Involvement of RhoA and its interaction with protein kinase C
and Src in CCK-stimulated pancreatic acini
Fumihiko
Nozu1,
Yasuhiro
Tsunoda1,
Adenike I.
Ibitayo2,
Khalil N.
Bitar2, and
Chung
Owyang1
Departments of 1 Internal
Medicine and 2 Pediatrics,
University of Michigan Medical Center, Ann Arbor, Michigan 48109
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ABSTRACT |
We evaluated intracellular pathways responsible
for the activation of the small GTP-binding protein Rho p21 in rat
pancreatic acini. Intact acini were incubated with or without CCK and
carbachol, and Triton X-100-soluble and crude microsomes were used for
Western immunoblotting. When a RhoA-specific antibody was
used, a single band at the location of 21 kDa was
detected. CCK (10 pM-10 nM) and carbachol (0.1-100 µM) dose
dependently increased the amount of immunodetectable RhoA with a peak
increase occurring at 3 min. High-affinity CCK-A-receptor agonists
JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities
of the RhoA band, suggesting that stimulation of RhoA is mediated by
the low-affinity CCK-A receptor. Although an increase in RhoA did not
require the presence of extracellular
Ca2+, the intracellular
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK
and carbachol. The Gq protein
inhibitor G protein antagonist-2A (10 µM) and the phospholipase C
(PLC) inhibitor U-73122 (10 µM) markedly reduced RhoA bands in
response to CCK. The protein kinase C (PKC) activator phorbol ester
(10-1,000 nM) dose dependently increased the intensities of the
RhoA band, which were inhibited by the PKC inhibitor K-252a (1 µM).
The
pp60c-src
inhibitor herbimycin A (6 µM) inhibited the RhoA band in
response to CCK, whereas the calmodulin inhibitor W-7 (100 µM) and
the phosphoinositide 3-kinase inhibitor wortmannin (6 µM) had no
effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK
stimulation. In permeabilized acini, the Rho inhibitor Clostridium
botulinum C3 exoenzyme dose
dependently inhibited amylase secretion evoked by a
Ca2+ concentration with an
IC50 of C3 exoenzyme at 1 ng/ml.
We concluded that the small GTP-binding protein RhoA p21 exists in
pancreatic acini and appears to be involved in the mediation of
pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways
are involved in the activation of PKC and Src cascades via
Gq protein and PLC.
cholecystokinin; pancreas
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INTRODUCTION |
THERE ARE MANY SMALL monomeric GTP-binding proteins
(molecular mass, 20-30 kDa) that show clear sequence
homology to the proteins encoded by the Ras oncogenes, and some 50 distinct mammalian proteins of this type have been characterized as
members of the Ras superfamily (2, 8, 17). Several different
subfamilies have been defined based on their primary sources (Ras
family, Rab family, Rho family, and others). These small GTP-binding
proteins play a pivotal role in the regulation of cell functions. In
general, the Ras family is involved in cell growth, cell proliferation,
and differentiation (8), whereas the Rab family regulates intracellular
vesicular transport during exocytosis and endocytosis (2), and the Rho family modulates the cytoskeletal systems (16).
Although several lines of evidence indicate that Ras, Rab3A, and Rab3D
are involved in stimulus-secretion coupling of pancreatic acinar cells
(10, 23, 24), the existence and functional role of the Rho proteins
have not yet been ascertained in this cell type. Because the
microtubular-microfilamentous systems participate in mediating
pancreatic exocytosis (19), it is likely that the Rho protein might be
involved during pancreatic enzyme secretion. Therefore, we attempted to
identify and characterize the functional role(s) of Rho in rat
pancreatic acini. Our results showed that the small GTP-binding protein
RhoA p21 exists and appears to mediate pancreatic enzyme secretion
evoked by cholecystokinin (CCK) and carbachol. The activation of RhoA
involves the sequential stimulation of protein kinase C (PKC) and Src
kinase cascades via the Gq protein and phospholipase C (PLC) pathway.
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METHODS |
Materials. Chemicals were purchased
from the following sources. CCK-8, carbachol, creatinine phosphatase,
creatinine phosphokinase, ATP,
12-O-tetradecanoylphorbol 13-acetate
(TPA), and wortmannin were from Sigma Chemical (St. Louis, MO). K-252a,
W-7, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) were from Calbiochem (La Jolla, CA).
Clostridium botulinum C3 exoenzyme,
U-73122, G protein antagonist-2A (GP antagonist-2A), and herbimycin A
were from Biomol (Plymouth Meeting, PA). Anti-RhoA mouse monoclonal
antibody 26C4 and anti-RhoB rabbit polyclonal antibody 119 were from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Src
(pp60c-src)
mouse monoclonal antibody was from Upstate Biotechnology (Lake Placid,
NY). Protein G-Sepharose was from Pharmacia Biotech (Uppsala, Sweden).
Streptolysin O (SLO) was from GIBCO BRL (Grand Island, NY). JMV-180 and
CCK-OPE were from Research Plus (Bayonne, NJ).
Isolation of pancreatic acinar cells.
Isolated rat pancreatic acini were prepared by collagenase digestion
with pancreas obtained from male Sprague-Dawley rats (33). Acini
obtained were suspended in a physiological salt solution (PSS). The PSS
contained 0.1% bovine serum albumin, 0.1 mg/ml soybean trypsin
inhibitor, and (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1 NaH2PO4, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 5.5 D-glucose, and
2 L-glutamine with Eagle's
minimum essential amino acid neutralized with NaOH. The PSS was
adjusted to pH 7.4 and equilibrated with 100%
O2. Isolated acini were
preincubated for 40 min at 37°C in 40 ml of PSS, washed twice, and
resuspended in 40 ml of fresh PSS.
Preparation of SLO-permeabilized pancreatic acinar
cells and measurement of amylase secretion.
Permeabilized pancreatic acinar cells were prepared as described
previously (31). Dispersed pancreatic acini suspended with the fresh
PSS were washed twice and resuspended with 40 ml of cytosol buffer. The
cytosol buffer (pH 7.2 at 37°C) contained 0.2% bovine serum
albumin, 1 mM ATP, 1 mM creatine phosphate, 50 µg/ml creatine
phosphokinase, and (in mM) 20 NaCl, 0.5 MgSO4, 100 KCl, 0.2 NaH2PO4,
0.8 Na2HPO4, 10 HEPES, and 10 glucose. One hundred nanomolar
free-Ca2+ solution
was prepared with
Ca2+-Mg2+-ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) buffer (in mM: 2.55 CaSO4, 1.64 MgSO4, and 5 EGTA). Solutions with
different Ca2+ concentrations
([Ca2+]; 100 pM to 2 µM) were prepared by altering concentrations of both
CaSO4 and
MgSO4 in the cytosol buffer, which
contained 5 mM EGTA. Aliquots of SLO (1 U/ml) were added to a 40-ml
cell suspension that was suspended in 100 nM
Ca2+ cytosol buffer, incubated for
20 min, and gassed with 100% O2. After centrifugation (50 g), cells
were washed twice, resuspended with a 20-ml fresh and SLO-free cytosol
buffer (100 nM Ca2+), and
incubated in the presence or absence of various concentrations of C3
exoenzyme for 30 min at 37°C. After centrifugation and removal of
C3 exoenzyme, aliquots of the acinar cell suspension (2 ml each × 10 samples) were then incubated with buffers containing various
[Ca2+] gassed with
100% O2 for 30 min at 37°C.
The incubation was terminated by centrifugation (10,000 rpm) for 50 s
at 4°C in a microcentrifuge. The amylase that released into the
supernatant (1 ml of PSS × 2 in 10 groups) and that remained in
the pellet (1 ml of Triton X-100 × 2 in 10 groups) in each
microcentrifuge was assayed by using procion yellow starch as a
substrate. Amylase secretion was expressed as the percentage of the
total content in each sample.
Immunoprecipitation. Acinar cells (2 × 106) in 1 ml of PSS were
incubated with reagents for the indicated periods. The incubation was
stopped with 1 ml of chilled 8 mM HEPES buffer (pH 7.4) containing (in
mM) 1 sodium orthovanadate, 0.5 Na2HPO4,
109.5 NaCl, 4.7 KCl, and 1.13 MgCl2. The suspension was
immediately centrifuged (10,000 rpm) for 15 s at 4°C. The
supernatant was discarded, and the resultant pellet was resuspended in
chilled lysis buffer (pH 7.4) containing (in mM) 25
-glycerophosphate, 0.2 sodium orthovanadate, 1 phenylmethylsulfonyl fluoride, 5 EDTA, 1 dithiothreitol, 150 NaCl, and 50 tris(hydroxymethyl)aminomethane (Tris), 25 sodium fluoride and 0.2%
Triton X-100, 10 mg/ml leupeptin, and 0.05 trypsin inhibitor units
(TIU)/ml aprotinin. Each suspension was sonicated,
vortexed for 30 s, and centrifuged (10,000 rpm) for 10 min at 4°C.
The supernatant (crude microsomal fractions) was diluted with lysis
buffer to 1 mg/ml. Protein was determined by the method of Bradford
(3). Five hundred microliters of crude microsomes (0.5 mg protein) were
incubated with anti-Src monoclonal antibody (4 µg/ml) or
anti-RhoA monoclonal antibody (1 µg/ml) overnight at
4°C and with 50 ml of protein G-Sepharose for 2 h
at 4°C. The immunoprecipitates were washed three times with 100 mM Tris buffer, pH 7.5, containing 150 mM NaCl and 1.5 ml/l
Tween 20 and then analyzed by SDS-PAGE and Western blotting.
Western immunoblotting. Acinar cells
(2 × 106) in 0.5 ml of PSS
were incubated with reagents as described previously (34). The
incubation was stopped with 1 ml of chilled 8 mM HEPES buffer (pH 7.4)
containing (in mM) 1 sodium orthovanadate, 0.5 Na2HPO4, 109.5 NaCl, 4.7 KCl, and 1.13 MgCl2. The suspension was
immediately centrifuged (10,000 rpm) for 15 s at 4°C. The
supernatant was discarded, and the resultant pellet was resuspended in
150 µl of lysis solution (pH 7.4) containing (in mM) 66.7
-glycerophosphate, 1 sodium orthovanadate, 1 phenylmethylsulfonyl
fluoride, 1.5 EGTA, 1 dithiothreitol, 1% Triton X-100, 10 mg/ml
leupeptin, and 0.05 TIU/ml aprotinin. Each suspension was sonicated,
vortexed for 30 s at 4°C, and centrifuged at 10,000 rpm for 10 min.
The supernatant (25 µl, ~80 µg protein), which contained crude
microsomes, was mixed with 2.5 ml of 100%
-mercaptoethanol and 12.5 µl of Laemmli buffer (pH 7.8), containing 62.5 mM Tris base, 2.3%
SDS, 7.5% glycerol, and 0.1% bromphenol blue. Protein was determined
by the method of Bradford (3). The solution was heated at 95°C for
5 min and separated by SDS-PAGE on 15% polyacrylamide gels and
electrophoretically transferred to nitrocellulose membrane (Bio-Rad,
Hercules, CA) for 1 h at 100 V at 4°C. Immunoblotting was performed
with a mouse monoclonal RhoA antibody or a rabbit polyclonal RhoB
antibody (1 µg/ml). The membrane was then incubated with
peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (1:3,000
dilution). The blots were developed and visualized with a
chemiluminescent horseradish peroxidase substrate (enhanced chemiluminescence, Amersham, Arlington Heights, IL).
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RESULTS |
CCK-8 and carbachol but not high-affinity
CCK-A-receptor agonists increased the density of the immunodetectable
RhoA band. When we applied the RhoA (26C4) mouse
monoclonal IgG, a single band at the location of 21 kDa was detected in
unstimulated rat pancreatic acinar cells (Fig.
1). Treatment of pancreatic acini with
CCK-8 (10 pM to 10 nM) for 20 min resulted in a quantitative increase
in the intensities of the RhoA p21 in the Triton X-100-soluble and
crude microsomal fractions (10,000 rpm supernatant of the cell
sonicates; Fig. 1A). Similarly,
carbachol (0.1-100 µM) also increased the immunodetectable RhoA
bands in a dose-dependent manner (Fig.
1B). On the other hand, the
high-affinity CCK-A agonists JMV-180 and CCK-OPE (1-1,000 nM) did
not increase the intensities of the RhoA band over basal (Fig. 1,
C and
D). Densitometry data from the
scanned gels are summarized in Fig. 2.
These results indicate that stimulation of the
M3 muscarinic receptor as well as
the low- (but not high-) affinity CCK-A receptor resulted in a
quantitative increase in RhoA in crude microsomes. CCK-8 at 1, 10, and
100 pM increased the density of the RhoA band with a peak increase
occurring at 3 min, which was sustained up to 20-30 min (Fig.
3). Note that all immunoblotting or
immunoprecipitation data presented in Figs. 1-9 were performed
with the same experimental conditions (e.g., exposure time and amounts
of protein). Each figure was prepared from data conducted on the same
series of experiments.

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Fig. 1.
Western immunoblotting of RhoA with monoclonal anti-RhoA antibody.
Pancreatic acini were treated with or without cholecystokinin (CCK)-8
(A), carbachol
(B), JMV-180
(C), and CCK-OPE
(D) for 20 min at 37°C at
indicated concentrations. Protein from Triton X-100-soluble and crude
microsomal fractions (10,000 rpm supernatant of sonicates) was resolved
on 15% SDS-PAGE gel and transferred to nitrocellulose membrane
electrophoretically. Membrane was blotted with monoclonal anti-RhoA
antibody. Immunoreactive bands were visualized by goat anti-mouse IgG
and by enhanced chemiluminescence. US, unstimulated cells. Similar
results were observed in 4 experiments.
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Fig. 2.
Intensities of RhoA band after CCK-8, carbachol (CCh), JMV-180, and
CCK-OPE stimulations. Quantification of intensities of RhoA band was
performed by scanning densitometry. Data are means ± SE from 4 separate experiments. Note that all densitometry data, including Figs.
2, 3, 8, and 9, were calculated as a percentage of basal conducted on
same series of experiments.
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Fig. 3.
Intensities of RhoA band after CCK-8 stimulation. Acini were treated
with CCK-8 for times indicated at concentrations of
10 12
(A),
10 11
(B), and
10 10
(C) M. Triton X-100-soluble and
crude microsomes (10,000 rpm supernatant of sonicates) were used for
immunoblotting with monoclonal RhoA antibody. Similar results were
shown in 3 experiments for each concentration of CCK-8 tested.
D: quantification of intensities of
RhoA band was performed by scanning densitometry. Data are means ± SE from 3 separate experiments.
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RhoB could not be detected in pancreatic
acini. To determine whether other members of the
subfamily of Rho proteins are present in rat pancreatic acini, we
performed Western immunoblotting using RhoB and RhoA antibodies. It
should be noted that the RhoA monoclonal antibody does not cross-react
with RhoC, RhoG, Rac1, Rac2, or Cdc42Hs proteins, whereas the RhoB
polyclonal antibody has no cross-reactivity with RhoA, RhoC, or other
members of the Ras gene superfamily (tested by Santa Cruz
Biotechnology). In contrast to RhoA, no RhoB bands were detected in
unstimulated and CCK-stimulated pancreatic crude microsomes (Fig.
4).

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Fig. 4.
Intensities of RhoA and RhoB bands after CCK-8 stimulation. Acini were
stimulated with various concentrations of CCK-8 for 20 min at 37°C.
Crude microsomes were subjected to immunoblotting with RhoA
(A) and RhoB
(B) antibodies. Experiments were
repeated 3 times with each antibody tested.
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Stimulation of RhoA was independent of extracellular
[Ca2+]
but required intracellular
[Ca2+].
We next examined the requirement of
[Ca2+] on
CCK-stimulated RhoA activation. Elimination of extracellular
Ca2+ (by the addition of 1 mM EGTA
plus 0 CaCl2 in the PSS) did not inhibit mass increases in RhoA in response to various concentrations of
CCK-8 and carbachol (Fig. 5,
A-D). On the other hand,
preloading of pancreatic acini with the intracellular
Ca2+ chelator BAPTA-AM at 100 µM
for 10 min abolished the immunodetectable RhoA band in CCK- and
carbachol-stimulated pancreatic crude microsomes (Fig. 5,
E and
F, respectively). These results
suggest that stimulation of RhoA is independent of extracellular
[Ca2+]
([Ca2+]o)
but requires intracellular
[Ca2+]
([Ca2+]i).

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Fig. 5.
Effects of extracellular Ca2+ and
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-AM (BAPTA-AM) on RhoA after CCK-8 or carbachol stimulation. Acinar
cells were stimulated with CCK-8 (A
and C) or carbachol
(B and
D) in presence or absence of
extracellular concentration of
Ca2+
([Ca2+]o)
for 20 min at 37°C. Crude microsomes were used for immunoblotting
with monoclonal RhoA antibody.
[Ca2+]o-free
solution was identical to physiological salt solution (PSS) except that
CaCl2 was replaced with 1 mM EGTA.
In E and
F, acini were preincubated with
BAPTA-AM (10 4 M) for 10 min
at 37°C and further coincubated with CCK-8 and carbachol,
respectively, for 20 min. These results are representative of 3 independent experiments.
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Increases in immunodetectable RhoA were mediated by the
Gq protein and PLC pathway.
We have recently demonstrated that, depending on the CCK agonists used,
the CCK-A receptor in rat pancreatic acini is coupled to three
different effectors and/or signal transduction pathways (30-32, 34, 35). The low-affinity CCK-A receptor is coupled to the
conventional Gq
/PLC-
pathway, resulting in the production of
D-myo-inositol
1,4,5-trisphosphate (to release
Ca2+ and activate calmodulin) and
diacylglycerol (to activate PKC). In addition, the
low-affinity CCK-A receptor also appears to be coupled to the
nonreceptor protein tyrosine kinase pathway, in which
pp60c-src-phosphoinositide
3 kinase (PI 3-kinase) are the key enzymes in mediating
[Ca2+]o-dependent
pancreatic exocytosis. On the other hand, the high-affinity CCK-A
receptor is coupled to the
Gq
-cytosolic phospholipase
A2 (cPLA2) pathway to produce
arachidonic acid, which enhances intracellular Ca2+ oscillations. To determine
which pathways are involved in functional roles of the RhoA protein, we
examined the effects of specific inhibitors of these three signal
transduction pathways. The specificity of these inhibitors has been
previously determined (30-32, 34, 35). The G protein antagonist GP
antagonist-2A (10 µM), which selectively inhibits the activation of
Gq, and the PLC inhibitor U-73122
(10 µM) inhibited the intensities of the RhoA band evoked by CCK-8
(Fig. 6, A
and B). In contrast, the calmodulin
kinase inhibitor W-7 (100 µM) and the PI 3-kinase inhibitor
wortmannin (6 µM) had no effect (Fig. 6,
C and
E). The Src inhibitor herbimycin A
(6 µM) decreased the RhoA band in response to CCK-8 (Fig.
6D). In separate experiments, we
showed that the PKC activator TPA (10-1,000 nM) increased the
intensities of the RhoA band, whereas the PKC inhibitor K-252a, a
substance closely related to staurosporine, inhibited the action of
CCK-8 (Fig. 7,
A and
B). Densitometry data from these
results are shown in Fig. 8. These results
suggest that increases in immunodetectable RhoA are mediated by the
stimulation of PKC and Src cascades via the
Gq protein and PLC pathway.

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Fig. 6.
Effects of various antagonists on RhoA. Acini were pretreated with (+)
or without ( ) various antagonists for 5 min and further
coincubated with various concentrations of CCK-8 for 20 min at
37°C. Crude microsomes were used for immunoblotting with monoclonal
RhoA antibody. A:
10 5 M G protein
antagonist-2A (GP). B:
10 5 M U-73122.
C:
10 4 M W-7.
D: 6 × 10 6 M herbimycin A (HA).
E: 6 × 10 6 M wortmannin (WM). Each
set of experiments was repeated 3 times with similar results.
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Fig. 7.
Effects of protein kinase C (PKC) agonist
12-O-tetradecanoylphorbol 13-acetate
(TPA) and PKC antagonist K-252a on RhoA.
A: acini were incubated with TPA for
20 min at 37°C. B: acini were
pretreated with (+) or without ( ) K-252a
(10 6 M) for 5 min and
further coincubated with various concentrations of CCK-8 for 20 min at
37°C. Crude microsomes were utilized for immunoblotting with
monoclonal RhoA antibody. Similar results were obtained in 3 separate
sets of experiments.
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Fig. 8.
Intensities of RhoA band after CCK-8
(A and
C) or TPA
(B) stimulation with indicated
antagonists. Quantification of intensities of RhoA band was performed
by scanning densitometry. Data are means ± SE from 3 separate
experiments.
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RhoA was immunoprecipitated with Src.
To investigate possible interactions between RhoA and Src, we performed
immunoprecipitation studies. Previously, we demonstrated that CCK
stimulated phosphotransferase activities of Src in rat pancreatic acini
(34). This study showed that immunoblotting with Src antibody revealed
the presence of a single band corresponding to p60 in crude microsomes
(Fig.
9A). This band was quantitatively increased in response to CCK-8 stimulation in a dose-dependent manner. Association of Src with RhoA
was subsequently examined by the probing of Src immunoprecipitates with
RhoA antibody or vice versa (i.e., probing of RhoA immunoprecipitates
with Src antibody). Each immunoprecipitation revealed the presence of
the band corresponding to RhoA p21 (Fig.
9B) and
pp60c-src
(Fig. 9C). CCK-8 (10 pM-10 nM)
caused a quantitative increase in these bands in a dose-dependent
manner (Fig. 9D). These observations suggest that Src and RhoA formed an immunocomplex and CCK-8 enhanced a
significant increase in this complex.

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Fig. 9.
Effects of CCK-8 on Src and association of Src with RhoA. Acini were
incubated with CCK-8 for 20 min at 37°C. Triton X-100-soluble and
crude microsomes (10,000 rpm supernatant of sonicates) were
immunoprecipitated (IP) with anti-Src antibody (AB), analyzed by
SDS-PAGE, and immunoblotted (IB) with either anti-Src AB (Src-AB;
A) or anti-RhoA AB (Rho-AB;
B).
C: RhoA-AB was immunoprecipitated and
then Src-AB was used for blotting.
D: quantification of intensities of
Src and RhoA bands was performed by scanning densitometry. Data are
means ± SE from 4 separate experiments.
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C3 exoenzyme inhibited amylase secretion in
SLO-permeabilized pancreatic acini. We next examined
the functional role of RhoA in the mediation of pancreatic enzyme
secretion. The Clostridium botulinum C3 exoenzyme
has been shown to selectively ADP-ribosylate RhoA, -B, and -C, but not
Rac and Cdc42, at the asparagine-41 residue, blocking their action (1,
20, 21, 26). In rat pancreatic acini, it has been shown that C3
exoenzyme inhibited the increase of immunodetectable RhoA in response
to CCK-8 without affecting the production of inositol polyphosphates
(15). Because C3 exoenzyme is not cell permeable (tested by Biomol), we
evaluated the effect of C3 exoenzyme in SLO-permeabilized pancreatic
acini. Increasing
[Ca2+] from 100 pM to
2 µM resulted in an increase in amylase secretion (in %total/30 min)
from 6.3 ± 0.6 (n = 13 determinations) to 9.7 ± 0.7 (P < 0.005; n = 17 determinations). Pretreatment of permeabilized acini with
an inhibitor of Rho, C3 exoenzyme, for 30 min followed by incubation
with 2 µM Ca2+ for 30 min at
37°C resulted in a concentration-dependent inhibition of amylase
secretion with an IC50 of C3
exoenzyme at 1 ng/ml and the maximal inhibition occurring at 10 ng/ml
(Fig.
10A).
This IC50 value is one log unit
lower than that required to inhibit activities of the Rho subfamily in
other cell types (1, 21, 26). On the other hand, C3 exoenzyme did not
affect the minimum amylase secretion stimulated by 100 pM
Ca2+, which was lower than the
basal cytosolic [Ca2+]
(Fig. 10A). Increasing
[Ca2+] from 0.2 to 2 µM in the absence of C3 exoenzyme resulted in a significant increase
in amylase secretion. This
[Ca2+]-dependent
amylase secretion was significantly inhibited by 10 ng/ml of C3
exoenzyme (Fig. 10B). These
observations suggest that RhoA is involved in
[Ca2+]-dependent
pancreatic exocytosis.

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Fig. 10.
Effects of Rho inhibitor Clostridium
botulinum C3 exoenzyme on amylase secretion with
streptolysin O (SLO)-permeabilized pancreatic acini.
A: intact acini were permeabilized by
1 U/ml SLO for 20 min at 37°C in
10 7 M
Ca2+ cytosol buffer. After washout
of SLO, acini were incubated with various concentrations of C3 (20 min;
37°C) at 10 7 M
Ca2+ and permeabilized cells were
resuspended in either 10 10
M or 2 × 10 6 M
Ca2+ cytosol buffer. Amylase
secretion was measured at 30 min.
* P < 0.05, ** P < 0.01, *** P < 0.001 compared with
control (C3 at 0 ng/ml). B: intact
acini were permeabilized by 1 U/ml SLO for 20 min at 37°C at
10 7 M
Ca2+ cytosol buffer. After washout
of SLO, acini were incubated with various concentrations of
Ca2+ (2 × 10 7 to 2 × 10 6 M) with (10 ng/ml) or
without C3. Data are means ± SE from 4-8 separate experiments
(8-17 determinations). * P < 0.05 compared with C3 control by unpaired
t-tests.
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DISCUSSION |
Several lines of evidence suggest that the monomeric small GTP-binding
protein Rho plays an important role in mediating signal transduction in
various cell types (16). In this study, we demonstrated the existence
of Rho protein in rat pancreatic acini and its functional role in
mediating pancreatic enzyme secretion. We showed that, with the use of
an anti-RhoA mouse monoclonal antibody (26C4), a single band (p21)
could be detected by Western immunoblotting during the basal state in
rat pancreatic acini. On stimulation with CCK-8 or carbachol, an
increase in RhoA band intensities was observed. Peak increases in the
p21 RhoA band were observed when intact acini were stimulated with
submaximal concentrations of CCK-8 (
1 nM). Reduction of the
intensities of the RhoA band occurred with supramaximal concentrations
of CCK-8 (>1 nM). This phenomenon is similar to the biphasic
pancreatic amylase secretion stimulated by CCK-8, suggesting a
functional relationship between RhoA and amylase secretion. If RhoA
mediates cytoskeletal assembly, it is conceivable that high
concentrations of CCK-8 may disrupt the cytoskeletal system, resulting
in the reduction of enzyme secretion (19, 22).
In several cell types, it has been shown that the Rho protein may
translocate from the cytosol to the plasma membrane during receptor
activation (13, 14, 27, 36). Because we used Triton X-100-soluble and
crude microsomal fractions (10,000 rpm, supernatant of the cell
sonicates) for immunoblotting, it is unclear whether quantitative
increases in the RhoA band during cell stimulation are due to
translocation of RhoA from the cytosol to the plasma membrane or actual
activation of membrane RhoA proteins. One possibility is that RhoA may
translocate from Triton X-100-insoluble to -soluble fractions after
cell stimulation. Because RhoA but not RhoB proteins were increased
during CCK and carbachol stimulation, it appears that activation of the
Rho family by these agonists is protein specific. Our studies also
demonstrated that
[Ca2+]o
is not necessary for increases in RhoA in response to either CCK or
carbachol. On the other hand, application of the intracellular Ca2+ chelator BAPTA-AM
significantly inhibited the appearance of the RhoA band in response to
CCK or carbachol stimulation, suggesting that
[Ca2+]i
at the basal level is required for stimulation of RhoA.
The interrelationship between Rho and other serine/threonine kinases or
tyrosine kinases may vary in different cell systems. For example, the
tyrosine kinase
pp60c-src
regulates rearrangement of actin cytoskeleton and
p190Rho-GTPase activating protein (GAP) after
stimulation by epidermal growth factor in fibloblast cell lines
(6). On the other hand, it has been shown that, in Swiss
3T3 cells, translocation of Src kinase to the cell periphery is
mediated by the actin cytoskeleton under the control of the Rho family
(12). Furthermore, activation of PI 3-kinase in Swiss 3T3 cells and
platelets is dependent on Rho, suggesting that PI 3-kinase lies
downstream of Rho (20, 36). These observations suggest that there is a
close interaction between Rho and protein tyrosine kinases. On the
other hand, in several cell types, the serine/threonine kinase PKC has
been shown to activate Rho (28, 29). Our studies demonstrated that, in rat pancreatic acini, increases in immunodetectable RhoA are mediated by Gq and PLC pathways via the PKC
cascade but not calmodulin pathways. Furthermore, we showed that, in
contrast to CCK-8 and carbachol, high-affinity CCK-A-receptor agonists,
which fail to stimulate PKC (35), do not increase Rho. Thus it appears
that RhoA activation may depend on PKC stimulation at the basal level of
[Ca2+]i
rather than on the Ca2+ signal
frequency and amplitude (Ref. 4 and this study). In addition, in
response to CCK-8 stimulation, PKC translocation (30 s) precedes Rho
activation (3 min), suggesting that PKC lies upstream of Rho (Refs. 4
and 35 and this study). It is still unknown, however, whether PKC
directly or indirectly mediates increases in immunodetectable Rho. In
rat pancreatic acini, it has been shown that CCK stimulates the
formation of Shc-Grb2-SOS complex through a PKC-dependent mechanism
(7). It is conceivable that this complex may activate Rho to form
"GTP-bound Rho" in a manner similar to Ras (7, 10). Because PKC
is a serine/threonine kinase and Shc is a tyrosine-phosphorylated
protein, the precise mechanism by which PKC activates Shc remains to be determined.
Alternatively, our study showed that RhoA is also regulated by
pp60c-src.
This is because herbimycin A, which abolishes Src kinase activities in
response to CCK in rat pancreatic acini (34), inhibits RhoA intensities
stimulated by CCK. Because RhoA was coimmunoprecipitated with Src, RhoA
may interact with Src. The precise mechanism by which Src forms the
immunocomplex with Rho remains to be determined. Because Rho does not
possess Src homology domains, this interaction may be mediated by
Rho-GAP, which possesses Src homology domains. Indeed,
p190Rho-GAP is considered to be a preferred substrate of
c-Src in fibroblast cell lines (6). This possibility is supported by
the fact that Ras-GAP is known to be associated with
pp60c-src,
and the observation that tyrosine phosphorylation of Ras-GAP (by Src)
decreases its ability to enhance the GTPase activity, maintaining Ras
in its activated GTP-bound state (11). Because Rho possesses the formin
homology (FH) 1 domain, which is enriched in proline, this domain may
directly interact with the Src homology (SH) 3 domain of Src.
Alternatively,
p34cdc-2,
a substrate for
pp60c-src,
may be an intermediate between Src and Rho (34). Our laboratory (34)
previously identified
pp60c-src in
rat pancreatic acini, characterized its functional role in mediating
pancreatic enzyme secretion, and suggested its association with PI
3-kinase in response to CCK. Several lines of evidence reveal that PI
3-kinase is a direct substrate of
pp60c-src
(5). In other cell types, PI 3-kinase binds directly to Rho-guanosine 5'-O-(3-thiotriphosphate)
(GTP
S) and is thereby activated, probably via the
Rho-GAP-like domain located on the p85 regulatory subunit of PI 3-kinase (36). These observations suggest possible interactions among Src, Rho, and PI 3-kinase. Furthermore, it has recently been reported (15) that, in rat pancreatic acini, Rho plays a role in
the ability of CCK to stimulate tyrosine phosphorylation of focal
adhesion kinase (p125FAK) and the cytoskeletal protein
paxillin utilizing PLC-dependent and -independent pathways. Because
pancreatic acini possess actin and myosin and actin-associated
proteins (e.g., vinculin and villin) that are preferred substrates of
Src (9, 18), it is conceivable that Src, RhoA, PI 3-kinase, and focal
adhesion kinase may reciprocally act on the actin cytoskeleton in
mediating pancreatic exocytosis.
Finally, we determined the functional role of RhoA in the mediation of
pancreatic enzyme secretion. We demonstrated that the Rho inhibitor C3
exoenzyme inhibited
[Ca2+]-dependent
amylase secretion in permeablized acini, suggesting that RhoA is likely
to play a role in pancreatic exocytosis. Although C3 exoenzyme
inhibited
[Ca2+]-dependent
enzyme secretion in permeabilized acini, the high-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE, which elicit intracellular Ca2+ oscillations (30-32,
35), did not increase the amount of Rho. This suggests that
[Ca2+] in permeable
cells may directly act on the Rho-associated actin cytoskeleton in
mediating enzyme secretion, in which small soluble proteins are
extruded from the cells. It has recently been reported that
pretreatment of intact rat pancreatic acini with the high concentration
of C3 exoenzyme (25 µg/ml) for 120 min resulted in a significant
inhibition of amylase secretion elicited by either CCK or TPA. This
result further supports the interaction between PKC and Rho on
pancreatic enzyme secretion (25). In conclusion, the small GTP-binding
protein RhoA p21 appears to be involved in the mediation of pancreatic
enzyme secretion. RhoA pathways are involved in the activation of
PKC and
pp60c-src
cascades via Gq protein and PLC.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Koji Nakao for valuable suggestions and technical advice.
 |
FOOTNOTES |
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-32830 and P30-DK-34933.
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: C. Owyang, Univ.
of Michigan Medical Center, 3912 Taubman Center, Box 0362, Ann Arbor,
MI 48109.
Received 20 April 1998; accepted in final form 30 December 1998.
 |
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