From the Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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Activation of phospholipase C (PLC)
is one of the earliest events in angiotensin II (Ang II) type 1 (AT1) receptor (R)-mediated signal transduction in
vascular smooth muscle cells (VSMCs). The coupling mechanisms of
AT1 Rs to PLC, however, are controversial, because both
tyrosine phosphorylation of PLC- and G protein-dependent PLC-
activation pathways have been reported. The expression of PLC-
1, furthermore, has not been consistently demonstrated in VSMCs.
Here we identified the PLC subtypes and subunits of heterotrimeric G
proteins involved in AT1 R-PLC coupling using cultured rat
VSMCs. Western analysis revealed the expression of PLC-
1, -
1, and
-
1 in VSMCs. Ang II-stimulated inositol trisphosphate
(IP3) formation measured at 15 s, which corresponds to
the peak response, was significantly inhibited by electroporation of
antibodies against PLC-
1, but not by anti-PLC-
and -
antibodies. Electroporation of anti-G
q/11 and
-G
12 antibodies also showed significant inhibition of
the Ang II-induced IP3 generation at 15 s, while
anti-G
i and G
13 antibodies were
ineffective. Furthermore, in VSMCs electroporated with anti-G
antibody and cells stably transfected with the plasmid encoding the
G
-binding region of the carboxyl terminus of
-adrenergic receptor kinase1, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP3 production after 30 s,
a time period which temporally correlated with PLC-
tyrosine
phosphorylation in response to Ang II. Moreover, electropor-ation
of anti-PLC-
antibody markedly inhibited the IP3
production measured at 30 s, indicating that tyrosine
phosphorylation of PLC-
contributes mainly to the later phase of PLC
activation. Thus, these results suggest that: 1) AT1
receptors sequentially couple to PLC-
1 via a heterotrimeric G
protein and to PLC-
via a downstream tyrosine kinase; 2) the initial
AT1 receptor-PLC-
1 coupling is mediated by
G
q/11
and G
12
; 3) G
acts as a signal transducer for activation of PLC in VSMCs. The
sequential coupling of AT1 receptors to PLC-
1 and
PLC-
, as well as dual coupling of AT1 receptors to
distinct G
proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling
network in VSMCs.
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INTRODUCTION |
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Angiotensin II (Ang II)1
plays an important role in controlling both contraction and growth of
vascular smooth muscle cells (VSMCs) through complex intracellular
signaling events involving pathways classically associated with both
G-protein coupled and tyrosine kinase-mediated responses (1). In VSMCs,
most of the Ang II effects are mediated by AT1 receptors
which belong to the 7-transmembrane spanning, heterotrimeric G
protein-coupled receptor family (2, 3). The rat AT1
receptor has been shown in various preparations to be capable of
coupling to various -subunits (Gq, Gq/11,
and Gi/o) (4, 5), which may provide insights into the
potential mechanism by which a single AT1 receptor
stimulates various signaling cascades.
Recently, it has become apparent that AT1 receptors also
couple to G13
1
3, a
heterotrimeric G protein whose
-subunit belongs to the nonpertussis
toxin-sensitive G
12 family. In rat portal vein myocytes,
the G
subunits derived from G
13 apparently mediate Ang II activation of an L-type Ca2+ channel (6, 7). In
general, however, the immediate effectors coupled to the
G
12 family of G proteins are unknown. Although G
12 transduces thrombin receptor activation of
AP-1-mediated gene expression (8), and both G
12 and
G
13 activate Jun kinase/stress-activated protein kinase,
the most proximal signals remain to be defined.
Ang II binding to AT1 receptors in VSMC causes a distinctly biphasic response, with a rapid and transient activation of phosphatidylinositol-specific PLC to produce inositol trisphosphates (IP3) and diacylglycerol, followed by prolonged activation of phospholipase D (9). IP3 formation is markedly increased within a few seconds, reaches a maximum at 15 s, and then gradually returns to control levels (10). Although activation of PLC is one of the earliest events in Ang II signaling (10), the mechanisms by which AT1 receptors couple to PLC in VSMCs are controversial.
Three families of mammalian PLC isozymes, PLC-, -
, and -
, have
been described based on their molecular structure and mechanism of
regulation (11). PLC-
isozymes have been shown to be activated by
G
and G
subunits of the heterotrimeric G proteins, while PLC-
isozymes are regulated by tyrosine phosphorylation (11). PLC-
isozymes are smaller (85 kDa) than PLC-
and -
(150 and 145 kDa, respectively) and their function remains unclear (11). In
general, G protein-coupled receptors are assumed to activate PLC-
isozymes by coupling to the heterotrimeric G proteins (11), while
growth factor receptors are proposed to activate PLC-
by tyrosine
phosphorylation (11). However, in rat VSMCs, Marrero et al.
(12) demonstrated that activation of the G protein-coupled AT1 receptor induced tyrosine phosphorylation and
activation of PLC-
, although potential involvement of G proteins was
not analyzed. We previously showed that Ang II-stimulated PLC
activation is mediated by a pertussis toxin-insensitive G protein (13),
at least in part represented by G
q/11 (4), in these same
cells. We also showed that prolonged incubation with Ang II causes
selective down-regulation of G
q/11, providing indirect
additional evidence that the AT1 receptor interacts with
G
q/11 in intact VSMCs as demonstrated in
vitro (4). Consistent with our reports, AT1 receptors
in other systems have been shown to couple to PLC-
via
G
q/11 (14, 15), although G
q/11-mediated
activation of PLC-
is only partial (4, 16). In rat and rabbit VSMCs,
however, PLC-
1 protein has been difficult to detect (12, 17). In
human aortic VSMCs, Schelling et al. (18) showed that both
PLC-
1 and PLC-
are expressed, and that Ang II-PLC signaling is
mediated by PLC-
1, but not by PLC-
. Thus, in VSMCs, it remains
unclear which PLC isozymes are expressed, and whether AT1
receptor-PLC activation is mediated by direct coupling to G protein
subunits or by stimulation of a downstream tyrosine kinase.
In addition to these uncertainties, it is unclear how tyrosine
phosphorylation of PLC-, which occurs at 30 s to 1 min (12), could
mediate the earliest measurable increase in IP3 formation (<5 s) (10) in response to Ang II. Since the most proximal signal transmission by G protein-coupled receptors is likely achieved through
the heterotrimeric G protein subunits, G
and G
, both of which
have been shown to stimulate PLC-
in other systems (11), we
hypothesized that the earliest activation of PLC by AT1
receptors occurs through the coupling to G
or G
, and the later
phase of IP3 generation involves tyrosine phosphorylation
of PLC-
. Thus, the present study was designed to clarify the role of
tyrosine kinases and G proteins in AT1 receptor-PLC
coupling, and to identify the PLC subtypes and the subunits of
heterotrimeric G proteins involved in their coupling in VSMCs. For this
purpose, we measured IP3 production by Ang II in cultured
rat VSMCs electroporated with specific antibodies against PLC isozymes
and G protein subunits, and in cells stably transfected with a plasmid
encoding the G
-binding region of the carboxyl terminus of
-adrenergic receptor kinase 1 (
ARK1ct) (19) to sequester free
G
. We provide evidence for the temporal dispersion of
AT1 receptor signals through the sequential activation of
PLC-
1 and PLC-
, and for a role for G
q/11 and
G
12 as well as their associated G
subunits in
activation of PLC-
1.
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EXPERIMENTAL PROCEDURES |
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Materials--
Tween 20, acrylamide, SDS, nonfat dry milk, low
molecular weight protein markers, and goat anti-rabbit IgG-horseradish
peroxidase (HRP)-conjugate were purchased from Bio-Rad. Protein A/G
Plus-agarose, anti-Gi, anti-G
q/11,
anti-G
common, anti-G
12,
anti-G
13, and anti-PLC-
1 antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PLC-
1 (IgG),
anti-PLC-
1 (IgG), and anti-phosphotyrosine (IgG) monoclonal
antibodies were purchased from Upstate Biotechnology (Lake Placid, NY).
Mouse monoclonal antibody against PLC-
(IgM) and rat pituitary
homogenate were obtained from Transduction Laboratories (Lexington,
KY). The enhanced chemiluminescence (ECL) Western blotting detection
system and goat anti-mouse IgG-HRP-conjugate were obtained from
Amersham Life Sciences. PVDF membranes (0.45 µm) and Nytran membranes
were purchased from Millipore (Bedford, MA) and Schleicher & Schuell,
Inc. (Keene, NH), respectively. The pcDNA3 vector was from
Invitrogen (San Diego, CA). Bovine serum albumin and
phenylmethanesulfonyl fluoride were from Boehringer Mannheim.
Lipofectin, geneticin, soybean trypsin inhibitor, glutamine, penicillin, streptomycin, Opti-MEM I reduced serum medium, and trypsin:EDTA were purchased from Life Technologies, Inc. (Gaithersburg, MD). TRI reagent was from Molecular Research Center (Cincinnati, OH).
The Prime-It II kit and QuikHyb solution were from Stratagene (Menasha,
WI). Monofluor was purchased from National Diagnostics (Atlanta, GA),
and myo-[3H]inositol (1000 µCi/ml) was from
NEN Life Science Products Inc. (Wilmington, DE). Common buffer salts
were obtained from Fisher (Pittsburgh, PA). All other chemicals and
reagents, including Dulbecco's modified Eagle's medium (DMEM) with 25 mM Hepes and 4.5 g/liter glucose and calf serum, were from
Sigma.
Cell Culture-- VSMCs were isolated from male Sprague-Dawley rat thoracic aortas by enzymatic digestion as described previously (20). Cells were grown in DMEM supplemented with 10% calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and were passaged twice a week by harvesting with trypsin:EDTA and seeding into 75-cm2 flasks. For experiments, cells between passages 6 and 15 were used at confluence.
Stable Transfection of ARK1ct Expression
Plasmid--
pRK/
ARK1ct (Gly495-Leu689) DNA
(19), a kind gift from Dr. Robert J. Lefkowitz, was digested with
EcoRI and XbaI and cloned into the eukaryotic
expression plasmid pcDNA3. Transcription of pcDNA3/
ARK1ct cDNA was under control of the cytomegalovirus immediate-early gene
enhancer/promoter. This vector also contains a neomycin-resistance gene, allowing selection of transfected cells with geneticin. Four µg
of purified pcDNA3 alone or pcDNA3/
ARK1ct plasmid in 100 µl of H2O were gently mixed with Lipofectin solution (100 µl). The DNA-liposome complex was added directly to 40-50%
confluent VSMCs plated in 60-mm dishes in Opti-MEM I reduced serum
medium and incubated for 18 h at 37 °C. The medium was then
changed to DMEM containing 20% fetal bovine serum. After 48 h,
transfected VSMCs were split 1:3 into 100-mm dishes and incubated in
DMEM containing 10% fetal bovine serum and 400 µg/ml geneticin.
Eight days after selection, geneticin-resistant colonies were isolated using cloning cylinders. Transfected cells were maintained in selection
medium until they were plated into 35- or 100-mm dishes for
experiments.
RNA Isolation and Northern Blot Analysis--
Total RNA was
extracted from cells as described previously (4). 10-µg RNA samples
were separated by electrophoresis in 1.0% agarose gels containing
6.6% formaldehyde. RNA was transferred onto a nylon membrane and
immobilized by UV cross-linking (Stratalinker, Stratagene). The probe,
ARK1ct cDNA derived from EcoRI/XbaI
digestion of pRK-
ARK1ct DNA (19), was labeled with
[
-32P]dCTP using a random primer labeling kit
(Prime-It II). After UV cross-linking, membranes were prehybridized at
68 °C for 2 h in QuikHyb solution (Stratagene). The
hybridization was performed for 2 h at 68 °C with
32P-labeled probe in the same solution. Membranes were
washed two times in 1 × SSC + 0.1% SDS at 50 °C and once in
0.2 × SSC + 0.1% SDS at 55 °C. After autoradiography, the
relative density of each band was determined using laser densitometry.
Staining of the 28 S rRNA band by ethidium bromide, after transfer to
the membrane, was used for normalization.
Measurement of IP3 Production-- Assay of PLC activity in intact VSMCs was performed as described previously (4). Cells grown on 35-mm dishes were labeled for 24 h with myo-[3H]inositol (15 µCi/ml) in 2 ml of culture medium. After washing, cells were incubated at 37 °C for 20 min in balanced salt solution buffer of the following composition (in mM, 130 NaCl, 5 KCl, 1 MgCl2, 1.5 CaCl2, 20 HEPES (buffered to pH 7.4 with Tris base)). Incubation buffer was removed and replaced with 1 ml of buffer with or without 100 nM Ang II for indicated times. The reaction was terminated by rapid aspiration of the buffer and addition of 1 ml of chloroform/methanol/HCl (1:2:0.05). Organic and aqueous phases of the extract plus a 0.5-ml rinse were separated by addition of 500 µl of chloroform and 900 µl of distilled water, followed by centrifugation for 10 min at 500 × g. Chloroform phases were removed and aqueous phases were washed with chloroform. IP, IP2, and IP3 fractions extracted into the aqueous phases were sequentially eluted from AG-1-X8 anion exchange columns using 180 mM NH4 formate, 5 mM sodium tetraborate; 400 mM NH4 formate, 100 mM formic acid; 1 M NH4 formate, 100 mM formic acid, respectively. All inositol phosphates were quantified by liquid scintillation spectroscopy.
Electroporation-- Cells were electroporated in 35-mm tissue culture dishes using a Petri dish electrode manufactured by BTX (San Diego, CA). The electrode is 35 mm in diameter with a 2-mm gap and plated with gold. Electroporation was performed in Hank's balanced salt solution, pH 7.4 (in mM, 5 KCl, 0.3 KH2PO4, 138 NaCl, 4 NaHCO3, 0.3 NaHPO4, 1.26 CaCl2·2H2O, 0.82 MgSO4), containing antibodies at a concentration of 5 µg/ml. Cells were exposed to 1 pulse at 90 V for 40 ms (square wave) using a BTX Model T820 ElectroSquarePorator, similar to conditions used for electroporation of VSMCs in 100-mm culture plates (21). The tissue culture dishes were then incubated for 30 min at 37 °C (5% CO2), and then washed once with DMEM and further incubated in this same medium for 30 min at 37 °C. The effectiveness of the electroporation procedure was verified by measuring the intracellular incorporation of 125I-labeled rabbit IgG after electroporation. We found that 58.5 ± 3.3% (n = 8) of extracellular 125I-labeled rabbit IgG was efficiently incorporated within the cells without adversely affecting their viability. In contrast, VSMCs exposed to a mock experiment without electroporation contained undetectable levels of 125I-labeled rabbit IgG.
Protein Purification and Immunoblot Analysis--
Confluent
VSMCs in 100-mm dishes were washed three times with ice-cold PBS. Cells
were scraped in 500 µl of ice-cold lysis buffer, pH 7.4 (in
mM, 50 HEPES, 5 EDTA, 50 NaCl), containing 1% Triton
X-100, protease inhibitors (10 µg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin), and phosphatase inhibitors (in mM, 50 sodium fluoride, 1 sodium
orthovanadate, 10 sodium pyrophosphate). Solubilized proteins were
centrifuged at 14,000 × g for 30 min, and supernatants
were stored at 80 °C. Extracted protein was quantified by the
Bradford assay. Proteins were separated on 5 or 10% polyacrylamide
gels using SDS-PAGE and transferred to PVDF membranes (0.45 µm)
overnight. Membranes were blocked for 1 h with PBS containing 5%
nonfat dry milk and 0.1% Tween 20, and were incubated with primary
antibody for 1 h in PBS containing 1% nonfat dry milk and 0.1%
Tween 20, washed three times with PBS containing 0.2% nonfat dry milk
and 0.1% Tween 20, and then incubated with HRP-conjugated goat
anti-rabbit secondary antibody for 1 h. The ECL Western blotting
detection kit (Amersham Corp.) was used for detection.
Analysis of Tyrosine Phosphorylation of PLC---
VSMCs in
100-mm dishes at near confluence were growth arrested in 0.1%
serum-containing DMEM for 24 h. After washing three times with 5 ml of balanced salt solution buffer, cells were incubated at 37 °C
for 20 min in balanced salt solution buffer. Incubation buffer was
removed and replaced with 5 ml of buffer with or without 100 nM Ang II for the indicated times. The reaction was
terminated by rapid aspiration of the buffer and addition of ice-cold
PBS. Each plate was then treated with 0.5 ml of ice-cold lysis buffer and placed on ice for 30 min with occasional shaking. The solubilized cells were immunoprecipitated with Protein A/G Plus-agarose and anti-PLC-
antibody (2 µg/mg) overnight at 4 °C. The
immunoprecipitates were then recovered by centrifugation and washed
five times with the lysis buffer. The immunoprecipitated proteins were
dissolved in 50 µl of Laemmli buffer, boiled for 5 min, and separated
by SDS-PAGE on 9% polyacrylamide gels and transferred to PVDF
membranes. Blots were then probed with anti-phosphotyrosine monoclonal
antibody (1:1000 dilution) and detected with HRP-conjugated goat
anti-mouse secondary antibody (1:2000 dilution). The ECL Western
blotting detection kit (Amersham Corp.) was used for detection.
Ang II Receptor Binding-- The Ang II receptor binding assay was performed as described previously (13). Kd and Bmax (maximum number of binding sites) were determined by Scatchard analysis.
Statistical Analysis-- Results are expressed as mean ± S.E. Statistical significance was assessed by analysis of variance, followed by comparison of group averages by contrast analysis, using the SuperANOVA statistical program (Abacus Concepts, Berkeley, CA). A p value of <0.05 was considered to be statistically significant.
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RESULTS |
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Phospholipase C Isozymes Expressed in Rat VSMCs--
To identify
the PLC isozymes expressed in rat aortic SMCs, immunoblot analysis was
performed using antibodies raised against PLC-1, -
1, and -
1.
Anti-PLC-
1 antibody reacted with a 145-kDa protein, consistent with
the reported molecular mass for PLC-
1 (Fig.
1A), and anti-PLC-
1 reacted
with an 85-kDa protein of the reported molecular mass for PLC-
1
(Fig. 1C). In contrast to previous reports (12, 17), but in
agreement with the findings of Schelling et al. (18, 22), an
anti-PLC-
1 antibody identified a full-length 150-kDa protein as well
as a 100-kDa fragment (Fig. 1B). Both bands detected with the
anti-PLC-
1 antibody disappeared in the presence of control peptide
used for antibody generation, indicating that this antibody
specifically detects PLC-
1 (data not shown). Both the full-length
150-kDa protein and the 100-kDa fragment were detected using rat
pituitary or rat-1 fibroblast homogenates as positive controls (Fig.
1B and data not shown). These results suggest that PLC-
1,
-
1, and -
1 are expressed in rat VSMCs.
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Effect of Electroporation of Antibodies against PLC Isozymes on Ang
II-stimulated IP3 Formation--
To determine which PLC
isozymes are involved in AT1 receptor-PLC coupling, we
measured IP3 production stimulated by Ang II at 15 s
(peak response) in VSMCs electroporated with specific antibodies
against PLC-1, -
1, and -
1 (Fig.
2). The electroporation of specific
antibodies against cellular proteins has been shown to be an effective
technique for interrupting Ang II-induced signal transduction cascades
in cultured VSMCs (21). IP3 production stimulated by Ang II
in cells electroporated in the absence of antibody (mock
electroporation) was increased by 119 ± 7% (n = 6), while that in cells without electroporation was increased by
126 ± 8% (n = 3). Cells electroporated with
non-immune rabbit IgG showed a decrease in the Ang II response (20%)
compared with the cells with mock electroporation; therefore, the
effects of specific antibodies were always compared with the response
in the presence of rabbit IgG. When VSMCs were electroporated with anti-PLC-
1 antibody, there was no inhibition of Ang II-induced IP3 formation (3 ± 0.1% inhibition,
n = 3). Electroporation of anti-PLC-
antibody showed
a small and insignificant inhibitory effect (10 ± 5% inhibition,
n = 4) at 15 s. In contrast, electroporation of an
antibody against PLC-
1 markedly inhibited IP3 production in response to Ang II (83 ± 5% inhibition, n = 3, p < 0.05). These data indicate a critical role for
PLC-
1 in the initial AT1 receptor-PLC coupling in
VSMCs.
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Effect of Tyrosine Kinase Inhibition on Ang II-stimulated
IP3 Formation--
Judging from the time course that has
been reported for PLC- phosphorylation by Ang II (12), we
hypothesized that tyrosine kinase-dependent PLC-
activation might be involved in IP3 generation during the
later phases of the AT1 receptor signaling events in VSMCs.
Therefore, we examined the effect of genistein, a tyrosine kinase
inhibitor, on the time course of Ang II-induced IP3
formation. As shown in Fig. 3, genistein
(100 µM) inhibited IP3 production only after
30 s of Ang II stimulation without affecting the peak response at
15 s, consistent with the inability of anti-PLC-
antibody to
inhibit maximum IP3 production during the initial phase of
signaling. This genistein-induced inhibition was temporally correlated
with the PLC-
tyrosine phosphorylation in response to Ang II (Fig.
3, inset). PLC-
tyrosine phosphorylation peaked at
30 s and returned to control levels by 10 min. Furthermore, Ang
II-induced IP3 formation measured at 30 s was markedly
inhibited by electroporation of anti-PLC-
antibody (40 ± 6%
inhibition, n = 3, p < 0.05). Taken
together, these data suggest that tyrosine phosphorylation-dependent PLC-
activation is involved
primarily in the later phase of PLC activation.
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Effect of Electroporation of Antibodies against G Protein Subunits
on Ang II-stimulated IP3 Formation--
Since PLC- has
been shown to be activated by G
and G
subunits of
heterotrimeric G proteins (11), we next investigated the primary
subunits involved in the early AT1 receptor-PLC-
coupling. For this purpose, we measured IP3 production by
Ang II at 15 s (peak response) in cells electroporated with
specific antibodies against G
i, G
q/11,
G
12, G
13, and G
. As shown in Fig.
4, electroporation of
anti-G
i and G
13 antibodies, as well as
rabbit IgG (negative control), had no effect on Ang II-induced IP3 production. In contrast, anti-G
q/11 and
G
12 antibodies significantly inhibited the Ang II
response (56 ± 4% inhibition, n = 5, p < 0.05 and 62 ± 5% inhibition,
n = 7, p < 0.05, respectively). This incomplete inhibition is not due to insufficient amounts of antibody, because doubling the antibody concentration did not cause any further
attenuation of the response (anti-
12 + anti-
12, Fig. 4). Furthermore, when
anti-G
q/11 and anti-G
12 were combined, their inhibitory effects were additive and nearly complete (93% inhibition, n = 3), suggesting that both G
subunits
can couple to the AT1 receptor. The partial involvement of
G
q/11 in PLC coupling is consistent with our previous
finding that in G
q/11 down-regulated cells, Ang
II-stimulated IP3 formation is inhibited by 30% (4). The
effectiveness of anti-G
q/11 and G
12
antibodies was abolished when they were boiled (100 °C for 30 min)
prior to electroporation, confirming that active antibody was required for the observed effect. Additionally, when VSMCs were electroporated with anti-G
antibody, the Ang II response was also significantly inhibited (75 ± 6% inhibition, n = 6, p < 0.05), and this inhibition was reversed by boiling
the antibody. Thus, these results suggest that early AT1
receptor-PLC coupling is mediated by the G
q/11
and
G
12
complex of heterotrimeric G proteins, and that
G
, as well as the G
subunits, may serve as an active molecule
for transducing the AT1 receptor signal to PLC.
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Effect of Overexpression of ARK1ct on Ang II-stimulated
IP3 Formation--
To further assess the role of G
in the early phase of AT1 receptor-PLC coupling, we stably
transfected the plasmid encoding the
ARK1ct (effectively a G
antagonist) (19) into cultured rat VSMCs. Control cells were
transfected with vector only. The efficacy of
ARK1ct cDNA
transfection was evaluated and confirmed by Northern analysis (Fig.
5, left panel). As shown in
Fig. 5, IP3 production by 100 nM Ang II at
15 s was significantly inhibited in cells stably overexpressing
ARK1ct compared with that in 2 different cell lines transfected with
vector alone (average inhibition 43 ± 4%, n = 3). We verified by measuring equilibrium binding of 3H-Ang
II that AT1 receptor expression was not different in
vector- and in
ARK1ct-overexpressing cells (data not shown). Thus,
these results strongly suggest that G
acts as a signal transducer for PLC activation.
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DISCUSSION |
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The mechanisms by which AT1 receptors activate PLC in
rat VSMCs are controversial, because the expression of PLC-1 protein has not been consistently detected (12, 17), and both direct coupling
to G proteins (4, 13) and downstream tyrosine
kinase-dependent activation mechanisms (12) have been
reported. Here, we provide evidence that PLC-
1 and PLC-
are both
functionally expressed in rat VSMCs and demonstrate that they may be
sequentially activated by Ang II. Their mechanisms of activation are
quite different: PLC-
1 coupling to the AT1 receptor
appears to be mediated by G
q/11
and
G
12
heterotrimeric G proteins, while PLC-
activation is dependent on a downstream tyrosine kinase. It appears
that the active subunit transducing PLC-
1 stimulation may include G
derived from a nonpertussis toxin-sensitive G protein
-subunit.
In the present study, we identified a full-length (150 kDa) PLC-1 as
well as a 100-kDa fragment in rat VSMCs using an antibody raised
against the carboxyl terminus of rat PLC-
1 (Santa Cruz) (Fig. 1).
These two major bands of PLC-
1 were also detected by a different
mouse monoclonal antibody raised against the amino terminus of PLC-
1
of rat origin (Transduction Laboratories) (data not shown), and
disappeared when the antibody was preincubated with the control peptide
used for antibody generation, suggesting that both bands represent
PLC-
1 isoforms or fragments. Schelling et al. (22) have
previously detected the 100-kDa PLC-
1 fragment in rat aortic VSMCs
using the same rabbit polyclonal PLC-
1 antibody. Thus, the 100-kDa
band may represent a PLC proteolytic fragment (22) or a truncated
PLC-
1 isoform reported in other species (23, 24). The discrepancies
regarding PLC-
1 detection between our results and those of others
(12, 17) may be due to variations in VSMC phenotype or differences in
the antibodies or the cell extraction methods used for
immuodetection.
We also provided direct evidence that PLC-1 plays an important role
in the early phase of AT1 receptor-PLC coupling in rat VSMCs, because electroporation of specific anti-PLC-
1 antibody markedly inhibited the maximum IP3 generation observed at
15 s after Ang II stimulation (Fig. 2). Consistent with our
results, Schelling et al. (18) have recently demonstrated
that introduction of PLC-
1 antibody inhibits Ang II-stimulated
inositol phosphate production in
-escin-permeabilized human aortic
VSMCs. We found that a tyrosine kinase is involved in the later phase
of AT1 receptor-PLC coupling, because the tyrosine kinase
inhibitor genistein attenuated only the later phase (
30 s) of Ang
II-stimulated IP3 formation (Fig. 3). This result is
consistent with the reports by Marrero et al. (12) who
showed inhibition of Ang II-induced IP3 formation by
genistein, but the earliest time point measured was 30 s. The genistein-induced inhibition of IP3 production is
temporally correlated with increased tyrosine phosphorylation of
PLC-
in response to Ang II (Fig. 3)(12). Furthermore, the later
phase of IP3 production (at 30 s) was markedly
inhibited by electroporation of anti-PLC-
antibody, indicating that
AT1 receptors couple to PLC-
in the later phase of PLC
activation. However, a partial involvement of PLC-
1 in the later
phase cannot be ruled out, because the inhibition by genistein was
incomplete and anti-PLC-
1 antibody also inhibited the Ang II-induced
IP3 production at 30 s by 38% (data not shown). In
contrast, Schelling et al. (18) failed to detect inhibition
of the Ang II-induced PLC activation by a tyrosine kinase inhibitor in
human VSMCs. This discrepancy may be due to the fact that they measured
total inositol phosphate accumulation during 10 min of Ang II
stimulation as PLC activation, which may mostly reflect the peak
PLC-
-mediated response and mask the contribution of PLC-
. Taken
together, these results suggest that AT1 receptors
sequentially couple to PLC-
1 (<30 s) and PLC-
(
30 s) to
generate IP3 in rat VSMCs. This idea is supported by the
observation that intracellular Ca2+ mobilization induced by
classical growth factors, which is mediated via tyrosine
phosphorylation of PLC-
without prior
G-protein-dependent PLC-
activation, occurs much more
slowly than that induced by Ang II in VSMCs (12).
Since PLC-1 is activated by G
or G
subunits of
heterotrimeric G proteins (11), we next investigated the subunits of G
proteins that couple AT1 receptors to PLC-
1. Our results
suggest that AT1 receptors activate PLC-
1 via dual
coupling to G
q/11 and G
12, based on the
observation that electroporation of anti-G
q/11 and
-G
12, but not anti-G
i and
-G
13, antibodies significantly inhibited Ang
II-stimulated IP3 production measured at 15 s (Fig. 4). We have verified that G
i, G
q/11,
G
12, and G
13 are ubiquitously expressed
in rat VSMCs by immuoblot analysis
(4).2 G
12 is a
pertussis toxin-insensitive G protein and has been shown to be involved
in growth-related signaling pathways; however, specific effectors
regulated by G
12 have not been previously identified
(25). The inability of the anti-G
i antibody to block IP3 production serves as a negative control, since we have
previously reported that Ang II-induced PLC activation is mediated by
pertussis toxin-insensitive G proteins (13). The failure of the
anti-G
13 antibody to inhibit Ang II-induced PLC
activation is unlikely to be due to an inability of the antibody to
block G
function, because Macrez-Leprêtre et al.
(6) recently showed that microinjection of the same G
13
antibody inhibits Ang II-induced Ca2+ mobilization in rat
portal vein myocytes. Although it is generally believed that
AT1 receptors activate PLC-
via G
q/11
protein (14), several reports showed the possible involvement of other G proteins in AT1 receptor-PLC coupling. Indeed, we have
previously reported that long-term treatment with vasopressin, which
selectively down-regulates G
q/11 by 90%, inhibits Ang
II-induced IP3 production by only 30% (4). Gutowski
et al. (16) demonstrated that in membranes derived from NG
108-15 cells and rat liver, Ang II-induced stimulation of
phosphatidylinositol hydrolysis was partially inhibited (30-60%) by
an anti-G
q/11 antibody (16). Thus, the present result is
the first demonstration that AT1 receptors couple not only
to G
q/11 but also to G
12 to activate
PLC-
1 in rat VSMCs. Furthermore, we recently found that
AT1 receptor coupling to tonic phospholipase D activation
is not mediated by G
q/11, but rather exclusively by
G
12 in rat VSMCs.2 Therefore, it is becoming
clear that AT1 receptors couple to both
G
q/11 and the G
12 family of
heterotrimeric G proteins, as has been reported for other G
protein-coupled receptors (26). In contrast to the G
q
class of
subunits, G
12 does not stimulate inositol
hydrolysis in vitro (25). Therefore, the role of
G
12 may be rather to provide specific receptor-G protein
coupling, thus promoting GTPase activity and release of G
subunits for activating PLC in VSMCs.
A critical role of G as a signal transducer for PLC activation
was demonstrated by the inhibition of Ang II-stimulated IP3 formation by electroporation of anti-G
antibody and by
overexpression of the
ARK1ct, which has been used as a specific
G
antagonist (19). Since it has been suggested that
G
-mediated activation of PLC-
is associated with a relatively
low increase in IP3 (27), it is possible that both G
and G
q/11 cooperatively activate PLC-
to produce
large amounts of IP3 in intact VSMCs.
Our present result extends the original concept that G transduces
the signal for pertussis toxin-sensitive pathways (28). G
has
recently been shown to mediate PTX-insensitive activation of
PLC in Xenopus oocytes (29), and to be involved in c-Jun kinase activation by G
q-coupled m1 muscarinic receptors
in COS-7 cells (30). We recently found that the G
derived from
G
12/13 transduces AT1 receptor-mediated
tonic phospholipase D activation in rat VSMCs.2 Thus,
G
appears to be a common signal transducer in both PTX-sensitive and -insensitive signaling pathways in certain cell types.
In summary, the present study demonstrates that the early phase of Ang
II-induced PLC activation occurs through coupling to PLC-1 via
G
q/11 and G
12 as well as their associated
G
subunits, and the later phase involves a downstream tyrosine
kinase, presumably via phosphorylation of PLC-
. The sequential
coupling of AT1 receptors to PLC-
1 and PLC-
suggests
a novel mechanism for a temporally controlled, highly organized and
convergent Ang II-signaling network in VSMCs.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert J. Lefkowitz for
providing the ARK1ct construct and Carolyn Morris for excellent
secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL47557.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.
To whom correspondence should be addressed: Div. of Cardiology,
Emory University School of Medicine, 1639 Pierce Dr., Rm. 319, Atlanta,
GA 30322. Tel.: 404-727-8142; Fax: 404-727-3330; E-mail:
mfukai{at}emory.edu.
1
The abbreviations used are: Ang II, angiotensin
II; VSMCs, vascular smooth muscle cells; PLC, phospholipase C;
IP3, inositol trisphosphate; ARK1ct, the carboxyl
terminus of
-adrenergic receptor kinase 1; DMEM, Dulbecco's
modified Eagle's medium; PBS, phosphate-buffered saline; HRP,
horseradish peroxidase; ECL, enhanced chemiluminescence; PVDF,
polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis;
PLC, phospholipase C.
2 M. Ushio-Fukai, R. W. Alexander, and K. K. Griendling, unpublished data.
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REFERENCES |
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