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J. Biol. Chem., Vol. 278, Issue 38, 36470-36475, September 19, 2003

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Role of ADP-ribosylation Factor 6 (ARF6) in Gastric Acid Secretion^*

Jun Matsukawa , Kazuhisa Nakayama , Taku Nagao ¶, Hidenori Ichijo  and Tetsuro Urushidani  ¶ ||

From the Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, the Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, and the ^¶National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

Received for publication, May 23, 2003 , and in revised form, July 10, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg²⁺-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Activation of acid secretion in parietal cell involves mainly two steps: (a) translocation of the intracellular vesicles containing proton pump, so-called tubulovesicles or tubulocisternae, to the apical membrane, called intracellular canaliculus, and (b) the acquisition of potassium and chloride permeabilities, essential for operation of the pump, on the canalicular membrane. On the molecular level, this process is still unclear, but evidence has been found that suggests that regulated membrane trafficking and fusion events play the central role (1, 2). It is widely accepted that intracellular membrane traffic requires small monomeric GTPases (small G proteins), especially rab family proteins in various secretory cells (3). On the other hand, there are some observations opposing this idea. It was previously reported that GTPS¹ strongly inhibited gastric acid secretion in -toxin (4) or -escin-permeabilized glands (5). These data indicate that the one or more G proteins that regulate membrane traffic in parietal cell require GTP hydrolysis for their function. Rab family proteins cannot explain the inhibitory effect of GTPS, because they do not require GTPase activity to promote membrane transport, and non-hydrolysable GTP analogues always work as agonist (3). For these, we hypothesized that there could be one or more G proteins other than rab family that regulate membrane traffic in gastric parietal cell.

ADP-ribosylation factor (ARF) family proteins are known to regulate endocytosis, exocytosis, and membrane recycling in various secretory cells. In particular, they need their GTP-hydrolyzing activity to promote membrane transport and therefore a non-hydrolysable analogue of GTP inhibits their functions (3). We previously reported that brefeldin A, an inhibitor of ARF-guanine nucleotide exchange factor (GEF), cannot inhibit acid secretion (5). These observations interested us in ARF6, because GEF proteins for ARF6 (including ARF nucleotide-binding site opener (ARNO) and cytohesin) are known to be insensitive to this inhibitor. Although ARF1 is mainly localized to the Golgi complex and is a common regulator of non-clathrin and clathrin coat recruitment, ARF6 regulates endocytosis or exocytosis with the organization of the actin cytoskeleton, and does not co-localize with Golgi (3, 6). ARF6 is considered to be a feasible target for GTPS, so we performed the present experiments to elucidate its possible role in regulation of membrane traffic and acid secretion in parietal cell.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Anti-ARF6 mouse monoclonal antibody (3A-1) was purchased from Santa Cruz Biotechnology. Anti-H⁺,K⁺-ATPase -subunit rat polyclonal antibody was raised as described previously (7). The construction of an expression vector for C-terminally HA-tagged ARF6 (pcDNA3-ARF6-HA) mutant (wild type, Q67L, N122I) was described previously (8, 9). Myristoylated (myr) N-terminal peptide of ARF6 (myr-GKVLSKIFGNKE) and its reversed sequence (myr-EKNGFIKSLVKG) were custom synthesized by Qiagen. All the chemicals were reagent grade and obtained from Sigma, Nacalai Tesque, or Invitrogen except where otherwise noted.

Isolation of Gastric Glands and Preparation of Permeabilized Glands—Gastric glands were isolated from Japanese White rabbits (Shiraishi, Tokyo, Japan) essentially by the method of Berglindh (10). Isolated glands, suspended in the normal medium containing (in millimolar) 132.6 NaCl, 5 Na₂HPO₄, 1 NaH₂PO₄, 5.4 KCl, 1.2 MgSO₄, 1.0 CaCl₂, 25 HEPES-Na, pH 7.4, and 11.1 glucose with 1 mg/ml bovine serum albumin, were washed and suspended in a high K⁺ medium containing (in millimolar) 20 NaCl, 100 KCl, 1.0 MgSO₄, 0.5 EGTA, 2 ATP, 10 sodium pyruvate, and 20 HEPES, pH 7.4. The free Ca²⁺ concentration in the high K⁺ medium was calculated to be as high as 90 nM by the computer program Chelator, assuming that the contaminated Ca²⁺ in the medium was as high as 10 µM. The formation of homogenous pores in the plasma membrane of gastric cells by -escin was achieved by the modified cold incubation method as described (5). The permeabilized glands were used immediately for assay without further incubation. Acid secretion of the glands was monitored by accumulation of a weak base, [¹⁴C]aminopyrine, setting water content of the glands at a constant of 2.0 ml/mg dry wt (5). The medium used for permeabilized glands was the high K⁺ medium described above. To avoid the possible involvement of endogenous histamine, 100 µM cimetidine was always included, except when the glands were stimulated by histamine.

Subcellular Fractionation of the Glands—Subcellular fractionations were prepared from the homogenate as described by Urushidani and Forte (11) with a slight modification. For Mg²⁺-dependent distribution assay, isolated glands were homogenized in the Mg²⁺-containing buffer, containing (in millimolar) 250 sucrose, 25 HEPES, 2.5 MgSO₄, pH 7.4, or in the Mg²⁺-free buffer, containing (in millimolar) 250 sucrose, 25 HEPES, 0.5 EDTA, pH 7.4. For GTPS-dependent distribution assay, -escin-permeabilized glands were incubated at 37 °C for 30 min in the high K⁺ medium in the presence of 100 µM cimetidine or 100 µM GTPS, and homogenized in the Mg²⁺ containing buffer. To examine the subcellular distribution of ARF6, each fraction was analyzed with SDS-PAGE according to Laemmli (12) and then blotted on a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) with a semidry apparatus. The membrane was probed with the anti-ARF6 monoclonal antibody and visualized by chemiluminescence (Renaissance Western blot chemiluminescence reagent, PerkinElmer Life Sciences) with the use of horseradish peroxidase conjugated anti-mouse IgG as a second antibody.

Immunostaining—The isolated glands were fixed with 10% formalin, permeabilized with 0.1% Triton X-100, and incubated with anti-ARF6 monoclonal antibody (1:30) or anti-H⁺,K⁺-ATPase -subunit rat polyclonal antibody (7). These glands were visualized by Cy3-anti-mouse IgG (1:50) and fluorescein isothiocyanate-anti-rat IgG (1:100), and examined by microscopy (Nikon Eclipse TE300) using a confocal laser scanning system (µRadiance, Bio-Rad). Negative controls using control mouse IgG as the first antibody showed signals lower than the detection limit under the conditions presently employed.

Cell Culture—Gastric glands were cultured basically according to the method described by Chew et al. (13) with slight modifications (14). Isolated gastric glands were washed four times and incubated for 10 min in medium B (Dulbecco's modified Eagle's medium/F-12, 2 mg/ml bovine serum albumin, 10 mM glucose, 100 µg/ml gentamicin, pH 7.4) plus 25 µg/ml amphotericin B, to prevent yeast infection. After the glands were washed once in medium A (medium B plus 8 nM EGF, 10 nM hydrocortisone, 800 nM insulin, 3.1 nM sodium selenite, 2.6 µg/ml transferrin, 0.93 µg/ml ethanolamine, 5 µg/ml Geneticin, 8 µg/ml amphotericin B), glands were incubated for 30 min in 10% fetal bovine serum/Dulbecco's modified Eagle's medium-pre coated flask to exclude fibroblasts. Plating onto Matrigel (Collaborative Biomedical, diluted 1:7)-coated coverslips followed, and cells were thereafter incubated at 37 °C in culture medium A.

Construction and Use of ARF6 Adenovirus—ARF6-HA (wild type, Q67L, N122I) plasmids in pcDNA3 were used to create a recombinant adenovirus with the AdEasy vector system (15). First, the ARF6-HA cDNA was isolated from its plasmid by HindIII and XbaI, and the insert DNA was ligated into the pShuttle-CMV plasmid. This pShuttle-CMV: ARF6-HA recombinant plasmid was linearized and recombined with the pAdEasy plasmid in Escherichia coli. Purified pAdEasy:ARF6-HA plasmids were then digested with PacI endonuclease and transfected into low passage HEK293 cells using SuperFect transfection reagent (Qiagen). Control cells were infected with vector expressing enhanced green fluorescent protein (EGFP). The cultured gastric glands (3.5 x 10⁴ cells/well) were infected with viruses at a multiplicity of infection of 300. Forty hours after infection, the cells were used for aminopyrine uptake assay (14).

Statistical Analysis—Parametric data are expressed as means ± S.E. Multiple comparisons were analyzed by analysis of variance and Fisher's post hoc test with the use of a computer program (Super ANOVA, Abacus Concepts, Berkeley, CA). The level of significance was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effects of GTP and GTPS on Gastric Acid Secretion in -Escin-permeabilized Gastric Glands—As shown in Fig. 1, we found that GTP in the range of 0.3–3 mM dose-dependently stimulated aminopyrine accumulation in -escin-permeabilized gastric glands. The secretagogue effect of 100 µM cAMP was completely inhibited by 100 µM GTPS. These data indicate that one or more G proteins, of which the GTP hydrolysis cycle is essential for activation, are involved in acid secretion.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Opposite effects of GTP and GTPS on [14C] aminopyrine accumulation in -escin-permeabilized gastric glands. Gastric glands were permeabilized with 50 µM -escin, and aliquots were assayed for aminopyrine accumulation for 30 min at 37 °C in the presence of indicated secretagogues. Values are means ± S.E. of three to four experiments performed in a duplicate manner. **, significantly different from cAMP alone at p < 0.01.

Cellular Localization of ARF6 in Gastric Glands—If ARF6 has a role in regulation of membrane traffic and acid secretion in gastric parietal cell, it should exist in the cell. Rabbit isolated gastric glands were incubated with 100 µM cimetidine (resting) or 100 µM histamine plus 30 µM isobutylmethylxanthine (maximally stimulated), fixed with formalin, and stained with anti-ARF6 monoclonal antibody (Fig. 2). ARF6 mainly located in parietal cells, but not in chief cells, in the gastric gland. Within the parietal cell, ARF6 showed a relatively even distribution (with little membranous structure) in the resting state, whereas it took a more membranous appearance upon stimulation. This point will be developed in the following section. We also concluded that any changes in ARF6 could be interpreted as changes in parietal cells even when using the heterogenous preparation of gastric glands.

View larger version (50K): [in this window] [in a new window]   FIG. 2. Cellular localization of ARF6 in rabbit gastric glands. Isolated rabbit gastric glands were fixed, permeabilized, and probed with anti-ARF6 monoclonal antibody (1/30). The first antibody was visualized by Cy3-anti-mouse antibody (1/50). A, resting rabbit gastric gland (incubated with 100 µM cimetidine for 15 min at 37 °C) with the positions of parietal cell (a) and chief cell (b) are shown. Note that ARF6 is almost exclusively exists in parietal cell. B, stimulated gastric gland (incubated with 100 µM histamine plus 30 µM isobutylmethylxanthine for 15 min at 37 °C) is shown. Note that the staining pattern of ARF in stimulated parietal cell appears more membranous than that in resting ones.

To determine the intracellular distribution of ARF6, gastric glands were homogenized and fractionated into apical membrane-rich, tubulovesicle-rich, and cytosolic fractions followed by immunoblotting using anti-ARF6 antibody (Fig. 3). Since Gaschet and Hsu (16) reported that the intracellular distribution of ARF6 between membrane and cytosol was sensitive to the concentration of Mg²⁺, we used either Mg²⁺-containing or Mg²⁺-free EDTA-containing buffer for homogenization. ARF6 was harvested mainly (70%) in the cytosolic fraction in Mg²⁺-containing buffer, whereas in Mg²⁺-free buffer, it distributed to the membrane fractions. The content of ARF6 was increased 2-fold in the tubulovesicles, whereas that was 3-fold in the apical membrane, suggesting its preference to the latter. To confirm this observation, we performed immunohistochemistry of -escin-permeabilized gastric glands. In the presence of Mg²⁺, ARF6 showed a diffuse staining pattern in the cytosol of parietal cell (Fig. 4A). Because Mg²⁺ concentration in mammalian cells is approximately a few millimolar, this staining pattern of ARF6 could reflect physiological localization. When the permeabilized glands were treated with membrane-impermeable Mg²⁺ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), the staining of ARF6 appeared to accumulate in the membranous structure, and that is characteristic for apical intracellular canaliculi (Fig. 4B, arrows).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Subcellular distribution of ARF6. Gastric glands were homogenized either in the presence of Mg2+ (2.5 mM) or EDTA (0.5 mM). The homogenate was fractionated into apical membrane-rich (AM), tubulovesicle-rich (TV), and cytosolic (C) fractions followed by immunoblotting using anti-ARF6 antibody. The amount applied to the gel was set proportional to the suspended sample volume such that the density of the bands expressed the distribution within the total. A, a representative blot showing ARF6-positive bands. B, the bands on the blot were quantified by the densitometry and expressed as the % of total (n = 3, mean ± S.E.). Note that ARF6 mainly distributes to the soluble, cytosolic fraction in Mg2+-containing buffer, whereas in Mg2+-free buffer, it moved to the membrane fraction, the apical membrane-rich, rather than tubulovesicle-rich fraction. *, significantly different from corresponding control value (Mg2+-containing) at p < 0.05.

View larger version (66K): [in this window] [in a new window]   FIG. 4. Mg2+ chelation changes in localization of ARF6 in rabbit gastric glands. -Escin-permeabilized gastric glands were incubated, fixed, and probed by the same protocol as in Fig. 2. A, resting (incubated with 100 µM cimetidine for 15 min at 37 °C) glands are shown. B, glands incubated with 5 mM CDTA for 15 min at 37 °C are shown. It appears that ARF6 translocates to the intracellular canaliculi (arrows) by CDTA treatment. Due to the treatment with -escin, the preservation of the gland structure is inferior to that of intact glands.

In the next experiment, we examined the distribution of ARF6 in the presence or absence of 100 µM GTPS. In this assay, we incubated and homogenized gastric glands using Mg²⁺-containing buffer. When treated with GTPS, cytosolic ARF6 decreased and mainly translocated to the tubulovesicular compartment, but not to the apical membrane, in contrast to the Mg²⁺-free condition (Fig. 5, A and B). We tried to confirm this result by immunohistochemistry. As expected from the biochemical data, it was observed that the staining pattern with anti-ARF6 was quite similar to that with anti-H,K-ATPase -subunit, the tubulovesicle marker protein, in permeabilized, GTPS-treated glands (Fig. 6).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Redistribution of ARF6 in gastric glands with GTPS. -Escin-permeabilized gastric glands were incubated in high K+ buffer in the presence (+) or absence (–) of 100 µM GTPS (37 °C, 15 min), homogenized in Mg2+-containing buffer, and fractionated into apical membrane-rich (AM), tubulovesicle-rich (TV), and cytosolic (C) fractions followed by immunoblotting using anti-ARF6 antibody. A, a representative blot showing ARF6-positive bands. B, the bands on the blot were quantified by the densitometry and expressed as the % of total (n = 3, mean ± S.E.). Note that ARF6 in the cytosolic fraction moved to the tubulovesicle-rich, rather than the apical membrane-rich, fraction by the treatment with GTPS. *, significantly different from corresponding control value at p < 0.05.

View larger version (73K): [in this window] [in a new window]   FIG. 6. Co-localization of ARF6 and H+,K+-ATPase in -escin-permeabilized, GTPS-treated rabbit gastric glands. Isolated glands, permeabilized with -escin, were treated with 100 µM GTPS (37 °C, 15 min), fixed, and stained for ARF6 (green, A) and H+,K+-ATPase (red, B). Stainings of both ARF6 and H+,K+-ATPase, the marker enzyme of tubulovesicles, appear diffuse but show some membranous structure in the cytosol of parietal cell. The merged image (C) shows that most of these two proteins are co-localized, suggesting that ARF6 is present on tubulovesicular membranes in GTPS-treated parietal cells.

ARF6 Translocates to the Intracellular Canaliculi Together with H⁺,K⁺-ATPase in Secretagogue-stimulated Gastric Parietal Cells—Resting (incubated with 100 µM cimetidine for 30 min) or maximally stimulated (incubated with 100 µM histamine plus 30 µM isobutylmethylxanthine) gastric glands were double-stained with anti-ARF6 and anti-H⁺,K⁺-ATPase -subunit. Under the resting condition, both ARF6 and H⁺,K⁺-ATPase appeared to be diffusely distributed in cytosol, whereas the merged image showed little co-localization of these proteins (Fig. 7, A–C). This observation is consistent with the result of biochemical data, i.e. ARF6 distributed to cytosol, whereas H⁺,K⁺-ATPase mainly existed in tubulovesicles (which appeared to be diffuse under light microscopy). When the glands were stimulated, H⁺,K⁺-ATPase translocated to the intracellular canaliculi (Fig. 7, D–F). The staining pattern of ARF6 became similar to that of H⁺,K⁺-ATPase, and this was evident from the merged image. We suggest that ARF6 translocated to the apical membrane together with H⁺,K⁺-ATPase when parietal cell was stimulated.

View larger version (75K): [in this window] [in a new window]   FIG. 7. Redistribution of H+,K+-ATPase and ARF6 in association with stimulation of rabbit gastric glands. Resting isolated glands (treated with 100 µM cimetidine) were fixed, permeabilized, and stained for ARF6 (green, A) and H+,K+-ATPase (red, B). The merged image (C) shows that both ARF6 and H+,K+-ATPase are diffusely present in the cytosol of parietal cell and that their co-localization was relatively poor. When the glands were stimulated with 100 µM histamine plus 30 µM IBMX (15 min at 37 °C) and stained for ARF6 (in green, D) and H+,K+-ATPase (red, E), it was obvious from the merged image (F) that both proteins considerably co-localized, especially at the intracellular canaliculi (arrows) of the parietal cell.

Effects of Myristoylated N-terminal Peptide of ARF on Acid Secretion—To investigate whether ARF6 directly regulates acid secretion, we tested the effect of N-terminal peptide of ARF6 on aminopyrine accumulation of rabbit isolated glands. Because the N-terminal of small G proteins is thought to interact with the effectors and the interaction was expected to occur within membranes, we obtained the myristoylated synthetic N-terminal peptide of ARF6 (myr-GKVLSKIFGNKE) (17). Gastric glands were permeabilized with 50 µM -escin, and aliquots were assayed for aminopyrine accumulation for 30 min at 37 °C. As shown in Fig. 8, the aminopyrine ratio stimulated by 3 mM GTP was significantly inhibited by the peptide at 30 µM. In contrast, myristoylated peptide with reversed sequence, myr-EKNGFIKSLVKG, used as a negative control, failed to affect the aminopyrine ratio.

View larger version (16K): [in this window] [in a new window]   FIG. 8. Effects of myristoylated peptides on [14C]aminopyrine accumulation in -escin-permeabilized gastric glands. Gastric glands were permeabilized with 50 µM -escin and stimulated with 3 mM GTP at 37 °C for 30 min. Acid secretion was monitored by [14C]aminopyrine accumulation. Aminopyrine ratio above resting (100 µM cimetidine alone) was calculated and expressed as the percentage of stimulated value (cimetidine plus 3 mM GTP). Myristoylated N-terminal peptide of ARF6 (myr-GKVLSKIFGNKE) and its negative control with reversed sequence (myr-EKNGFIKSLVKG) were both used at 30 µM. Values are means ± S.E. of three separate experiments. *, significantly different from control at p < 0.05.

Effects of Expression of ARF6 and Its Mutants on Aminopyrine Accumulation in Cultured Gastric Glands—To investigate the direct role of ARF6 in acid secretion, we constructed adenoviruses, i.e. HA-tagged ARF6 wild type (WT) and its mutants (Q67L, and N122I), which were transiently expressed in cultured gastric glands. The expression of each mutant was estimated to be more than 10 times as much as the endogenous ARF6 assessed by immunoblotting (data not shown). The acid secretion was monitored by [¹⁴C]aminopyrine accumulation, and the results are shown in Fig. 9. ARF6 Q67L, a GTP hydrolysis-deficient, constitutive active mutant, significantly reduced acid secretion, whereas N122I, a mutant defective in GTP binding, tended to inhibit secretion but was not statistically significant.

View larger version (18K): [in this window] [in a new window]   FIG. 9. Effects of expression of ARF6 mutants on [14C]aminopyrine accumulation in cultured gastric glands. Gastric glands were cultured and infected with adenoviruses carrying wild type ARF6 (WT), GTPase-defective mutant (Q67L), GTP-binding defective mutant (N122I), as described under "Experimental Procedures" (multiplicity of infection = 300). [14C]Aminopyrine uptake was used to assess acid secretion. Data are expressed as values relative to histamine (3 µM)-stimulated cells expressing EGFP. Values are means ± S.E. of five separate experiments. *, significantly different from control at p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
At present, the generally accepted model for vesicular fusion events essential for acid secretory process includes rab family proteins, especially rab11a. There is no doubt of the importance of rab11a, because: (a) rab11a exists on the tubulovesicular membrane, (b) it translocates to the apical secretory membrane together with the proton pump, and (c) the dominant negative mutant of rab11a inhibits acid secretion (18, 19). However, there has been some evidence that another small G protein might be involved in the process: Miller and Hersey (4) reported that GTPS acted as a potent inhibitor of acid secretion in -toxin-permeabilized gastric glands. We also confirmed their results by using the -escin-permeabilized gland model and observed that translocation of tubulovesicles to the apical membrane was inhibited by GTPS, suggesting that one of the sites of action of GTPS is the fusion events (or earlier) in the sequence of activation (5). It was also observed that ARF-like immunoreactivity accumulated in the membrane fraction of GTPS-treated glands using non-selective anti-ARF antibody (5). In the same report, we applied various functional peptides, including rab and ARF, and found that the N-terminal peptide of ARF 1, but not of rab, showed biological activity, i.e. inhibition of acid secretion (5).

In the present study, we observed that GTP by itself behaved as an agonist for acid secretion in permeabilized glands and that the stimulatory effect of cAMP was abolished by GTPS. The inhibitory effect of GTPS cannot be explained by the involvement of rab members, because GTPS works as an agonist for most small G proteins in the secretory process. In fact, GTPS stimulates pepsinogen secretion from chief cells that are also contained in the gastric gland (4). Our results in turn clearly indicate a possible involvement of ARF family members in acid secretion, because ARF is the only family on which GTPS causes an inhibitory effect in the process involved (3). Because brefeldin A, an ARF-GEF inhibitor, was ineffective in acid secretion (5), only ARF6, of which GEF is known to be insensitive, remained as a candidate. In the day when these experiments were done, it was considered that ARF6 was a permanent resident on the membrane, and its localization was unaffected by GTPS. Therefore, we were reluctant to conclude that ARF6 is involved in acid secretion. However, it was recently revealed that ARF6 exists in both soluble and membrane-bound forms similar to other ARF family proteins (16, 20). We then embarked on the present study to elucidate whether ARF6 is involved in the acid secretory process.

ARF activity appears to be regulated by guanine nucleotide exchange and hydrolysis like other small G proteins, because cytosolic ARF is in a GDP-bound state, and it is GTP-bound ARF that associates with cellular membranes (21). It was reported that, unlike other ARF, membrane distribution of ARF6 expressed in various cell lines is affected by Mg²⁺ concentration (16). In gastric glands, ARF6 mainly exists in parietal cells and is located in both cytosol and membrane fractions in the presence of physiological concentration of Mg²⁺(2.5 mM). In the EDTA- or CDTA-containing buffer, it translocated from cytosol to intracellular canaliculi. Gaschet and Hsu (16) postulated that the translocation is due to the Mg²⁺ dependence of GAP activity specific for ARF6 on the membrane, i.e. removal of Mg²⁺ results in loss of GAP activity to hold ARF-GTP in the membrane. According to their interpretation, ARF6 existing in the cytosol in the GDP form moves to the apical membrane in an activated GTP-binding form and goes back to cytosol when GTP hydrolysis is activated by GAP on the membrane.

The GTPS-dependent recruitment of ARF6 from cytosol to the vesicular compartment supports the GTPase cycle-dependent translocation of ARF6 in parietal cell. In several secretory cells, GTPS-dependent translocation of ARF to secretory granules has also been observed (25). However, it is presently unclear why ARF6 distributes to different membrane compartments, i.e. to the apical membrane by Mg²⁺ chelator and to the tubulovesicles by GTPS in parietal cell. It could be possible that the Mg²⁺ sensitivity of GAP is different between these membranes or that the effect of Mg²⁺ chelating is not related to GAP but to another unknown mechanism. In any case, it could be concluded that ARF6 redistributes among cytosol, tubulovesicles, and apical secretory membranes depending upon its GTPase activity. In intact, highly stimulated parietal cells, ARF6 and H⁺,K⁺-ATPase co-localized on the apical intracellular canaliculi. Based on these data, the whole scenario would be that the activated GTP-bound form of ARF6 binds to tubulovesicular membrane, and subsequently this ARF6-tubulovesicle complex translocates to the apical membrane.

In addition to its translocation within parietal cell, ARF6 appears to play a role in acid secretion. In the present study, the myristoylated N-terminal fragment of ARF6, but not its reversed sequence, partially inhibited GTP-stimulated acid secretion in permeabilized glands. To confirm this, we employed an expression system using adenovirus as more specific probes. It was found that expression of ARF6Q67L, a GTP hydrolysis-deficient mutant, and not the wild type of ARF, inhibited histamine-induced acid secretion. This strongly supports our hypothesis that the GTP-hydrolysis cycle of ARF6 is essential for acid secretion. There have been several reports that overexpression of wild type ARF6 does not affect the physiological functions in which ARF6 was proved to be involved (23, 24). Zhang et al. (25) reported that constitutive active ARF1 (Q71L) caused vesiculation of the Golgi apparatus and expansion of the ER lumen, leading to inhibition of constitutive protein secretion in NRK cells. Although the mechanism whereby GTP-bound ARF inhibits secretion has not been fully elucidated, it is postulated that continuous association of ARF with specific membrane components arrests the events of membrane fusion (26). Considering the observation that ARF6 was accumulated in the tubulovesicular compartment in the presence of GTPS, it could be postulated that GTP-bound ARF6 translocates to tubulovesicles from cytosol, and also, if GTP hydrolysis is obstructed on the membrane, ARF6-tubulovesicle complex cannot translocate to the apical membrane. Myristoylated N-terminal fragment of ARF6 might weakly mimic this process with its accessibility to the membranes in parietal cell.

We have recently demonstrated that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and its carrier, phosphatidylinositol transfer protein, are essential components for acid secretion (27, 28). It was reported that ARF6 increased PI(4,5)P₂ production through activation of phospholipase D (29) or phosphatidylinositol-4-phosphate 5-kinase (30). Therefore, it would be reasonable to postulate that ARF6 works by producing PI(4,5)P₂, which is a key regulator of both membrane traffic and proton pumping in parietal cell. Obviously, further study is needed to elucidate the link between activation of protein kinase A and ARF6, as well as that between ARF6 and fusion events in parietal cell.

The main point of the present study is that we propose a reasonable answer for the long standing riddle: the potent inhibitory effect of GTPS on acid secretion. This successfully clarifies the important role of ARF6 in the physiologically normal cell, i.e. acutely isolated or primary cultured parietal cell, not in the transformed cell lines.

	FOOTNOTES

 
^* This study was supported in part by the Japanese Ministry of Education, Science, Sports and Culture Grants 13470511 and 13557220. The costs of publication of this article were defrayed in part by the payment of page charges. This 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. Tel.: 81-3-3700-1986; Fax: 81-3-3700-9647; E-mail: urusidani{at}nihs.go.jp.

¹ The abbreviations used are: GTPS, guanosine 5'-3-O-(thio)triphosphate; ARF, ADP-ribosylation factor; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; EGFP, enhanced green fluorescent protein; myr, myristoylated; CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; PI(4,5)P₂, phosphatidylinositol 4,5-diphosphate; HA, hemagglutinin; CMV, cytomegalovirus; WT, wild type.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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