Responsiveness of beta -escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments

Keiko Akagi, Taku Nagao, and Tetsuro Urushidani

Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We established a beta -escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [14C]aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of H+-K+-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTPgamma S, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the beta -escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function.

parietal cell; acid secretion; small GTP-binding protein; protein kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ON THE BASIS OF RECENT PROGRESS in cell biology, intracellular signal transduction has been clarified in various cell types. In acid-secreting parietal cells, many candidate components have been suggested (28). The main disadvantage of the parietal cell is that cell-free reconstitution of the secretory machinery has not been successful, in contrast to many other secretory cells. Moreover, we recently reported that a large number of the pharmacological probes, frequently used to analyze intracellular signal transduction, showed nonspecific effects on parietal cell function (25). This clearly shows that the analysis of intracellular signal transduction with cell-permeable probes has its limitations, at least in the acid-secretory process. It is thus desirable to develop a model system that enables the use of more specific probes, e.g., functional peptide fragments, functional protein itself, or antibody against the protein.

In various secretory cells, it is known that guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S), a nonhydrolyzable GTP analog, accelerates the secretory response. Experiments that use specific peptide fragments support the hypothesis that this phenomenon is due to the effects of GTPgamma S on the secretory granule-associated Rab protein, a member of the small GTP-binding protein family. For example, in rat adipocytes, the hypervariable region in the COOH terminal of Rab4 (191-210) interferes with insulin-mediated translocation of the glucose transporter via inhibition of the translocation of Rab4 (24). In pancreatic acinar cells, Rab3AL, the effector domain of Rab3, was reported to stimulate amylase secretion (21, 32). In contrast to these secretory cells, GTPgamma S inhibits acid secretion by the parietal cell, but the mechanism of action is unknown (17). Recently, it has been revealed that Rab11 colocalizes in the tubulovesicles as a parietal cell-specific small GTP-binding protein (4, 9, 10). However, its functional role has not yet been elucidated. Because the pharmacological tools to distinguish between the various small G-proteins are limited at present, it is again necessary to use a system allowing the introduction of molecules at least the size of peptide fragments into the cell.

In this respect, the permeabilized cell is an indispensable system for analysis of intracellular signal transduction. In gastric glands, Hersey and Steiner (11) first introduced digitonin-permeabilized glands. However, digitonin-treated glands lost their ability to respond to any stimulus after permeabilization, thus limiting their application in the study of signal transduction. Thibodeau et al. (26) developed the alpha -toxin-permeabilized cell. This model has a big advantage in that it retains an ability to respond to receptor-mediated stimuli (17). However, the permeability of the membrane is restricted to molecules <1,000 molecular weight This limits its utilization in that it works only when small but impermeable molecules, e.g., cAMP, labeled ATP (31), or GTPgamma S (17), are introduced into the cell. It is thus necessary to develop another system where molecules >2,000 molecular weight can be introduced into the cell (corresponding to peptides with ~20 amino acids).

In the present study, we developed a beta -escin-permeabilized system following the case of smooth muscle cells (13). It was reported that the saponin ester beta -escin rendered the cell membrane permeable to relatively large molecules, e.g., calmodulin (molecular weight 14,000), and preserved cell responsiveness via receptor activation (13). We now show that beta -escin-permeabilized isolated rabbit gastric glands retain their ability to respond to not only the second messenger, cAMP, but also to forskolin and histamine. With the use of this model, we report the effects of specific peptide fragments to elucidate the role of various putative components of signal transduction in gastric parietal cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Protein kinase C inhibitory peptide (pseudosubstrate for protein kinase C; 19---31) was obtained from Sigma Chemical (St. Louis, MO). Calmodulin-dependent protein kinase II inhibitory peptide (pseudosubstrate for calmodulin-dependent protein kinase II; 281-301), calmodulin inhibitory peptide (calmodulin binding domain of calmodulin-dependent protein kinase II; 290-304), and myosin light-chain kinase inhibitory peptide (pseudosubstrate for myosin light-chain kinase; 480-501) were obtained from Calbiochem (La Jolla, CA). The cAMP-dependent protein kinase inhibitory peptide (5---24) was from Promega (Madison, WI). The Rab-related peptides, based on the sequence of rat Rab3, i.e., Rab3AL (VSALGIDFKVKTIYRN; 27---42), and of rabbit Rab11, i.e., Rab11AL (KSALGVEFATRSIQVD; 41---56), Rab11NT (KSTIGVEFATRSIQVD; 41---56), and Rab11CT (SQKQMSDRRENDMSPSNNVVP; 177-197), and of rabbit Rab25, i.e., Rab25AL CRTALGVEFSTRTVLLG; 42---57, and the inhibitory peptide of ADP-ribosylation factor 1 (Arf1p; GNIFANLFKGLFGKKE; 2---17) were synthesized by Takara (Kusatsu, Japan).

Isolation and permeabilization of rabbit gastric glands. Gastric glands were isolated from Japanese White rabbits (Shiraishi, Tokyo, Japan) essentially by the method of Berglindh (3). Isolated glands, suspended in the normal medium, containing (in mM) 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 BSA, were washed and suspended in a high-K+ medium containing (in mM) 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 is as high as 10 µM.

The formation of homogenous pores in the plasma membrane of the gastric cells was achieved by the modified cold incubation method described by Iizuka et al. (13). Briefly, the glands were suspended in high-K+ medium with a 100-mg wet wt/ml and incubated with 50 µM or the indicated concentration of beta -escin (Sigma) for 120 min at 4°C. After that, the temperature was increased rapidly to 30°C by exchanging the cold medium with the prewarmed medium containing no beta -escin, and incubation was continued for 10 min at 30°C. The permeabilized glands were used immediately for assay without further incubation. Under humid conditions, beta -escin easily transforms to the inactive form, alpha -escin; therefore, extremely dry conditions were necessary for storage.

Acid secretion of the glands was monitored by accumulation of a weak base, [14C]aminopyrine, setting the water content of the glands at a constant of 2.0 µl/mg dry wt (3). The medium used for the 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 fractions were prepared from the homogenate as described by Urushidani and Forte (27). Permeabilized glands were incubated at 37°C for 30 min in the presence of 100 µM cimetidine (resting), 100 µM cimetidine plus 100 µM cAMP (stimulated), or 100 µM cimetidine plus 100 µM cAMP and 100 µM GTPgamma S. The glands were then pelleted at 1,000 rpm for 1 min and homogenized in a buffer containing (in mM) 113 mannitol, 37 sucrose, 0.5 EDTA, and 5 PIPES, pH 6.7. After removal of cell debris (40 g for 5 min), the homogenate was sequentially fractionated into three pellets: P1 (4,000 g for 10 min), P2 (14,500 g for 10 min), and P3 (100,000 g for 45 min). The low-speed pellet (P1) was further purified with a density gradient using 18% Ficoll and then used as the apical membrane-rich fraction. The microsomal fraction (P3) was used as the tubulovesicle-rich fraction.

To examine the subcellular distribution of H+-K+-ATPase or Arf, each fraction was analyzed with 7.5% (H+-K+-ATPase) or 13% (Arf) SDS-PAGE according to Laemmli (15) and then blotted on a polyvinylidene difluoride membrane (BioRad, Hercules, CA) with a semidry apparatus. The membrane was probed with anti-H+-K+-ATPase alpha -subunit monoclonal antibody (29) or anti-Arf3 monoclonal antibody (Transduction Laboratories, Lexington, KY) and visualized by chemiluminescence (Renaissance Western blot chemiluminescence reagent; DuPont NEN) with the use of horseradish peroxidase-conjugated anti-mouse IgG as a second antibody. The amount of H+-K+-ATPase alpha -subunit in each fraction was quantified by densitometry, multiplied by a volume factor, and summed, and percentage of total amount was calculated for P1 and P3 fractions.

To examine the subcellular distribution of small GTP-binding proteins, each fraction was analyzed on a 13% SDS-PAGE and blotted on a nitrocellulose membrane. The transblot was subjected to [gamma -35S]GTPgamma S overlay assay (2). Briefly, the equilibrated membrane was incubated for 30 min in a buffer (50 mM Tris, pH 7.5, 0.3% Tween 20, 12 µM MgCl2, and 1 mM dithiothreitol) containing 10 µM ATP and 1 nM [gamma -35S]GTPgamma S (1,320 Ci/mmol; DuPont NEN). After that, the sheet was washed three times and dried, and the radioactivity was detected by an imaging analyzer (Fuji Film, BAS-2000). Specificity of the binding was confirmed by incubating the sheets in the presence of 10 µM unlabeled GTP.

Miscellaneous. Lactate dehydrogenase was assayed with a clinical assay kit (kit CII, Wako Pure Chemical, Tokyo, Japan). Acridine orange quenching of purified rabbit gastric microsomes was performed as described (25).

Statistical analysis. Parametric data were expressed as means ± SE. Multiple comparisons were analyzed by ANOVA and Dunnet's post hoc test with a computer program (Super ANOVA, ABACUS Concepts, Berkeley, CA). The level of significance was uniformly set at P < 0.05, and no further calculation of P value was performed.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Permeabilization of gastric glands by treatment with beta -escin. To determine the optimal concentration of beta -escin, the aminopyrine accumulation stimulated by several agonists was measured after treatment with various concentrations of beta -escin (Fig. 1A). In the intact glands, 100 µM cAMP showed little stimulatory effect. When the glands were treated with beta -escin, the responsiveness to cAMP increased up to 40 µM of the agent and then it began to decrease in the higher concentration range. In contrast, the responsiveness to 100 µM histamine decreased with the concentration of beta -escin, and it almost disappeared at 100 µM or higher. The responsiveness to 10 µM forskolin was observed to be similar to that of histamine, except in the higher concentration range of beta -escin, where small but significant responses still remained. In this experiment, the response of untreated glands was assayed in the normal medium, whereas the response of beta -escin-treated glands was assayed in high-K+ medium. We recently found that cAMP-mediated acid secretion was markedly potentiated in high-K+ medium (1). Therefore, it could not be simply concluded that the optimal permeabilization occurred at 40-60 µM. To clarify this point, we performed further measurements with the use of forskolin and an H+-K+-ATPase inhibitor (Mg2+ chelator), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA). The response to 10 µM forskolin was not inhibited by CDTA in the intact preparation because of its impermeability. When the glands were treated with increasing concentrations of beta -escin, the response to forskolin was progressively inhibited and was abolished at ~40-60 µM of beta -escin (Fig. 1A). We considered that the net inhibition by CDTA of the forskolin-stimulated aminopyrine ratio reflected the permeability of the basolateral membrane of the parietal cell, whereas the decrease in the aminopyrine ratio in the higher concentrations of beta -escin without CDTA indicated the disturbance of the secretory machinery. To find out the optimal concentration of beta -escin, 40, 50, and 60 µM beta -escin were tested (Fig. 1B). The inhibitory effect of CDTA on forskolin-stimulated secretion was sometimes incomplete at 40 µM, whereas the agonist-stimulated secretion was profoundly attenuated at 60 µM beta -escin. Therefore, we chose 50 µM for the routine assays. The release of a large molecule, lactate dehydrogenase (140 kDa), was also monitored, and we found that only 17% of the total enzyme was released at this concentration of beta -escin (Fig. 1A) (100% release was obtained by the treatment with 1% Triton X-100). Therefore, it appears that the leakage of large molecules is negligible under the condition employed. Under this condition, all the cells in the glands were stained with trypan blue. With the use of FITC-phalloidin (molecular weight ~1,200) as a probe, intracellular canaliculi were clearly visible in >80% of the parietal cells.


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Fig. 1.   Concentration dependency of beta -escin for permeabilization of isolated glands. Gastric glands were permeabilized with indicated concentration of beta -escin for 120 min at 4°C, followed by a postincubation for 10 min at 30°C in high-K+ medium. A: aliquots were assayed for aminopyrine accumulation for 30 min at 37°C in absence or presence of indicated secretagogues. Results were expressed as aminopyrine ratio above value without secretagogue. An aliquot of gland media was also assayed for released amount of lactate dehydrogenase (LDH). Results are expressed as a percentage of maximum, which was designated as total cellular LDH released by 1% Triton X-100. A representative of 2 identical experiments with full measurements is shown. CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid. B: gastric glands were permeabilized with 40, 50, or 60 µM beta -escin and aliquots were assayed for aminopyrine accumulation as described above. A representative of 3 identical experiments is shown.

Acid-secretory responses of beta -escin-permeabilized glands. In the beta -escin-permeabilized glands, the second messenger cAMP, the adenylate cyclase activator forskolin, and the H2 receptor agonist histamine stimulated [14C]aminopyrine accumulation, the indicator of acid secretion (Fig. 2). The muscarine receptor agonist carbachol failed to cause any increase in the aminopyrine ratio. This was in contrast to what was reported by Miller and Hersey (17) and in our own unpublished observation that the alpha -toxin-permeabilized model retained its sensitivity to the cholinergic agonist. The response to forskolin (Fig. 2) as well as that to histamine (not shown) or cAMP (see Fig. 6) was abolished by CDTA or GTPgamma S, both of which produce no effect (impermeable) in the intact glands. The catalytic subunit of cAMP-dependent protein kinase (40 kDa) up to 1,000 U/ml failed to stimulate acid secretion as expected from the putative size of the pore, as high as 15 kDa (13).


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Fig. 2.   Effects of various stimulants on [14C]aminopyrine accumulation in beta -escin-permeabilized glands. Values are means ± SE of 3-6 separate experiments in a duplicate manner. GTPgamma S, guanosine-5'-O-(3-thiotriphosphate). * P < 0.05 vs. forskolin alone.

In the routine assay condition, the medium contained 0.5 mM EGTA and the free Ca2+ concentration was estimated to be as high as 90 nM. Increments of free Ca2+ concentration up to 0.1 mM showed no effect on the aminopyrine ratio with or without cAMP (data not shown), and this result is consistent with that in alpha -toxin-permeabilized glands (17). Although the free Ca2+ concentration was decreased to <0.1 nM by adding 10 mM EGTA, stimulation with 100 µM cAMP was unaffected (aminopyrine ratios of resting, 40.2 ± 11.9; stimulated by 100 µM cAMP in 0.5 mM EGTA, 181 ± 19; means stimulated by 100 µM cAMP in 10 mM EGTA, 187 ± 23; means ± SE, n = 3).

Translocation of the H+-K+-ATPase and effects of GTPgamma S. To elucidate the site of action of GTPgamma S in the sequence of the activation of acid secretion, the redistribution of H+-K+-ATPase was quantified. beta -Escin-permeabilized glands were incubated with or without 100 µM cAMP for 30 min, homogenized, and then fractionated. Each fraction was analyzed by SDS-PAGE and Western blotting with anti-H+-K+-ATPase alpha -subunit monoclonal antibody. As in the intact glands (27) and alpha -toxin-permeabilized glands (31), redistribution of the proton pump from tubulovesicle (microsomal fraction) to the apical membrane (low-speed pellet) occurred in association with stimulation. When 100 µM GTPgamma S was included, the translocation was blocked (Fig. 3). The density of the H+-K+-ATPase alpha -subunit was quantified, and percentage of total amount was calculated for the microsomal fraction (P3) and low-speed pellet (P1) in four separate experiments. In the resting glands, 53.5 ± 1.1% of total H+-K+-ATPase was recovered in the microsome, whereas 14.7 ± 0.3% was in the low-speed pellet. In the stimulated glands, the amount of alpha -subunit in the microsome significantly decreased to 43.4 ± 1.6% of total (P < 0.05 vs. resting), and the amount in the low-speed pellet concomitantly increased to 21.4 ± 1.3% (P < 0.05 vs. resting). When the glands were stimulated in the presence of GTPgamma S, those values approached the resting level (54.5 ± 4.3% and 15.7 ± 1.1% for the microsome and the low-speed pellet, respectively). These results suggest that the site of action of GTPgamma S is in the fusion events or earlier in the sequence of the activation process.


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Fig. 3.   Redistribution of H+-K+-ATPase in beta -escin-permeabilized glands. Resting (lanes 1 and 4), 100 µM cAMP-stimulated (lanes 2 and 5), and 100 µM cAMP plus 100 µM GTPgamma S-treated (lanes 3 and 6) glands were homogenized and separated into fractions, including low-speed pellet (P1; 15 µg protein/lane) and microsomal fraction (P3; 3 µg protein/lane). Each fraction was separated on an SDS-PAGE and blotted on a polyvinylidene difluoride (PVDF) membrane, and H+-K+-ATPase was visualized by enhanced chemiluminescence with anti-H+-K+-ATPase alpha -subunit (1/50,000 dilution for first antibody and 1/10,000 dilution for second antibody, horseradish peroxidase conjugated anti-mouse IgG). Note that, when stimulated, amount of H+-K+-ATPase was increased in P1 and correspondingly was decreased in P3 and that this change was inhibited by treatment with GTPgamma S. A representative of 4 experiments with essentially same results is shown. Density of H+-K+-ATPase alpha -subunit was quantified and percentage of total amount was calculated in 4 separate experiments. From lane 1 to lane 6, values were 14.7 ± 0.3, 21.4 ± 1.3, 15.7 ± 1.1, 53.5 ± 1.1, 43.4 ± 1.6, and 54.5 ± 4.3, respectively. Increase in P1 and decrease in P3 by stimulation were both statistically significant (P < 0.05).

Effects of various inhibitory peptides on the aminopyrine accumulation in beta -escin-permeabilized glands. Aminopyrine accumulation stimulated by 100 µM cAMP was dose-dependently inhibited by cAMP-dependent protein kinase inhibitory peptide and almost abolished at 20 µM or more, as shown in Fig. 4A. This peptide also inhibited acid secretion stimulated by forskolin and by histamine (Fig. 4B). It was confirmed that cAMP-dependent protein kinase inhibitory peptide was ineffective on the aminopyrine accumulation stimulated by dibutyryl cAMP in intact glands (data not shown).


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Fig. 4.   Inhibitory effects of cAMP-dependent protein kinase inhibitor peptide (5---24) on aminopyrine accumulation in beta -escin-permeabilized gastric glands. A: dose-response curve of cAMP-dependent protein kinase inhibitor peptide for aminopyrine ratio stimulated by 100 µM cAMP. Aminopyrine ratio without secretagogue is also indicated by open circle. B: effects of 30 µM cAMP-dependent protein kinase inhibitor peptide on aminopyrine ratio stimulated by 100 µM histamine or 10 µM forskolin. Values are means ± SE of at least 3 separate experiments in a duplicate manner. * P < 0.05 vs. control.

In the next series of experiments, the effect of peptides related to other protein kinases on cAMP-stimulated aminopyrine accumulation were examined and summarized in Fig. 5A. The following peptides were without effect up to 50 µM: the pseudosubstrate peptide (19---31) of protein kinase C (the specific inhibitor of protein kinase C), pseudosubstrate (281---301) for calmodulin-dependent protein kinase II (the specific inhibitor of calmodulin-dependent protein kinase II), and the calmodulin binding domain (290---309) of calmodulin kinase II (the specific inhibitor of calmodulin). In contrast, the pseudosubstrate peptide (480---501) of myosin light-chain kinase (the specific inhibitor of myosin light chain kinase) showed a potent inhibitory activity at 30 µM. As shown in Fig. 5B, the dose-response curve of the myosin light-chain kinase inhibitory peptide on cAMP-stimulated aminopyrine accumulation was steep, and the inhibitory effect was evident at 20 µM or more. It was also confirmed that the myosin light-chain kinase inhibitory peptide was without effect on dibutyryl cAMP-stimulated aminopyrine accumulation in the intact glands up to 100 µM (data not shown).


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Fig. 5.   Effects of various inhibitor peptides on aminopyrine accumulation in beta -escin-permeabilized gastric glands. A: effects of protein kinase C inhibitory peptide (PKC; 50 µM), calmodulin inhibitory peptide (CaM; 50 µM), calmodulin dependent kinase II inhibitory peptide (CaMKII; 50 µM), and myosin light-chain kinase inhibitory peptide (MLCK; 30 µM) on aminopyrine ratio stimulated by 100 µM cAMP. Values are means ± SE of 3-4 separate experiments in a duplicate manner. * P < 0.05 vs. control. B: dose-response curve of myosin light-chain kinase inhibitory peptide for aminopyrine accumulation stimulated by 100 µM cAMP. Aminopyrine ratio without secretagogue is also indicated as open circle. Values are means ± SE of 3 separate experiments in a duplicate manner.

Effects of various peptides related to small GTP-binding proteins. As shown in Fig. 6, 100 µM GTPgamma S inhibited aminopyrine accumulation stimulated by 100 µM cAMP in beta -escin-permeabilized glands. On the basis of the assumption that the target of GTPgamma S is one of the small GTP-binding proteins, the effects of several functional fragments of small GTP-binding proteins were examined. The fragments employed were as follows: the inhibitory peptide (2---17) of Arf1 (Arf1p), the Rab3 effector domain (Rab3AL), the NH2-terminal peptides of Rab11 (Rab11NT) and Rab25 (Rab25AL), the fragments in which the sequence of the NH2-terminal peptide of Rab11 was replaced with AL to strengthen the effector (Rab11AL), and the COOH-terminal hypervariable region of Rab11 (Rab11CT). Rab3AL was demonstrated to be effective in pancreatic acinar cells (21). Rab11 and Rab25 were both reported to exist on the tubulovesicle membranes (5), and the COOH terminal of the Rab family proteins has been proven to play an important role in the binding to target membranes in other systems (7). None of the peptides tested in the present study showed stimulatory effects by themselves or potentiating effects on the 10 µM cAMP (submaximal concentration)-stimulated secretion (data not shown). Moreover, all of the Rab-related peptides up to 100 µM failed to show any inhibitory effect on the aminopyrine accumulation stimulated by 100 µM cAMP in beta -escin-permeabilized glands (Fig. 6). In the case of the Arf1p, a significant inhibition was observed at 100 µM, although the inhibition was less potent than 100 µM GTPgamma S.


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Fig. 6.   Effects of GTPgamma S and various small GTP-binding protein-related peptides on aminopyrine accumulation stimulated by 100 µM cAMP in beta -escin-permeabilized glands. Concentrations of GTPgamma S and peptides were all 100 µM. Values are means ± SE of at least 3 separate experiments in a duplicate manner. * P < 0.05 vs. 100 µM cAMP alone.

To check the possibility that Arf1p has inhibitory activity on H+-K+-ATPase and/or protonophoric activity, acridine orange quenching assay was performed. When the purified gastric microsomes were incubated with acridine orange in the presence of ATP, KCl, and valinomycin, quenching of acridine orange occurred because of the formation of a proton gradient. It was observed that Arf1p up to 100 µM did not affect the quenching, confirming that the peptide neither inhibits the proton pump nor cancels the formed proton gradient (data not shown).

Search for small GTP-binding proteins whose location is changed by GTPgamma S. If the mode of action of GTPgamma S is fixing a certain small GTP-binding protein to GTP form, the protein is expected to accumulate in certain compartment(s). To examine this hypothesis, beta -escin-permeabilized glands were treated with 100 µM cAMP in the presence or absence of 100 µM GTPgamma S, homogenized, and then fractionated. Each fraction was analyzed on an SDS-PAGE, blotted to a nitrocellulose membrane, and then subjected to [gamma -35S]GTPgamma S overlay assay to visualize the GTP-binding protein. The most prominent changes were observed in the microsomal fraction and the apical membrane fraction, as shown in Fig. 7. By GTPgamma S overlay assay, the binding was markedly increased in the region corresponding to 17 kDa by treatment with GTPgamma S in both microsomes and apical membranes. Because the apparent molecular mass was somewhat lower than the known Rab proteins and was close to Arf, we probed the transblot with anti-Arf3 antibody. It is clearly shown in Fig. 7 (bottom) that the position of 17 kDa was positive to anti-Arf3 antibody. Arf1 and Arf3 are classified as class I (18). According to the manufacturer's data, the antibody also recognizes Arf1 to some extent. It was thus concluded that Arf1 and/or Arf3 became accumulated in the membranes by treatment with GTPgamma S in permeabilized gastric glands.


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Fig. 7.   Search for GTP-binding proteins in fractions from beta -escin-permeabilized glands. Resting (lanes 1 and 4), 100 µM cAMP-stimulated (lanes 2 and 5), and 100 µM cAMP plus 100 µM GTPgamma S-treated (lanes 3 and 6) glands were homogenized and fractionated to obtain tubulovesicle-rich microsomes (A) and apical membrane-rich fractions (B). Each fraction (50 µg protein/lane) was separated on an SDS-PAGE and blotted on a nitrocellulose or PVDF membrane. Nitrocellulose membrane was subjected to [gamma -35S]GTPgamma S overlay assay (top). Note that [gamma -35S]GTPgamma S-binding was increased by treatment with GTPgamma S at position indicated by arrowhead (~17 kDa). PVDF membrane (bottom) was probed with anti-Arf3 monoclonal antibody (1/500 dilution for first antibody, 1/5,000 dilution for second antibody, horseradish peroxidase-conjugated anti-mouse IgG) and visualized by enhanced chemiluminesence. Note that same positions where labeling was increased by GTPgamma S were positive to antibody.

Because the potential involvement of Arf in acid secretion was suggested, we examined the effects of brefeldin A, which is known to inhibit the action of Arf by interfering with the GDP/GTP exchange factor (6, 22). However, 50 µM brefeldin A failed to affect aminopyrine accumulation in the cAMP-stimulated permeabilized glands (aminopyrine ratios at resting, 51.6 ± 9.8; stimulated by 100 µM cAMP, 164 ± 7.7; cAMP plus brefeldin A, 155 ± 10; means ± SE, n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have established a new model of isolated gastric gland permeabilized with beta -escin. In this system, cAMP, forskolin, and even histamine can activate acid secretion after the permeabilization step. This property is similar to that of the alpha -toxin-permeabilized model (17). The major advantage of the present system over the alpha -toxin model is that relatively large molecules, e.g., peptides consisting of >20 amino acids, are able to be introduced into the cell. The cAMP-stimulated acid secretion, monitored by [14C]aminopyrine accumulation, was dose-dependently inhibited by cAMP-dependent protein kinase inhibitor peptide (molecular weight 2,222.4). There seems to be consensus that activation of cAMP-dependent protein kinase is involved in the acid-secretory process. This is based mainly on two observations: 1) the activation of cAMP-dependent protein kinase by histamine was demonstrated in parietal cells, and 2) H-89, a "specific" inhibitor of cAMP-dependent protein kinase, inhibited acid secretion (28). However, the former does not demonstrate a direct connection between cAMP-dependent protein kinase activation and acid secretion, and the latter is now equivocal because we have shown that H-89 works as a protonophore in parietal cells (25). In contrast to previous studies with this pharmacological agent of questionable specificity, the present findings provide strong evidence that cAMP-dependent protein kinase plays a role in the regulation of acid secretion via an inhibitory peptide that is widely accepted as a specific inhibitor of the kinase in permeabilized cell models.

The inhibitory peptides for protein kinase C, calmodulin kinase II, and calmodulin were all ineffective. These data are not surprising because we found no potentiating interaction between the stimulation by cAMP and Ca2+, and thus the observed activation in the present model was expected to be a part of the secretory process, i.e., the cAMP-dependent protein kinase pathway, which is independent of the intracellular Ca2+. We do not know why the obvious cross talk between cAMP and Ca2+ observed in the intact cell (16, 19) disappeared in the permeabilized glands, but a similar phenomenon was also observed in alpha -toxin-permeabilized glands where no large molecule was expected to be lost (17). The results with calmodulin inhibitor peptide support our opinion (25) that the putative involvement of calmodulin in the final step of acid secretion, suggested by use of the so-called calmodulin antagonists (23), is not real but is instead an erroneous conclusion based on the protonophoric nature of these drugs.

With the use of ME-3407 and wortmannin as enzyme inhibitors, we previously postulated that myosin light-chain kinase might be involved in acid secretion (29). However, the inhibitory profile of ME-3407 for kinases is wide (29), and wortmannin inhibits phosphatidylinositol-3-kinase in the far lower concentration ranges (30). We also found that "specific" myosin light-chain kinase inhibitors ML-7 and ML-9 are both protonophores and useless for this purpose (25). Therefore, it should be concluded that the involvement of myosin light-chain kinase in acid secretion has not been demonstrated yet. In the present study, however, it was shown that the inhibitor peptide of myosin light-chain kinase almost abolished cAMP-stimulated aminopyrine accumulation in beta -escin permeabilized glands. If this effect is due exclusively to inhibition of myosin light-chain kinase, it supports the hypothesis that acid secretion is regulated by myosin light chain kinase, as suggested for insulin secretion from rat pancreatic beta -cell (12) and for catecholamine release from bovine adrenal gland (14). Our observations apparently contradict the generally believed sequence that myosin light-chain kinase is a Ca2+/calmodulin-dependent enzyme and is inhibited when phosphorylated by cAMP-dependent protein kinase. This gave us the idea that regulation of putative myosin light-chain kinase in the parietal cell has a unique regulatory mechanism different from conventional myosin light-chain kinase.

Miller and Hersey (17) first showed that GTPgamma S inhibited acid secretion in alpha -toxin-permeabilized glands. The mechanism of inhibition has not been elucidated, but it is reasonable to postulate that some GTP-binding protein(s), e.g., Rab, are involved. In the present study, it was demonstrated that the site of action of GTPgamma S was in the membrane fusion process or earlier, because stimulation-associated translocation of H+-K+-ATPase from the tubulovesicles to the apical membrane fraction was blocked by GTPgamma S. If GTPgamma S works as the activator of Rab protein, it would be expected that the effector domain of Rab protein works as an inhibitor, and that the COOH-terminal hypervariable domain works as an activator of acid secretion in the parietal cell. We tested the following peptides in the present study: 1) the Rab3 effector domain (Rab3AL), 2) the NH2-terminal peptides of Rab11 (Rab11NT) and Rab25 (Rab25AL), 3) the fragment in which the sequence of the NH2-terminal peptide of Rab11 was replaced with AL to strengthen the effector (Rab11AL), and 4) the COOH-terminal hypervariable region of Rab11 (Rab11CT). However, none of these peptides showed a significant effect on beta -escin-permeabilized glands, irrespective of stimulation with cAMP. These results suggest that Rab11 is constitutively associated with the effector proteins if this protein is functionally involved in the secretory machinery. This might reflect the observation that Rab11 in the tubulovesicle translocated to the apical membrane without dissociation (5), in contrast to Rab3, which was reported to be "cycling off" from the secretory membrane to the cytosol in the course of the activation of secretion (21). It is also possible that these peptides did not exhibit the presumed effects in the parietal cell.

In contrast to the Rab proteins, Arf1 inhibitory peptide significantly attenuated cAMP-stimulated aminopyrine accumulation in beta -escin-permeabilized glands. It was also shown that treatment with GTPgamma S caused an accumulation of Arf1 and/or 3 in the membrane containing H+-K+-ATPase. Okamoto et al. (20) recently demonstrated that tubulovesicular membranes contained clathrin as well as the AP1 adapter complex. In another cell system, it was shown that binding of these components to the membrane was affected by GTPgamma S and Arf (22). It could be possible that in the gastric parietal cell, GTPgamma S also fixed Arf in a membrane-bound state to prevent the release of clathrin coat from the vesicles and subsequently inhibited the delivery of the proton pump to the apical membrane. However, there was a clear discrepancy for the involvement of Arf, in that brefeldin A, which inhibits the GDP/GTP exchange factor of Arf and subsequent association of Arf to the membrane, fails to inhibit aminopyrine accumulation stimulated by cAMP. It is unreasonable to suppose that the activation of acid secretion requires only dissociation, but not association, of Arf to the membrane.

There is a brefeldin A-insensitive subtype, Arf6, that is postulated to be involved in the secretory process of the chromaffin cell. However, Arf6 is a resident in the membrane, and thus its localization is affected neither by brefeldin A nor by GTPgamma S (6). Therefore, Arf6 should be excluded from the candidate proteins as the target of GTPgamma S. On the basis of these facts, we postulate that the accumulation of Arf in the membrane is not directly related to the inhibitory effect of GTPgamma S. For the inhibitory effect of the NH2-terminal peptide of Arf, a possible explanation is that the peptide also has the property of interacting with Arf-independent pathways, e.g., phospholipases (8, 18), although any target candidate in the parietal cell is presently unknown. Further work employing a new strategy is necessary to elucidate the possible role of small GTP-binding proteins in the activation of acid secretion.

In conclusion, our new model, the beta -escin-permeabilized gastric gland, is quite a useful model for analyzing signal transduction in parietal cells with the use of highly specific functional peptide fragments as probes. Although it is inevitable that these fragments will show unexpected pharmacological effects, the reliability of the results has been greatly improved. A breakthrough in the study of parietal cell function could be expected using this model for future work.


    ACKNOWLEDGEMENTS

This work was supported in part by Japanese Ministry of Education, Science, Sports, and Culture Grants 09672216 and 10557219.


    FOOTNOTES

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 and other correspondence: T. Urushidani, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The Univ. of Tokyo, Tokyo 113-0033, Japan (E-mail: urushi{at}mol.f.u-tokyo.ac.jp).

Received 19 January 1999; accepted in final form 24 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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