Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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We established a
-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)
(GTP
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
GTP
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
-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
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INTRODUCTION |
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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)
(GTPS), 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 GTP
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, GTP
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 -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 GTP
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 -escin-permeabilized system
following the case of smooth muscle cells (13). It was reported that
the saponin ester
-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
-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.
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MATERIALS AND METHODS |
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Materials.
Protein kinase C inhibitory peptide (pseudosubstrate for protein kinase
C; 1931) 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 ofSubcellular 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 GTPS. 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.
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.
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RESULTS |
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Permeabilization of gastric glands by treatment with
-escin.
To determine the optimal concentration of
-escin, the aminopyrine
accumulation stimulated by several agonists was measured after
treatment with various concentrations of
-escin (Fig.
1A). In the intact glands, 100 µM cAMP showed little stimulatory effect. When the glands were treated with
-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
-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
-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
-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
-escin, the
response to forskolin was progressively inhibited and was abolished at
~40-60 µM of
-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
-escin without CDTA indicated the disturbance of
the secretory machinery. To find out the optimal concentration of
-escin, 40, 50, and 60 µM
-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
-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
-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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Acid-secretory responses of -escin-permeabilized
glands.
In the
-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
-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 GTP
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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Translocation of the
H+-K+-ATPase
and effects of GTPS.
To elucidate the site of action of GTP
S in the sequence of the
activation of acid secretion, the redistribution of
H+-K+-ATPase
was quantified.
-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
-subunit monoclonal antibody. As in the intact glands (27) and
-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 GTP
S was included, the translocation was blocked (Fig.
3). The density of the
H+-K+-ATPase
-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
-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 GTP
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 GTP
S is in the fusion events or
earlier in the sequence of the activation process.
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Effects of various inhibitory peptides on the aminopyrine
accumulation in -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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Effects of various peptides related to small GTP-binding proteins.
As shown in Fig. 6, 100 µM GTPS
inhibited aminopyrine accumulation stimulated by 100 µM cAMP in
-escin-permeabilized glands. On the basis of the assumption that the
target of GTP
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
-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 GTP
S.
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Search for small GTP-binding proteins whose location is changed by
GTPS.
If the mode of action of GTP
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,
-escin-permeabilized glands were treated with 100 µM cAMP in the presence or absence of
100 µM GTP
S, homogenized, and then fractionated. Each fraction was
analyzed on an SDS-PAGE, blotted to a nitrocellulose membrane, and then
subjected to
[
-35S]GTP
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
GTP
S overlay assay, the binding was markedly increased in the region corresponding to 17 kDa by treatment with GTP
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 GTP
S in
permeabilized gastric glands.
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DISCUSSION |
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In the present study, we have established a new model of isolated
gastric gland permeabilized with -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
-toxin-permeabilized model (17). The major advantage of the present
system over the
-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 -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 -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
-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 GTPS inhibited acid
secretion in
-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 GTP
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
GTP
S. If GTP
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
-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
-escin-permeabilized glands. It was also shown that treatment with
GTP
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 GTP
S and Arf (22). It could be possible
that in the gastric parietal cell, GTP
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 GTPS (6). Therefore, Arf6
should be excluded from the candidate proteins as the target of
GTP
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 GTP
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 -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.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Japanese Ministry of Education, Science, Sports, and Culture Grants 09672216 and 10557219.
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FOOTNOTES |
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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.
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