Sequential activation of heterotrimeric and monomeric G
proteins mediates PLD activity in smooth muscle
K. S.
Murthy,
H.
Zhou,
J. R.
Grider, and
G. M.
Makhlouf
Departments of Medicine and Physiology, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia
23298
 |
ABSTRACT |
The identity of G
proteins mediating CCK-stimulated phospholipase D (PLD) activity was
determined in intestinal smooth muscle cells. CCK-8 activated
Gq/11, G13, and G12, and the
monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation
factor (ARF). Activation of RhoA, but not ARF, was mediated by
G13 and inhibited by G
13 antibody.
CCK-stimulated PLD activity was partly mediated by RhoA and could be
inhibited to the same extent (47 ± 2% to 53 ± 6%) by
1) a dominant negative RhoA mutant, 2) RhoA antibody or G
13 antibody, and 3)
Clostridium botulinum C3 exoenzyme. PLD activity was also
inhibited by ARF antibody, and the effect was additive to that of RhoA
antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C,
bisindolylmaleimide I, and a selective protein kinase C (PKC)-
inhibitor; the inhibition was additive to that of ARF and RhoA
antibodies and C3 exoenzyme. In contrast, activated G12 was
not coupled to RhoA or ARF, and G
12 antibody augmented
PLD activity. Thus agonist-stimulated PLD activity is mediated
additively by G13-dependent RhoA and by ARF and PKC-
and
is modulated by an inhibitory G12-dependent pathway.
G12; protein kinase C; phospholipase D; intestinal
smooth muscle
 |
INTRODUCTION |
A PHOSPHOLIPASE
D (PLD) GENE superfamily characterized by a number of conserved
structural domains and sequence motifs has been identified in various
species (6, 7, 12). PLD is widely distributed in mammalian
tissues and located in cellular membranes and the cytosol. The specific
substrate for the main mammalian isoforms, PLD1a and PLD1b, is
phosphatidylcholine (PC). Phosphatidic acid (PA) and choline, the
primary products of PC hydrolysis by PLD, are rapidly converted to
diacylglycerol (DAG) and phosophocholine by phosphatidate
phosphohydrolase and choline kinase, respectively (9, 19).
Dephosphorylation of PA is a major source of agonist-stimulated,
sustained DAG production in various tissues, including smooth muscle,
resulting in sustained activation of protein kinase C (PKC) (9,
26). PLD also catalyses transphosphatidylation, in which a
phosphatidyl group of PC is transferred to glycerol or a primary
alcohol (9, 26). This PLD-specific reaction has
facilitated analysis of the regulation of PLD.
We (26) have previously shown that in intestinal smooth
muscle, agonist-stimulated PLD activity is preceded by, but independent of, phosphatidylinositol (PI) hydrolysis via PLC-
. PLD activity was
modulated by PKC but did not require an increase in resting intracellular Ca2+ levels (26). Sustained PLD
and PKC activities were abolished by guanosine
5'-O-(2-thiodiphosphate) (GDP
S) and appeared to be
regulated by a G protein(s) distinct from members of the Gq or Gi/o families (25, 26). In various cells,
PLD activity can be modulated by phosphatidylinositol 4,5-bisphosphate
and/or fatty acids, such as oleate (9, 21, 31).
Recent studies (3, 5, 13, 18-20, 22, 23, 31, 34) have
examined the upstream pathways linking the receptor to PLD activity, in
particular the participation of heterotrimeric and monomeric G
proteins. Two monomeric G proteins, the Ras-homology protein
(RhoA) and the ADP ribosylation factor (ARF), have been identified that
vary in their ability to activate PLD in different tissues (3, 5,
18-20, 31, 34). In the resting state, RhoA, like other Rho
family G proteins, is bound to a GDP-dissociation inhibitor in the
cytoplasm. Agonist-receptor binding activates a specific guanine
nucleotide exchange factor (Rho-GEF) that promotes dissociation of the
inhibitor from RhoA, translocation of RhoA to membranes, and activation
of RhoA by exchange of GTP for GDP (13). A similar process
promotes the translocation of ARF to membranes and its activation by
GTP/GDP exchange (22, 23). There is also substantial
evidence that either or both G13 and G12 are
involved in mediating receptor-dependent activation of RhoA (10,
18, 28). The involvement of either G protein is often cell and
agonist specific. Microinjection of G
12 or
G
13 into Swiss 3T3 fibroblasts stimulates the formation
of stress fibers, a process mediated by RhoA (4).
Thrombin, however, stimulates stress fiber formation via
G12 only and lysophosphatidic acid via G13 only
(18). In CCL39 fibroblasts, Rho-dependent stimulation of
Na+/H+ exchange is activated by G13
but inhibited by G12 (17).
The existence of pathways involving sequential coupling of
heterotrimeric (G13 and G12) and monomeric
(RhoA and ARF) G proteins to activation of PLD in smooth muscle has not
been determined. In a recent study of cultured vascular smooth muscle,
PLD activity induced by angiotensin II was shown to be partly inhibited
by RhoA and G
12 antibodies, as well as by G
and
pp60src antibodies, suggesting involvement of
G
-Src and G12-RhoA pathways (34). In the present study, we examined the roles
of G13 and G12 in activation of RhoA and ARF
and of both monomeric G proteins in activation of PLD. CCK-8 was shown
to stimulate PLD activity additively via RhoA, ARF, and PKC-
. CCK
activated both G13 and G12, but only
G13 was coupled to activation of RhoA, Rho kinase (ROK),
and PLD. ARF activation was not mediated by either G13 or
G12, and activation of G12 resulted in
inhibition of PLD.
 |
MATERIALS AND METHODS |
Dispersion of intestinal smooth muscle cells.
Smooth muscle cells were isolated from the circular muscle layer of
rabbit intestine by sequential enzymatic digestion, filtration, and
centrifugation as described previously (24-26).
Muscle strips were incubated for 60 min at 31°C in 15 ml of HEPES
medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin
inhibitor with no added Ca2+. The composition of the medium
was 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's
essential amino acid mixture. The partly digested tissue was washed
with 100 ml of enzyme-free medium and reincubated for 40-60 min to
allow spontaneous dispersion of muscle cells. The cells were harvested
by filtration through 500-µm Nitex mesh, centrifuged twice for 10 min
at 350 g, and resuspended in HEPES medium containing 2 mM
Ca2+. In some experiments, the cells were permeabilized by
incubation for 5 min with saponin (35 µg/ml) in a
low-Ca2+ (100 nM) medium as described previously (24,
25) and resuspended in saponin-free medium with 1.5 mM ATP and
ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase).
Identification of receptor-activated G proteins in membranes.
G proteins selectively activated by CCK-8 were identified by an
adaptation of the method of Okamoto et al. (29) as
described previously (24). Muscle cells were homogenized
in 20 mM HEPES medium (pH 7.4). After centrifugation at 25,000 g for 15 min, the membranes were solubilized at 4°C in 20 mM HEPES medium (pH 7.4) and 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The
membranes were incubated with 60 nM 35S-labeled guanosine
5'-O-(3-thiotriphosphate) ([35S]GTP
S) in a
medium containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM
MgCl2 for 20 min at 37°C in the presence or absence of
CCK-8 (1 nM). The reaction was stopped with 10 vol of 100 mM Tris · HCl medium (pH 8.0) containing 10 mM MgCl2,
100 mM NaCl, and 20 µM GTP, and the solubilized membranes were
incubated for 2 h on ice in wells precoated with specific
antibodies to G
q/11, G
13, and
G
12. The wells were washed three times with phosphate buffer containing 0.05% Tween 20, and the radioactivity in each well
was counted.
Measurement of expression, translocation, and activation of RhoA
and ARF.
Muscle cells were homogenized in a solution containing 10 mM
Tris · HCl (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 250 mM
sucrose, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride,
20 µg/ml leupeptin, and 20 µg/ml aprotinin. The suspension was
centrifuged at 100,000 g for 30 min at 4°C, and the
supernatant was collected as the cytosolic fraction. Pellets were
resuspended, and proteins were extracted by incubation for 30 min in
the homogenization buffer containing 1% Triton X-100 and 1% sodium
cholate. The extract was centrifuged at 1,000 g for 10 min,
and the supernatant was collected as the particulate fraction. Proteins
(80-100 µg) were resolved by 12% SDS-PAGE and
electrophoretically transferred to nitrocellulose membranes. After
incubation in 5% nonfat dry milk to block nonspecific antibody
binding, the blots were incubated first with antibodies to RhoA or ARF
and then with secondary antibodies conjugated with horseradish
peroxidase. The bands were identified by enhanced chemiluminescence.
RhoA and ARF activities were measured in muscle cells incubated for
3 h in low-phosphate (0.12 mM NaH2PO4)
buffer containing 10 mM HEPES, 2.5 mM glucose, 1% BSA, and 10 mCi of
32PO4. Aliquots (2 × 106
cells) were treated with CCK-8 (1 nM) for 10 min, and the reaction was
stopped with lysis buffer containing 20 mM Tris · HCl (pH 7.4),
250 mM sucrose, 150 mM NaCl, 2 mM EGTA, 10 mM MgCl2, 1 mM Na2P2O7, 1 mM NaF, 1 mM
Na3VO4, 1% Triton X-100, 0.5% Nonidet, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 20 µg/ml
aprotinin. RhoA and ARF were immunoprecipitated separately, using
specific antibodies, washed three times with lysis buffer, and boiled
for 20 min at 68°C in buffer containing 5 mM EDTA, 2 mM DTT, 0.2%
SDS, 0.5 mM GTP, and 0.5 mM GDP. GTP and GDP were separated on
polyethylene-cellulose plates developed with 1 M KH2PO4 (pH 3.4) and measured by autoradiography.
Assay for PLD activity.
PLD activity was determined by the formation of phosphatidylethanol
(PEt), a specific product of PLD activity in the presence of ethanol.
Muscle cells (2 × 106 cells/ml) were incubated with
[3H]myristic acid (2 µCi/ml) for 3 h and then with
150 mM ethanol for 15 min at 31°C in HEPES medium. The cells were
then centrifuged at 350 g for 10 min to remove excess
[3H]myristic acid and resuspended in fresh medium. CCK-8
(1 nM) was added for 10 min, and the reaction was terminated by the
addition of 1.8 ml of chloroform-methanol-HCl (100:200:2, vol/vol/vol) and extracted by the method of Bligh and Dyer (2) as
described previously (26). The organic phase was dried
under N2 and analyzed for [3H]PEt by TLC on
silica gel plates (dipped in 1% potassium oxalate), with ethyl
acetate-2,2,4-trimethylpentane-acetic acid-water (13:2:3:10) as a
running solvent. [3H]PEt was identified using unlabeled
standards, which were sprayed with 0.1% 1,2-dichlorofluorescein in
isopropyl alcohol and visualized under ultraviolet light at 357 nm. The
spots corresponding to PEt were scraped and counted by liquid scintillation.
Transfection of dominant negative RhoA cDNA into cultured smooth
muscle.
Dominant negative RhoA cDNA was subcloned into the multiple cloning
site (EcoR I) of the eukaryotic expression vector pEXV. A
myc tag was incorporated into the NH2 terminus.
Recombinant plasmid DNAs were transiently transfected into the muscle
cells in primary culture using Lipofectamine Plus reagent. Cells were cotransfected with 2 µg of pEXV-myc tag RhoA dominant
negative and 1 µg of pGreen Lantern-1 for 48 h. Control cells
were cotransfected with 2 µg pEXV vector and 1 µg of pGreen
Lantern-1 DNA. Transfection efficiency was monitored by the expression
of the green fluorescent protein using FITC filters. In the RhoA
dominant negative mutant, asparagine was substituted for serine at
position 19 (N19RhoA).
Materials.
[3H]myristic acid (22.4 Ci/mmol) and carrier-free
[32P]Pi were obtained from NEN Life Science
Products (Boston, MA). Collagenase type II and soybean trypsin
inhibitor were from Worthington Biochemicals (Freehold, NJ). Polyclonal
antibodies to G
13, G
12,
G
q/11, RhoA, and ARF were from Santa Cruz Biotechnology,
(Santa Cruz, CA), and all other chemicals were from Sigma Chemical (St.
Louis, MO). Dominant negative RhoA cDNA was a gift of Dr. Andrea
Todisco, University of Michigan. Myristoylated pseudosubstrate peptide inhibitors of PKC isoforms were a gift from Drs. A. Dartt and D. Zoukhri, Harvard Medical School.
 |
RESULTS |
Expression and receptor-mediated activation of G13 and
G12 in intestinal smooth muscle.
Previous studies (24) have shown that several G proteins
(Gq/11, Gs, Gi-1, Gi-2,
and Gi-3) are expressed in intestinal smooth muscle, where
they are coupled to various receptors. Western blot analysis in the
present study showed that G13 and G12 are also expressed in intestinal smooth muscle cells (Fig.
1). CCK-8, a ligand previously shown to
activate Gq/11, also activated G13 and
G12, significantly increasing the binding of
[35S]GTP
S to G
q/11,
G
13, and G
12 by 75 ± 4%
(P < 0.01), 86 ± 17% (P < 0.01), and 102 ± 14% (P < 0.01),
respectively (Fig. 1).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Expression and activation of G 13,
G 12, and G q/11 in intestinal smooth
muscle. A: Western blot analysis was performed in
homogenates prepared from dispersed intestinal circular muscle cells
using specific antibodies to G 13, G 12,
and G q/11. B: agonist-induced activation of G
proteins was measured by the increase in binding of guanosine
5'-O-(3-thiotriphosphate) (GTP S) to G 13,
G 12, and G q/11. Intestinal muscle cell
membranes were solubilized with 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and
incubated with 60 nM [35S]GTP S in the presence or
absence of 1 nM CCK for 20 min. Aliquots were added to wells precoated
with G 13, G 12, or G q/11
antibody for 2 h, and bound radioactivity was measured and
expressed as counts/min (cpm)/mg protein. CCK-8 caused a significant
increase in the binding of [35S]GTP S to
G 13, G 12, and G q/11.
Values are means ± SE of 4 experiments. ** P < 0.01.
|
|
Receptor-mediated translocation and activation of RhoA and ARF.
Western blot analysis showed that the monomeric G proteins RhoA and ARF
were present mainly in the cytosolic fraction in the resting state, but
increased significantly in the membrane fraction after stimulation of
the muscle cells with CCK-8 (Figs. 2 and 3).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
CCK-induced translocation and activation of Ras-homology
protein (RhoA) in intestinal smooth muscle. A: Western blot
analysis using RhoA antibody was performed on cytosolic (C) and
particulate (M) fractions prepared from muscle cell homogenates before
and after treatment with 1 nM CCK-8. An increase in binding of RhoA to
membranes was observed after treatment of the cells with CCK-8.
B: activation of RhoA was measured in permeabilized muscle
cells labeled for 3 h with 32PO4 and
treated for 10 min with CCK-8 (1 nM). Experiments were done in control
cells and in cells incubated for 1 h with antibody (Ab) to
G 13 or G 12. [32P]GTP
binding in RhoA immunoprecipitates was determined by TLC followed by
autoradiography. Results are expressed as %GTP incorporation. GTP
binding to RhoA was blocked by pretreatment of cells with antibody to
G 13 but not G 12. Values are means ± SE of 4-5 experiments. ** P < 0.01.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
CCK-induced translocation and activation of ADP
ribosylation factor (ARF) in intestinal smooth muscle. A:
Western blot analysis using ARF antibody was performed on cytosolic and
particulate fractions prepared from muscle cell homogenates before and
after treatment with 1 nM CCK-8. An increase in binding of ARF to
membranes was observed after treating the cells with CCK-8.
B: activation of ARF was measured in permeabilized muscle
cells labeled for 3 h with 32PO4 and
treated for 10 min with CCK-8 (1 nM). Experiments were done in control
cells and in cells incubated for 1 h with antibody to
G 13 or G 12. [32]GTP binding
in ARF immunoprecipitates was determined by TLC followed by
autoradiography. Results are expressed as %GTP incorporation. GTP
binding to ARF was not blocked by pretreatment of cells with
G 13 or G 12 antibody. Values are
means ± SE of 4-5 experiments. ** P < 0.01.
|
|
CCK-induced translocation of RhoA and ARF to the membrane was
accompanied by a significant increase in the activities of both G
proteins as indicated by the increase in the incorporation of [32P]GTP (Figs. 2 and 3). RhoA activity was inhibited
74 ± 9% (P < 0.01) by preincubation of
permeabilized muscle cells for 1 h with G
13
antibody (5 µg/ml), whereas ARF activity was not affected (Figs. 2
and 3). Preincubation with G
12 antibody had no effect on
either RhoA or ARF activity (Figs. 2 and 3).
G13-dependent activation and G12-dependent
inhibition of PLD.
CCK caused a sustained fourfold increase in PLD activity as determined
by the formation of [3H]PEt [basal: 455 ± 71 counts/min (cpm)/106 cells; CCK-8: 2,415 ± 158 cpm/106 cells]. CCK-stimulated PLD activity was inhibited
in a concentration-dependent fashion by the PLD inhibitor PCCG-16 with
an EC50 of 0.1 µM. PLD activity was also inhibited in a
concentration-dependent fashion by preincubation of permeabilized
muscle cells for 1 h with G
13 antibody (0.1-10
µg/ml); a maximal inhibition of 47 ± 2% (P < 0.001) was elicited with 5 µg/ml of antibody (Fig.
4). In contrast, preincubation with
G
12 antibody (10 µg/ml) increased PLD activity by
33 ± 2% (P < 0.001). Preincubation with
antibodies to G
q/11, Gi
1-2, Gi
3,
and G
had no effect on PLD activity.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibition of CCK-stimulated phospholipase D (PLD)
activity in intestinal smooth muscle by G 13 and RhoA
antibodies. Muscle cells were labeled with [3H]myristic
acid for 3 h, and CCK (1 nM)-induced PLD activity was determined
by the formation of phosphatidylethanol ([3H]PEt) in the
presence of ethanol. Results are expressed as cpm/106
cells. Preincubation of permeabilized muscle cells for 1 h with
G 13 antibody or RhoA antibody (0.1-10 µg/ml)
inhibited PLD activity in a concentration-dependent fashion. Values are
means ± SE of 4 experiments.
|
|
RhoA- and ARF-dependent activation of PLD.
The ability of RhoA to activate PLD was examined 1) in
cultured smooth muscle cells transfected with a dominant negative
mutant of RhoA (N19RhoA), 2) in permeabilized, freshly
dispersed smooth muscle cells incubated for 1 h with RhoA
antibody, and 3) in intact smooth muscle cells incubated for
3 h with the Clostridium botulinum C3 exoenzyme.
Transfection of cultured muscle cells with a dominant negative RhoA
mutant inhibited CCK-stimulated PLD activity by 49 ± 8% (P < 0.01; n = 5) (Fig.
5). Preincubation of freshly dispersed permeabilized smooth muscle cells with RhoA antibody (0.1-10
µg/ml) inhibited CCK-stimulated PLD activity in a
concentration-dependent fashion with a maximal inhibition of 53 ± 6% (P < 0.01) at 5 µg/ml (Fig. 4). Preincubation of
freshly dispersed intact smooth muscle cells with C3 exoenzyme
(0.2-2 µg/ml), which inactivates RhoA by ADP ribosylation of
Asn41, inhibited CCK-stimulated PLD activity in a
concentration-dependent fashion with a maximal inhibition of 52 ± 3% (P < 0.001) at 2 µg/ml (Fig.
6). The inhibition was similar to that
elicited by RhoA antibody or by transfection of the dominant negative
RhoA mutant. A combination of RhoA antibody (5 µg/ml) and C3
exoenzyme (2 µg/ml) was not additive (54 ± 5% inhibition). A
similar degree of inhibition by G
13 antibody and RhoA
antibody or C3 exoenzyme was consistent with the ability of
G13 to stimulate PLD by activating only RhoA. HA-1077,
which preferentially inhibits RhoA kinase activity, inhibited
CCK-stimulated PLD activity by 29 ± 3% (P < 0.01) when used at an EC50 of 10 µM. At this
concentration, HA-1077 has only a minimal effect on PKC activity
(<10%) (33). PLD activity stimulated by GTP
S (100 µM) was inhibited 49 ± 4% (P < 0.01) by RhoA
antibody (5 µg/ml) and to the same extent (53 ± 4%;
P < 0.01) by C3 exoenzyme. A combination of C3
exoenzyme and RhoA antibody did not elicit greater inhibition (54 ± 6%). PLD activity stimulated by phorbol 12-myristate 13-acetate (1 µM) was not affected by pretreating the cells with either C3
exoenzyme or RhoA antibody.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of CCK-stimulated PLD activity in cultured
smooth muscle cells expressing a dominant negative mutant of RhoA
(N19RhoA). Muscle cells were labeled with [3H]myristic
acid for 3 h, and PLD activity induced by CCK-8 (1 nM) was
determined by the formation of [3H]PEt in the presence of
ethanol. Results are expressed as cpm/106 cells. In muscle
cells transfected with vector only, basal and CCK-stimulated PLD
activities were similar to those in freshly dispersed muscle cells.
CCK-stimulated PLD activity was inhibited 49 ± 8% in cells
expressing N19RhoA. Values are means ± SE of 5 experiments.
** P < 0.01, significant inhibition.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of CCK-stimulated PLD activity in intestinal
smooth muscle by Clostridium botulinum C3 exoenzyme. Muscle
cells were labeled with [3H]myristic acid for 3 h,
and PLD activity induced by CCK-8 (1 nM) was determined by the
formation of [3H]PEt in the presence of ethanol. Results
are expressed as cpm/106 cells. Preincubation of muscle
cells for 3 h with C3 exoenzyme inhibited PLD activity in a
concentration-dependent fashion. Maximal inhibition was similar to that
obtained with RhoA and G 13 antibodies (see Fig. 4).
Values are means ± SE of 4 experiments.
|
|
Preincubation of permeabilized smooth muscle cells for 1 h with
ARF antibody (5 µg/ml) inhibited CCK-stimulated PLD activity by
29 ± 9% (P < 0.05) (Fig.
7). The inhibition was additive to that
elicited by RhoA antibody (5 µg/ml) or C3 exoenzyme (2 µg/ml) (78 ± 3% and 75 ± 6% inhibition, respectively) (Fig. 7).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of CCK-stimulated PLD activity in intestinal
smooth muscle by combinations of ARF antibody with RhoA antibody or C3
exoenzyme (C3). PLD activity was measured as described in the Fig. 4
legend, and the results are expressed as cpm/106 cells. A
combination of ARF antibody (5 µg/ml) with either RhoA antibody (5 µg/ml) or C3 exoenzyme (2 µg/ml) caused additive inhibition. A
combination of C3 exoenzyme and RhoA antibody was not additive (see
RESULTS). Values are means ± SE of 5-6
experiments. ** P < 0.01, significant inhibition
from control CCK response; ## P < 0.01, significantly different from inhibition by each agent alone.
|
|
PKC-dependent activation of PLD.
Calphostin C, which blocks the DAG-binding site of PKC, and
bisindolylmaleimide I, which blocks the ATP-binding site, inhibited CCK-stimulated PLD activity by 30 ± 3% and 30 ± 2%,
respectively (P < 0.01) (Fig.
8). A selective myristoylated
pseudosubstrate peptide inhibitor of PKC-
and a common inhibitor of
PKC-
,
,
inhibited PLD activity to the same extent as calphostin
C (32 ± 3% and 29 ± 2%, respectively; P < 0.01); a selective pseudosubstrate inhibitor of PKC-
had no
effect (1 ± 2%) (Fig. 8). A combination of calphostin C with
either PKC-
or PKC-
,
,
inhibitors was not additive (30 ± 4% and 31 ± 5%, respectively). The pattern implied that
PKC-dependent activation of PLD was mediated by PKC-
.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 8.
Inhibition of CCK-stimulated PLD activity in intestinal
smooth muscle by selective protein kinase C (PKC) inhibitors. PLD
activity was measured as described in the Fig. 4 legend, and the
results are expressed as cpm/106 cells. PLD activity was
significantly inhibited by bisindolylmaleimide (Bis, 1 µM),
calphostin C (CalC, 1 µM), and myristoylated pseudosubstrate peptide
inhibitors of PKC- and PKC , , (1 µM). Values are
means ± SE of 4 experiments. ** P 0.01, significantly different from control CCK response.
|
|
The ability of PKC to activate PLD was additive to that of either RhoA
or ARF. A combination of calphostin C with RhoA antibody or C3
exoenzyme inhibited CCK-stimulated PLD activity by 75 ± 5% and
73 ± 4%, respectively, and a combination with ARF antibody inhibited PLD activity by 58 ± 3% (Fig.
9). Combining calphostin C with both RhoA
and ARF antibodies inhibited PLD activity by 83 ± 2% (Fig. 9),
whereas a combination of calphostin C with both C3 exoenzyme and ARF
antibody inhibited PLD activity by 86 ± 2%.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 9.
Inhibition of CCK-stimulated PLD activity in intestinal
smooth muscle by combinations of calphostin C with ARF and RhoA
antibodies. PLD activity was measured as described in the Fig. 4
legend, and the results are expressed as cpm/106 cells.
Inhibition induced by RhoA or ARF antibody was additive to that of
calphostin C. Values are means ± SE of 5-6 experiments.
** P < 0.01, significant inhibition from control
CCK response; ## P < 0.01, significantly different from inhibition by each agent alone.
|
|
 |
DISCUSSION |
This study shows that agonist-induced, sustained activation of PLD
involves a distinct set of heterotrimeric and monomeric G proteins. The
pathways involved are depicted schematically in Fig.
10. CCK-8 activated the heterotrimeric
G proteins G13 and G12 and the monomeric G
proteins RhoA and ARF. The
-subunit of G13, but not
G12, was coupled to sequential activation of RhoA and PLD.
RhoA was the dominant activator of PLD, accounting for 50% of the
response, and its effect appeared to be mediated by ROK. ARF also
activated PLD, but its effect was not mediated by either
G13 or G12. The effects of RhoA and ARF were
additive to those of PKC-
, the specific isoform that mediates
activation of PLD by PKC. Unexpectedly, the activation of
G12 by CCK-8 resulted in inhibition of PLD. The evidence is
summarized as follows.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 10.
Schema depicting the role of heterotrimeric G proteins
(G13 and G12), monomeric G proteins (RhoA and
ARF), and PKC- in the regulation of agonist-stimulated sustained PLD
activity in intestinal smooth muscle. Receptor-linked agonists (e.g.,
CCK-8) activate both G13 and G12.
G 13 initiates a cascade involving sequential activation
of a Rho-specific, guanine nucleotide exchange factor (Rho-GEF), RhoA,
Rho-associated kinase (ROK), and PLD. G 12 mediates an
inhibitory pathway that attenuates agonist-induced activation of PLD;
steps in the pathway initiated by G12 have not been
identified. Agonist-induced activation of ARF enhances PLD activity;
steps in the pathway involving ARF have not been identified.
Dephosphorylation of the phosphatidic acid (PA), the primary product of
PLD, yields diacylglycerol (DAG), which activates
Ca2+-dependent and -independent PKC isozymes, one of which,
the Ca2+-dependent PKC- , acts to enhance PLD activity.
Thus agonist-stimulated, sustained PLD activity is mediated additively
by G13-dependent RhoA and by ARF and PKC- and is
modulated by an inhibitory G12-dependent pathway
(stimulation and inhibition denoted by + and , respectively).
|
|
Receptor-mediated activation of G13 and G12
and its relation to PLD activity.
CCK-8 activated three heterotrimeric G proteins (Gq/11,
G13, and G12), only one of which,
G13, was coupled to sustained activation of PLD.
G
13 antibody inhibited CCK-stimulated PLD activity,
whereas G
12 antibody increased CCK-stimulated PLD
activity, suggesting that G12 mediated an inhibitory
pathway. Antibodies to the
-subunits of Gq/11,
Gi1-2, Gi3, and Gs, and a
common antibody to G
had no effect.
Coupling of G13 to RhoA but not ARF.
CCK-8 induced translocation of RhoA and ARF to the membrane and
activated both monomeric G proteins as indicated by the increase in GTP
binding to RhoA and ARF. G
13 antibody but not
G
12 antibody inhibited activation of RhoA; neither
G
13 nor G
12 antibody had any effect on
ARF. Thus in intestinal smooth muscle, only G13 and RhoA
were sequentially coupled.
Activation of PLD via RhoA and ARF.
Agonist-stimulated PLD activity was mediated additively by RhoA and
ARF. RhoA antibody, the Clostridium botulinum C3 exoenzyme, and a dominant negative RhoA mutant transfected into cultured muscle
cells inhibited agonist-stimulated PLD activity to the same extent
(49% to 53%). The extent of inhibition was similar to that obtained
with G
13 antibody (47%), consistent with sequential activation of G13, RhoA, and PLD. ARF antibody inhibited
PLD activity to a lesser extent (29%), and its effect was additive to
that of RhoA antibody or C3 exoenzyme (75% to 78%), suggesting that RhoA and ARF activate PLD via distinct mechanisms. The effect of RhoA
appeared to be mediated by ROK and was inhibited by HA-1077, a
preferential inhibitor of ROK (30, 33).
Neither the mechanism by which CCK induced activation of ARF and its
translocation to membranes nor the mechanism by which ARF activated PLD
was identified. As noted above, G
12 and
G
13 antibodies did not block receptor-mediated
activation of ARF. It seemed unlikely that G
subunits that can
bind to ARF were involved in its activation, because a common G
antibody had no effect on CCK-stimulated PLD activity.
Activation of PLD by PKC.
Activation of PLD by PKC has been demonstrated in various cell types
and represents a feedback mechanism, because DAG, the main activator of
PKC, is largely generated by dephosphorylation of PA, the primary
product of PLD activity (1, 9, 32). Previous studies
(26) on intestinal circular and longitudinal smooth muscle
have shown that agonist-stimulated PLD activity was partly inhibited by
calphostin C. The present study confirmed that PLD activity was partly
inhibited by calphostin C, as well as by selective inhibitors of
PKC-
. The involvement of PKC-
, and to a lesser extent PKC-
I
and -
II, has been demonstrated in other cell types (1, 9,
32). In the present study, the inhibition of PLD activity by
calphostin C was additive to that of RhoA or ARF antibodies, applied
separately or in combination.
The mechanism by which PKC activates PLD has not been fully
established. In vitro studies suggest that the stimulatory activity of
PKC resides in its regulatory domain, because PKC fragments devoid of
catalytic domain retain their stimulatory activity in vitro, and
inhibitors of catalytic activity appear to be ineffective (8). In vivo, however, as in the present study, blockers
of the regulatory and catalytic domains of PKC inhibited PLD activity (1, 8, 9). Because no evidence exists for direct
phosphorylation of PLD by PKC, the effectiveness of both types of
inhibitors suggests that PKC may act indirectly on PLD via an
intermediate susceptible to stimulatory phosphorylation.
A recent study by Ushio-Fukai et al. (34) showed that
angiotensin II-stimulated PLD activity in cultured vascular smooth muscle was inhibited by antibodies to G
12,
G
, RhoA, and c-src, suggesting involvement of
G
12-RhoA and G
-Src pathways. However, the roles of
G13, ARF, or PKC and their interplay with the RhoA/ROK
pathway were not examined. In intestinal smooth muscle, G12, unlike G13, mediated an inhibitory PLD
response, whereas in cultured vascular smooth muscle, G12
mediated a stimulatory response. Differential involvement of
G12 and G13 in activation of monomeric G
proteins has been reported (4, 10, 17, 18, 28) in other
cell types, and as noted earlier, appears to be both cell and agonist specific.
The functional significance of agonist-stimulated, sustained activation
of PLD resides in the ability of its primary product, PA, to generate
DAG and thus activate PKC. We and others (15, 16, 25, 35)
have provided evidence that specific isoforms of PKC are involved in
sustained contraction of vascular and visceral smooth muscle. Sustained
contraction of intestinal smooth muscle induced by G protein-coupled
agonists is mediated by the Ca2+-independent isoform,
PKC-
, whereas sustained contraction induced by phorbol esters and
growth factors (e.g., epidermal growth factor) is mediated by PKC-
,
and possibly other Ca2+-dependent isoforms
(25). Preliminary evidence suggests that agonist-stimulated PKC-
activity and sustained contraction of intestinal smooth muscle are mediated by a pathway involving sequential activation of G13, RhoA, and PLD and could be inhibited by
GDP
S, G
13 and RhoA antibodies, and by PLD and PKC
inhibitors (27). Recent studies (11, 14, 16,
33) have provided further evidence of a functional linkage
between RhoA, PKC, and sustained muscle contraction; activation of ROK
inhibits myosin light chain phosphatase via the PKC target protein
CPI-17, resulting in phosphorylation of myosin light chain and
sustained contraction.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-15564.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: G.M.
Makhlouf, PO Box 980711, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, Virginia 23298-0711 (E-mail:
makhlouf{at}hsc.vcu.edu).
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. Section 1734 solely to indicate this fact.
Received 13 July 2000; accepted in final form 11 September 2000.
 |
REFERENCES |
1.
Balboa, MA,
Firestein BL,
Godson C,
Bekk KS,
and
Insel PA.
Protein kinase C
mediates phospholipase D activation by nucleotides and phorbol esters in Madin-Darby canine kidney cells. Stimulation of phospholipase D is independent of activation of polyphospho-inositide-specific phospholipase C and phospholipase A2.
J Biol Chem
269:
10511-10516,
1994[Abstract/Free Full Text].
2.
Bligh, EG,
and
Dyer WJ.
A rapid method of total lipid extraction and purification.
Can J Biochem Physiol
37:
911-917,
1959[ISI].
3.
Brown, HA,
Gutowski S,
Moomaw CR,
Slaughter C,
and
Sternweis PC.
ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity.
Cell
75:
1137-1144,
1993[ISI][Medline].
4.
Buhl, AM,
Johnson NL,
Dhanasekaran N,
and
Johnson GL.
G
12 and G
13 stimulate Rho-dependent stress fibre formation and focal adhesion assembly.
J Biol Chem
270:
24631-24634,
1995[Abstract/Free Full Text].
5.
Cockcroft, S,
Thomas GMH,
Fensome A,
Geny B,
Cunningham E,
Gout I,
Hiles I,
Totty NF,
Truong O,
and
Hsuan JJ.
Phospholipase D: a downstream effector of ARF in granulocytes.
Science
263:
523-526,
1994[ISI][Medline].
6.
Colley, WC,
Altshuller YM,
Sue-Ling CK,
Copeland NG,
Gilbert DJ,
Jenkins NA,
Branch KD,
Tsirka SE,
Bollag WB,
and
Frohman MA.
Cloning and expression analysis of murine phospholipase D1.
Biochem J
326:
745-753,
1997[ISI][Medline].
7.
Colley, WC,
Sung TC,
Roll R,
Jenco J,
Hammond SM,
Altshuller YM,
Bar-Sagi D,
Morris AJ,
and
Frohman MA.
Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.
Curr Biol
7:
191-201,
1997[ISI][Medline].
8.
Conricode, KM,
Brewer KA,
and
Exton JH.
Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism.
J Biol Chem
267:
7199-7202,
1992[Abstract/Free Full Text].
9.
Exton, JH.
Phospholipase D: enzymology, mechanisms of regulation, and function.
Physiol Rev
77:
303-320,
1997[Abstract/Free Full Text].
10.
Ghola, A,
Harhammer R,
and
Schultz G.
The G protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho.
J Biol Chem
273:
4653-4659,
1998[Abstract/Free Full Text].
11.
Gong, MC,
Fujihara H,
Somlyo AV,
and
Somlyo AP.
Translocation of RhoA associated with Ca2+ sensitization of rabbit smooth muscle.
J Biol Chem
272:
10704-10709,
1997[Abstract/Free Full Text].
12.
Hammond, SM,
Altshuller YM,
Sung TC,
Rudge SA,
Rose K,
Engebrecht JA,
Morris AJ,
and
Frohman MA.
Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.
J Biol Chem
270:
29640-29643,
1995[Abstract/Free Full Text].
13.
Hart, MJ,
Jiang X,
Kozasa T,
Roscoe W,
Singer WD,
Gilman AG,
Sternweis PC,
and
Bollag G.
Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G
13.
Science
280:
2112-2114,
1998[Abstract/Free Full Text].
14.
Kitazawa, T,
Eto M,
Woodsome TP,
and
Brautigan DL.
Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility.
J Biol Chem
275:
9897-9900,
2000[Abstract/Free Full Text].
15.
Lee, YH,
Kim I,
Laporte R,
Walsh MP,
and
Morgan KT.
Isozyme-specific inhibitors of protein kinase C translocation: effects on contractility of single permeabilized vascular smooth muscle cells of the ferret.
J Physiol (Lond)
517:
709-720,
1999[Abstract/Free Full Text].
16.
Li, L,
Eto M,
Lee MR,
Morita F,
Yazawa M,
and
Kiatzawa T.
Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit smooth muscle.
J Physiol (Lond)
508:
871-881,
1998[Abstract/Free Full Text].
17.
Lin, X,
Voyno-Yasenetskaya T,
Hooley R,
Lin CY,
Orlowski J,
and
Barber DL.
G
12 differentially regulates Na+-H+ exchanger isoforms.
J Biol Chem
271:
22604-22610,
1996[Abstract/Free Full Text].
18.
Majumdar, M,
Seasholtz TM,
Buckmaster C,
Toksoz D,
and
Brown JH.
A rho exchange factor mediates thrombin and G
12-induced cytoskeletal responses.
J Biol Chem
274:
26815-26821,
1999[Abstract/Free Full Text].
19.
Malcolm, KC,
Elliott CM,
and
Exton JH.
Evidence for Rho-mediated agonist stimulation of phospholipase D in fibroblasts. Effects of Clostridium botulinum C3 exoenzyme.
J Biol Chem
271:
13135-13139,
1996[Abstract/Free Full Text].
20.
Malcolm, KC,
Ross AH,
Qiu RG,
Symons M,
and
Exton JH.
Activation of rat liver phospholipase D by the small GTP binding protein RhoA.
J Biol Chem
269:
25951-25954,
1994[Abstract/Free Full Text].
21.
Massenburg D, Han JS, Liyanage M, Patton WA, Rhee SG, Moss J, and
Vaughan M. Activation of rat brain phospholipase D by
ADP-ribosylation factors 1, 5, and 6: separation of ADP-ribosylation
factor-dependent and oleate-dependent enzymes. Proc Natl Acad Sci
USA 93: 4300-4304.
22.
Meacci, E,
Vasta V,
Moorman JP,
Bobak DA,
Bruni P,
Moss J,
and
Vaughan M.
Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in human adenocarcinoma A549 cells.
J Biol Chem
274:
18605-18612,
1999[Abstract/Free Full Text].
23.
Moss, J,
and
Vaughan M.
Molecules in the ARF orbit.
J Biol Chem
273:
21431-21434,
1998[Free Full Text].
24.
Murthy, KS,
Coy DH,
and
Makhlouf GM.
Somatostatin receptor-mediated signaling in smooth muscle: activation of phospholipase C-
3 by G
and inhibition of adenylyl cyclase by G
i1 and G
o.
J Biol Chem
271:
23458-23463,
1996[Abstract/Free Full Text].
25.
Murthy, KS,
Grider JR,
Kuemmerle JF,
and
Makhlouf GM.
Sustained muscle contraction induced by agonists, growth factors, and Ca2+ mediated by distinct PKC isozymes.
Am J Physiol Gastrointest Liver Physiol
279:
G201-G210,
2000[Abstract/Free Full Text].
26.
Murthy, KS,
and
Makhlouf GM.
Agonist-mediated activation of phosphatidylcholine-specific phospholipase C and D in intestinal smooth muscle.
Mol Pharmacol
48:
293-304,
1995[Abstract].
27.
Murthy, KS,
and
Makhlouf GM.
G13-mediated activation of PLD induces translocation of PKC-
and sustained muscle contraction (Abstract).
Gastroenterology
114:
A1167,
1998.
28.
Needham, LK,
and
Rozengurt E.
G
12 and G
13 stimulate Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate.
J Biol Chem
273:
14626-14632,
1998[Abstract/Free Full Text].
29.
Okamoto, T,
Ikezu T,
Murayama Y,
Ogata E,
and
Nishimoto I.
Measurement of GTP
S binding to specific G proteins in membranes using G protein antibodies.
FEBS Lett
305:
125-128,
1992[ISI][Medline].
30.
Schmidt, M,
Vob M,
Weernik PAO,
Wetzel J,
Amano M,
Kaibuchi K,
and
Jakobs KH.
A role for Rho-kinase in Rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor.
J Biol Chem
274:
14648-14654,
1999[Abstract/Free Full Text].
31.
Seasholtz, TM,
Majumdar M,
and
Brown JH.
Rho as a mediator of G protein-coupled receptor signaling.
Mol Pharmacol
55:
949-956,
1999[Free Full Text].
32.
Singer, WD,
Brown HA,
Jiang X,
and
Sternweis PC.
Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase C activity.
J Biol Chem
271:
4504-4510,
1996[Abstract/Free Full Text].
33.
Sward, K,
Dreja K,
Susnjar M,
Hellstrand P,
Hartshorne DJ,
and
Walsh MP.
Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea pig ileum.
J Physiol (Lond)
522:
33-49,
2000[Abstract/Free Full Text].
34.
Ushio-Fukai, M,
Wayne Alexander R,
Akers M,
Lyons PR,
Lassegue B,
and
Griendling KK.
Angiotensin II receptor coupling to phospholipase D is mediated by the 
subunits of heterotrimeric G proteins in vascular smooth muscle cells.
Mol Pharmacol
55:
142-149,
1999[Abstract/Free Full Text].
35.
Wang, P,
and
Bitar KN.
RhoA regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27.
Am J Physiol Gastrointest Liver Physiol
275:
G1454-G1462,
1998[Abstract/Free Full Text].
Am J Physiol Gastrointest Liver Physiol 280(3):G381-G388
0193-1857/01 $5.00
Copyright © 2001 the American Physiological Society