the cycling of Rab proteins on and off their associated vesicle membranes is critical for their function. These studies have relied substantially on the analysis of Rab3 family members in neuronal, endocrine, and exocrine systems (12, 14, 26, 27). Although the predominance of reports supports the dissociation of Rab3 from vesicle membranes during the process of vesicle fusion, Bielinski et al. (5) were unable to observe any movement of Rab3a into a cytosolic fraction with depolarization of synaptosomes. Nevertheless, the vast majority of these studies suggested a model for Rab function in which Rab proteins would cycle off vesicles on fusion with target membranes (33). Because Rab-GDI can remove Rab proteins from membranes (37, 45, 49), by implication it has been hypothesized that Rab-GDI would cycle Rab proteins back to mature secretory vesicles through a cytosolic intermediate.

The findings presented here suggest a model for the function of Rab11a different from that for Rab3. The data presented here show more definitively that Rab11a traffics to the canalicular membrane in a sustained fashion without detectable cycling through the cytosol. Isolation of SA vesicles demonstrated that all the tubulovesicle-associated membrane proteins showed similar increases in immunoreactivity after stimulation of secretion in vivo with histamine. Immunoreactivity for Rab11a and Rab25, as well as for SCAMPs and syntaxin 3, showed increases similar to that for H-K-ATPase. Furthermore, immunocytochemical examination of stimulated parietal cells in primary culture also demonstrated a clear and sustained redistribution of Rab11a immunoreactivity to the canalicular membrane. Unfortunately, antibodies against other tubulovesicle components are not of suitable sensitivity for immunocytochemical analysis, so we have not been able to validate a similar redistribution for other tubulovesicle components. These results indicate that Rab11a does not redistribute to the cytosol during tubulovesicle fusion with the intracellular canaliculus.

Recent investigations have implicated Rab11a as a marker for plasma membrane recycling systems. Ullrich et al. (46) and others (23) demonstrated that Rab11a is associated with the plasma membrane recycling system in nonpolarized cells. In polarized cells the analogous structure, the apical recycling system, is responsible for processing of apical recycling and basolateral-to-apical transcytosis (2). We also recently observed that Rab11a and Rab25 are associated with apical membrane recycling systems in polarized MDCK cells (19). Thus the presence of Rab11a and Rab25 on tubulovesicles indicates that this vesicle population is part of an active recycling system. In the case of parietal cells, however, this recycling appears to be highly regulated along its exocytic and endocytic pathways.

The results presented above also provide the first demonstration of dynamin in gastric parietal cells. Dynamin is a large GTPase, and its activity is required for clathrin-dependent endocytosis. Although dynamin is not necessary for the assembly of coatamer complex, it does appear to act as a critical regulator of vesicle budding back from the plasma membrane (13, 48). Thus oligomers of dynamin may form "rings" that pinch off vesicle buds during reinternalization (42, 43). Although previous investigations have failed to identify classical coated vesicles in parietal cells (6, 16), clathrin immunoreactivity is present in parietal cells, and gamma -adaptin immunoreactivity is associated with a population of tubulovesicles (35). In addition, the parietal cell coatamer complex associates with the beta -subunit of H-K-ATPase (35). These results, along with the presence of a prominent concentration of dynamin at the canalicular surface, implicate the canaliculus as a site for membrane recycling. Indeed, Gottardi and Caplan (22) noted that the beta -subunit of H-K-ATPase contains a critical endocytotic recognition sequence of FRHY in its cytoplasmic tail. In transgenic animals expressing a mutation of the critical tyrosine, the proton pump was present constitutively on the canalicular surface (9). All these results demonstrate the importance of membrane recycling to the proper functioning of the parietal cell.

In summary, the results presented here indicate that Rab11a does not cycle off tubulovesicle membranes of parietal cells during fusion with the apical target surface. In contrast to the results obtained with Rab3 family members, the Rab proteins associated with apical recycling in polarized epithelial cells may remain with the component vesicle membranes during the process of fusion and likely retrieval. Further studies are required to discern whether similar principles are also at work in other systems of regulated epithelial ion transporter recycling to the apical membrane of polarized epithelial cells.

    ACKNOWLEDGEMENTS

We thank Dr. David Castle for the gift of antibody and Dr. Mark Knepper for sharing results before publication.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48370 and DK-43405 and a Veterans Administration Merit Award (J. R. Goldenring) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31900 (C. S. Chew)

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. §1734 solely to indicate this fact.

Address for reprint requests: J. R. Goldenring, Institute for Molecular Medicine and Genetics, CB-2803, Medical College of Georgia, 1120 Fifteenth St., Augusta, GA 30912-3175.

Received 21 January 1998; accepted in final form 8 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adrian, T. E., J. R. Goldenring, M. Oddsdottir, M. J. Zdon, K. A. Zucker, J. J. Lewis, and I. M. Modlin. A micro-method for the assay of cellular secretory physiology: application to rabbit parietal cells. Anal. Biochem. 182: 346-352, 1989[Medline].

2.   Apodaca, G., L. A. Katz, and K. E. Mostov. Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J. Cell Biol. 125: 67-86, 1994[Abstract].

3.   Balch, W. Small-GTP-binding proteins in vesicular transport. Trends Biochem. Sci. 15: 473-477, 1990[Medline].

4.   Berglindh, T., and K. J. Obrink. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol. Scand. 96: 150-159, 1976[Medline].

5.   Bielinski, D. F., H. Y. Pyun, K. Linko-Stentz, I. G. Macara, and R. E. Fine. Ral and Rab3A are major GTP-binding proteins of axonal rapid transport and synaptic vesicles and do no redistribute following depolarization-stimulated synaptosomal exocytosis. Biochim. Biophys. Acta 1151: 246-256, 1993[Medline].

6.   Black, J. A., T. M. Forte, and J. G. Forte. Structure of oxyntic cell membranes during conditions of rest and secretion of HCl as revealed by freeze-fracture. Anat. Rec. 196: 163-172, 1980[Medline].

7.   Calhoun, B. C., and J. R. Goldenring. Two Rab proteins, VAMP-2 and SCAMPs, are present on immunoisolated gastric tubulovesicles. Biochem. J. 325: 559-564, 1997[Medline].

8.   Chew, C. S., M. Ljungstrom, A. Smolka, and M. R. Brown. Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G254-G263, 1989[Abstract/Free Full Text].

9.   Courtois-Coutry, N., D. Rousch, V. Rajendran, J. B. McCarthy, J. Geibel, M. Kashgarian, and M. J. Caplan. A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion. Cell 90: 501-510, 1997[Medline].

10.   Crothers, J. M., Jr., D. C. Chow, and J. G. Forte. Omeprazole decreases H+-K+-ATPase protein and increases permeability of oxyntic secretory membranes in rabbits. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G231-G241, 1993[Abstract/Free Full Text].

11.   Crothers, J. M., Jr., W. W. Reenstra, and J. G. Forte. Ontogeny of gastric H+-K+-ATPase in suckling rabbits. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G913-G921, 1990[Abstract/Free Full Text].

12.   Darchen, F., A. Zahraoui, F. Hammel, M. P. Monteils, A. Tavitian, and D. Scherman. Association of GTP-binding protein Rab3A with bovine adrenal chromaffin granules. Proc. Natl. Acad. Sci. USA 87: 5692-5696, 1990[Abstract].

13.   De Camilli, P., K. Takie, and P. S. McPherson. The function of dynamin in endocytosis. Curr. Opin. Neurobiol. 5: 559-565, 1995[Medline].

14.   Fischer von Mollard, G., T. C. Sudhof, and R. Jahn. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349: 79-80, 1991[Medline].

15.   Forte, J. G., and D. X. Yao. The membrane recruitment-and-recycling hypothesis of gastric HCl secretion. Trends Cell Biol. 6: 45-48, 1996.

16.   Forte, T. M., T. E. Machen, and J. G. Forte. Ultrastructural changes in oxyntic cells associated with secretory function: a membrane recycling hypothesis. Gastroenterology 73: 941-955, 1977[Medline].

17.   Gaisano, H. Y., M. Ghai, P. N. Malkus, L. Sheu, A. Bouquillon, M. K. Bennett, and W. S. Trimble. Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells. Mol. Biol. Cell. 7: 2019-2027, 1996[Abstract].

18.   Goldenring, J. R., V. A. Asher, M. F. Barreuther, J. J. Lewis, S. M. Lohmann, U. Walter, and I. M. Modlin. Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G763-G773, 1992[Abstract/Free Full Text].

19.   Goldenring, J. R., S. Bhartur, J. Navarre, X. Wang, and J. Casanova. Rab11 and Rab25 localize to the apical recycling system in MDCK cells. Mol. Biol. Cell. 8: 408a, 1997.

20.   Goldenring, J. R., J. Smith, H. D. Vaughan, P. Cameron, W. Hawkins, and J. Navarre. Rab11 is an apically located small GTP-binding protein in epithelial tissues. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G515-G525, 1996[Abstract/Free Full Text].

21.   Goldenring, J. R., C. J. Soroka, K. R. Shen, L. H. Tang, W. Rodriguez, H. D. Vaughan, S. A. Stoch, and I. M. Modlin. Enrichment of rab11, a small GTP-binding protein, in gastric parietal cells. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G187-G194, 1994[Abstract/Free Full Text].

22.   Gottardi, C. J., and M. J. Caplan. An ion-transporting ATPase encodes multiple apical localization signals. J. Cell Biol. 121: 283-293, 1993[Abstract].

23.   Green, E. G., E. Ramm, N. M. Riley, D. J. Spiro, J. R. Goldenring, and M. Wessling-Resnick. Rab11 is associated with transferrin-containing recycling compartments in K562 cells. Biochem. Biophys. Res. Commun. 239: 612-616, 1997[Medline].

24.   Hanzel, D., T. Urushidani, W. R. Usinger, A. Smolka, and J. G. Forte. Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G1082-G1089, 1989[Abstract/Free Full Text].

25.   Hay, J. C., and R. H. Scheller. SNAREs and NSF in targetted membrane fusion. Curr. Opin. Cell Biol. 9: 505-512, 1997[Medline].

26.   Jena, B. P., F. D. Gumkowski, E. M. Konieczko, G. F. von Mollard, R. Jahn, and J. D. Jamieson. Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis. J. Cell Biol. 124: 43-53, 1994[Abstract].

27.   Johnston, P. A., B. T. Archer, K. Robinson, G. A. Mignery, R. Jahn, and T. C. Sudhof. Rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence. Neuron 7: 101-109, 1991[Medline].

28.   Low, S.-H., S. J. Chapin, T. Weimbs, L. G. Komuves, M. K. Bennett, and K. E. Mostov. Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol. Biol. Cell 7: 2007-2018, 1996[Abstract].

29.   Mandon, B., S. Nielson, B. K. Kishore, and M. A. Knepper. Expression of syntaxins in rat kidney. Am. J. Physiol. 273 (Renal Physiol. 42): F718-F730, 1997[Medline].

30.   Marples, D., M. A. Knepper, E. I. Christensen, and S. Nielsen. Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct. Am. J. Physiol. 269 (Cell Physiol. 38): C655-C664, 1995[Abstract].

31.   Morris, A., and R. A. Frizzell. Vesicle targetting and ion secretion in epithelial cells: implications for cystic fibrosis. Annu. Rev. Physiol. 56: 371-397, 1994[Medline].

32.   Nielsen, S., C.-L. Chou, D. Marples, E. I. Christensen, B. K. Kishore, and M. A. Knepper. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc. Natl. Acad. Sci. USA 92: 1013-1017, 1995[Abstract].

33.   Novick, P., and M. Zerial. The diversity of Rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9: 496-504, 1997[Medline].

34.   Nuoffer, C., and W. E. Balch. GTPases: multifunctional molecular switches regulating vesicular traffic. Annu. Rev. Biochem. 63: 949-990, 1994[Medline].

35.   Okamoto, C. T., S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. R. Goldenring. Identification of clathrin adapters on tubulovesicles of gastric acid secretory (oxyntic) cells. Am. J. Physiol. 274 (Cell Physiol. 43): C1017-C1029, 1998[Abstract/Free Full Text].

36.   Peng, X.-P., X. Yao, D.-C. Chow, J. G. Forte, and M. K. Bennett. Association of syntaxin 3 and vesicle associated membrane protein (VAMP) with H+/K+-ATPase-containing tubulovesicles in gastric parietal cells. Mol. Biol. Cell 8: 399-407, 1997[Abstract].

37.   Raffaniello, R. D., J. Lin, and J. P. Raufman. Actions and expression of RAB-GDP dissociation inhibitor in dispersed chief cells from guinea pig stomach. Biochem. Biophys. Res. Commun. 225: 232-237, 1996[Medline].

38.   Rothman, J. E., and G. Warren. Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr. Biol. 4: 220-233, 1994[Medline].

39.   Sabolic, L., T. Katsura, J. M. Verbabatz, and D. Brown. The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats. J. Membr. Biol. 143: 165-177, 1995[Medline].

40.   Sogaard, M., K. Tani, R. R. Ye, S. Geromanos, P. Tempst, T. Kirchhausen, J. E. Rothman, and T. Sollner. A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell 78: 937-948, 1994[Medline].

41.   Soroka, C. J., C. S. Chew, I. M. Modlin, D. Hanzel, A. Smolka, and J. R. Goldenring. Characterization of membrane and cytoskeletal compartments in cultured parietal cells using immunofluorescence and confocal microscopy. Eur. J. Cell Biol. 60: 76-87, 1993[Medline].

42.   Takei, K., P. S. McPherson, S. L. Schmid, and P. DeCamilli. Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 374: 186-192, 1995[Medline].

43.   Takei, K., O. Mundigl, L. Daniell, and P. De Camilli. The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin. J. Cell Biol. 133: 1237-1250, 1996[Abstract].

44.   Tousson, A., C. M. Fuller, and D. J. Benos. Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments. J. Cell Sci. 109: 1325-1334, 1996[Abstract/Free Full Text].

45.   Ullrich, O., H. Horiuchi, C. Bucci, and M. Zerial. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 368: 157-160, 1994[Medline].

46.   Ullrich, O., S. Reinsch, S. Urbe, M. Zerial, and R. G. Parton. Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135: 913-924, 1996[Abstract].

47.   Urushidani, T., and J. G. Forte. Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): G458-G465, 1987[Abstract/Free Full Text].

48.   Vallee, R. B., and P. M. Okamoto. The regulation of endocytosis: identifying dynamin's binding partners. Trends Cell Biol. 5: 43-47, 1995.

49.   Yang, C., V. I. Slepnev, and B. Goud. Rab proteins form in vivo complexes with two isoforms of the GDP-dissociation inhibitor protein (GDI). J. Biol. Chem. 269: 31891-31899, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(1):C163-C170
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society