(Received for publication, November 15, 1996, and in revised form, January 23, 1997)
From the Laboratoire de Physiologie Cellulaire et
Pharmacologie Moléculaire, CNRS ESA 5017, Université de
Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
and the ¶ Institut für Pharmakologie, Freie
Universität Berlin, Thielallee 69/73,
14195 Berlin, Federal Republic of Germany
The subunit composition of angiotensin
AT1 receptor-activated G protein was identified by
using antisense oligonucleotide injection into the nucleus of rat
portal vein myocytes. In these cells, we have previously shown that
increases in the cytoplasmic calcium concentration
([Ca2+]i) induced by activation of angiotensin
AT1 receptors were dependent on extracellular
Ca2+ entry by L-type Ca2+ channels and
subsequent Ca2+-induced Ca2+ release from the
intracellular stores. The angiotensin AT1
receptor-activated increases in [Ca2+]i were
selectively inhibited by injection of antisense oligonucleotides
directed against the mRNAs coding for the 13,
1, and
3 subunits. A correlating
reduction in G
13, G
1, and G
3 protein expression was confirmed by
immunocytochemistry. In addition, anti-
13 antibody and
synthetic peptide corresponding to the carboxyl terminus of the
G
13 subunit inhibited, in a
concentration-dependent manner, the angiotensin
AT1 receptor-mediated Ca2+ response. Reverse
transcription-polymerase chain reaction analysis showed that only the
angiotensin AT1A receptor was expressed in rat portal vein
smooth muscle. Furthermore, injection of anti-AT1A oligonucleotides selectively inhibited the angiotensin II-induced increase in [Ca2+]i. We conclude that the
receptor-activated signal leading to increases in
[Ca2+]i is transduced by the heterotrimeric
G13 protein composed of
13/
1/
3 subunits and that
the carboxyl terminus of the G
13 subunit interacts with
the angiotensin AT1A receptor.
In a variety of cells including smooth muscle cells, angiotensin
II (AII)1 appears to stimulate
voltage-dependent Ca2+ channels (1-5). In rat
portal vein myocytes, activation of angiotensin AT1
receptors induces an increase in the cytoplasmic Ca2+
concentration ([Ca2+]i) that depends on
activation of L-type Ca2+ channels and subsequent
Ca2+-induced Ca2+ release from the
intracellular stores without involving the inositol 1,4,5-trisphosphate
receptor (6). In contrast to data that have shown AII-induced
activation of phosphatidylinositol-specific phospholipase
C1 in vascular smooth muscle (7), other authors have
proposed that AII may induce hydrolysis of phosphatidylcholine by other
phospholipases C (5) or phospholipase D (8, 9), thereby leading to
generation of diacylglycerol and activation of protein kinase C. The
rat angiotensin AT1 receptor has been proposed to couple
with three distinct G proteins (Gq, Gi, and Go) on the basis of measurements of cAMP or inositol
1,4,5-trisphosphate accumulation and release of intracellularly stored
Ca2+ (10-12). However, in vascular myocytes, the G protein
that couples angiotensin AT1 receptors to
voltage-dependent Ca2+ channels and increases
in [Ca2+]i is insensitive to cholera toxin and
pertussis toxin (4, 5) and to intracellular application of an
anti-G
q/11 antibody (5).
Direct evidence that a G protein couples a receptor can be obtained by
using antibodies or antisense oligonucleotides targeting G protein
subunits (13, 14) and peptides that mimic the carboxyl-terminal region
of the G protein subunit, thus interacting with the receptors (15).
Information on receptor-G protein coupling has also been obtained by
studying the effects of synthetic peptides representing different
domains of the receptor on GTP
S binding to purified G proteins (16).
Although many articles have reported AII-induced increases in inositol
phosphate generation, only one has shown that the stimulation of
phosphatidylinositol hydrolysis by AII in membranes derived from NG
108-15 cells and rat liver appears to be attenuated (30-60%) but not
completely suppressed by an anti-
q/11 antibody (17). In
rat portal vein myocytes, intracellular application of an
anti-
q/11 antibody is ineffective with regard to
AII-induced increases in [Ca2+]i (5). Among the
pertussis toxin-insensitive G proteins, the functions of the two
members of the G12 subfamily (G12 and G13 proteins) are only beginning to be understood.
G
13 protein is widely expressed in mammalian tissues
(18, 19) and has been shown to be coupled to thromboxane and thrombin
receptors (20). Recently, expression of G
13 protein has
been reported to increase both Na+/H+ exchanger
and inducible nitric-oxide synthase activities (21-23) and
Ca2+ release-activated Ca2+ influx (24).
Furthermore, G
13 protein mediates inhibition of
voltage-dependent Ca2+ channels by bradykinin
(25).
The goal of this study was to determine the subunit composition of the
G protein that couples the angiotensin AT1 receptor to an
increase in [Ca2+]i in single myocytes isolated
from rat portal vein. Experiments with antisense oligonucleotides
designed to block synthesis of G protein subunits and angiotensin
AT1 receptor subtypes revealed that the AII-induced
elevation of [Ca2+]i was specifically inhibited
by antisense oligonucleotides directed against G13,
G
1, G
3, and angiotensin AT1A
receptors. Anti-
13 antibody and synthetic peptide
corresponding to the carboxyl terminus of the G
13
subunit also abrogated the angiotensin AT1A receptor-mediated Ca2+ response.
Isolated myocytes from
rat portal vein were obtained by enzymatic dispersion as described
previously (26). Cells were seeded at a density of ~103
cells/mm2 on glass slides imprinted with squares for
localization of injected cells and maintained in short-term primary
culture in medium M199 containing 2% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 20 units/ml
penicillin, and 20 µg/ml streptomycin; they were kept in an incubator
gassed with 95% air and 5% CO2 at 37 °C. The sequences of the oligonucleotides used in this study were determined by sequence
comparison and multiple alignment using Mac Molly Tetra software (Soft
Gene, Berlin). Oligonucleotides were from MWG-Biotech (Ebersberg,
Germany) or were synthesized in a DNA synthesizer (Milligen Model
8600); for synthesis of phosphorothioate oligonucleotides, the method
described by Iyer et al. (27) was used. Oligonucleotides were injected into the nuclei of myocytes by a manual injection system
(Eppendorf, Hamburg, Germany). The injection solution contained 10 µM oligonucleotides in water; ~10 fl were injected with
commercially available microcapillaries (Femtotips, Eppendorf) with an
outlet diameter of 0.5 µm. In some control experiments, myocytes were injected only with water and tested in comparison with non-injected cells and cells injected with nonsense or antisense oligonucleotides. The myocytes were cultured for 3-4 days in culture medium, and the
glass slides were transferred into a perfusion chamber for intracellular Ca2+ measurements. The sequences of
anti-ocom, anti-
q,
anti-
11, anti-
14, anti-
1,
anti-
2, anti-
3, anti-
4,
anti-
1, anti-
4, and anti-
5
antisense oligonucleotides have been previously published (14, 28). The
sequence of anti-
q/11com is ATGGACTCCAGAGT, corresponding to nucleotides 4-17 of
q cDNA (29);
those of anti-
12.1 and anti-
12.2 are
CTCCGGCCTCGGCCGGCAGCAAGC and CTAAGGGTCCGCACCACCCCGGACATG, respectively,
corresponding to nucleotides 32-55 and nucleotides
1 to +25 of
12 cDNA; those of anti-
13.1 and
anti-
13.2 are TGGTCGAAGTCCTGGCCGTGG and
CGACGGCAGGAAGTCCGCCATCTTG, respectively, corresponding to nucleotides
213-233 and nucleotides
4 to +21 of
13 cDNA; that
of nonsense
13.2 is GTTCTACCGCCTGAAGGACGGCAGC, corresponding to nucleotides
4 to +21 of
13 cDNA
(18); that of anti-
15 is CGTTATTGCTCAATCTCGGGTGGC,
corresponding to nucleotides
177 to
156 of
15
cDNA (30); that of anti-
5 is
TGCCATCTTCGTCCGGATGCAGCC, corresponding to nucleotides
18 to +6 of
5 cDNA (31); that of anti-
2 is
TTCCTTGGCATGCGCTTCAC, corresponding to nucleotides 122-141 of
2 cDNA (32); that of anti-
3 is
GTTCTCCGAAGTGGGCACAGGGGT, corresponding to nucleotides 165-188 of
3 cDNA (33); that of anti-
7 is
CTGGGCGACGTTGTTAGTACCTGA, corresponding to nucleotides 7-30 of rat
7 cDNA (34); that of anti-
8 is
GCGGGCCTCAGCGATCTTGGCCAT, corresponding to nucleotides 13-36 of
8 cDNA (35); those of anti-AT1A and
nonsense AT1A are GGCCATTTTGTTTTTCTGGGTTG and
GTTGGGTCTTTTTGTTTTACCGG, respectively, corresponding to nucleotides
17 to +6 of AT1A cDNA (36); that of scrambled
anti-AT1A is GCTGGTTTTTCTTTTGGATTGGC, corresponding to a
scrambled sequence of nucleotides
17 to +6 of AT1A
cDNA; and that of anti-AT1B is GGTCATGTCTCCCTTGGCGACGT, corresponding to nucleotides
17 to +6 of AT1B cDNA
(37).
Total RNA from rat portal vein myocytes was
purified using the Micro-Scale total RNA separation kit (CLONTECH, Palo
Alto, CA) following the instructions of the supplier. The resulting RNA
pellet was washed with 70% ethanol and resuspended in RNase-free water. The reverse transcription reaction was performed using the
Advantage RT-for-PCR kit (CLONTECH) following the instructions of the
supplier. The resulting cDNA was finally dissolved in 100 µl of
water. A volume of 10 µl was used to perform the PCR (first amplification) in 50 mM KCl, 2.5 mM
MgCl2, 20 mM Tris-HCl (pH 8.4), a 200 µM concentration of each dNTP, 200 ng of each primer (for
amplification of angiotensin AT1 receptor DNA fragments, a
200 nM concentration of each primer was used), and 2.5 units of Taq polymerase. For angiotensin AT1
receptors, amplification of DNA fragments was performed with 40 cycles
using the following conditions for each cycle: 90 s at 94 °C,
90 s at 55 °C, and 120 s at 72 °C. In each cycle, the
last step at 72 °C was extended by 5 s. For and
subunits, two subsequent amplifications of DNA fragments were
performed. In the first amplification, primers 1 and 3 were used. In
the second amplification, primer 2 (an internal primer for the
amplificates of the first amplification) and primer 3 or primer 1 were
used to confirm the specificity of the first amplification. To amplify
the fragments of the
subunits, the first amplification was
performed with 25 cycles using the following conditions for each cycle:
90 s at 95 °C, 90 s at 55 °C, and 120 s at
72 °C. The second amplification was performed using the same conditions and cycle numbers except that the annealing temperature was
increased to 60 °C. To amplify the fragments of the
subunits, the first amplification was performed with 30 cycles using the following conditions for each cycle: 90 s at 95 °C, 90 s
at 58 °C, and 120 s at 72 °C. The second amplification was
performed using the same conditions and cycle numbers except that the
annealing temperature was increased to 60 °C and the
MgCl2 concentration was decreased to 1.5 mM. As
an internal control, the expression of the
2-microglobulin single copy gene was tested in each
experiment. The amplified DNA fragments of cDNA were separated on a
2% agarose gel (Separide gel matrix, Life Technologies, Inc.) and
visualized by staining with ethidium bromide. Table I
indicates the primers used for PCRs and the references for cDNA
sequences (31, 32, 34-45).
|
Cells were loaded by incubation in physiological solution containing 1 µM fura-2/AM for 30 min at room temperature. These cells were washed and allowed to cleave the dye to the active fura-2 compound for at least 1 h. Fura-2 loading was usually uniform over the cytoplasm, and compartmentalization of the dye was never observed. Measurement of cytosolic Ca2+ concentration was carried out by the dual-wavelength fluorescence method as described previously (26). Briefly, fura-2-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope (Diaphot, Nikon, Tokyo). Single cells were alternately excited with UV light at 340 and 380 nm through a 10× oil immersion objective, and fluorescent light emitted from the Ca2+-sensitive dye was collected through a 510-nm long-pass filter with a charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan). The signal was processed (Hamamatsu DVS 3000) by correcting each fluorescence image for background fluorescence and calculating 340/380 nm fluorescence ratios on a pixel-to-pixel basis. Averaged frames were usually collected at each wavelength every 0.5 s. [Ca2+]i was calculated from mean ratios using a calibration for fura-2 determined in loaded cells (26). In some experiments, cells were loaded through a patch-clamp pipette filled with a solution containing 140 mM CsCl, 10 mM HEPES, and 0.05 mM Indo-1 (pH 7.3). The cell studied was illuminated at 360 nm, and emitted light was counted simultaneously at 405 and 480 nm by two photomultipliers (P1, Nikon). [Ca2+]i was estimated from the 405/480 nm fluorescence ratio using a calibration determined within cells (6). All measurements were made at 25 ± 1 °C.
The normal physiological solution contained 130 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 11 mM glucose, and 10 mM HEPES (pH 7.4) with NaOH. Substances were applied to the cells by pressure ejection from a glass pipette for the periods indicated on the figures. Before each experiment, a fast application of physiological solution was tested, and cells with movement artifacts were excluded.
Results are expressed as means ± S.E. Significance was tested by Student's t test. p values < 0.05 were considered as significant.
ImmunocytochemistryThree days after injection, myocytes were washed with phosphate-buffered saline solution, fixed with 3% (v/v) formaldehyde for 30 min at room temperature, and permeabilized in phosphate-buffered saline containing 3% fetal calf serum and 0.01 (w/v) saponin for 30 min. Cells were incubated with the same buffer containing 5% fetal calf serum, 0.01 (w/v) saponin, and the anti-G protein antibody at 1:100 dilution overnight at 4 °C. Then, cells were washed (4 × 10 min) in phosphate-buffered saline containing 3% fetal calf serum and 0.01 (w/v) saponin and incubated with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (diluted 1:200) in the same solution for 8 h at 4 °C. Thereafter, cells were washed (4 × 10 min) in phosphate-buffered saline and mounted in Moviol (Hoechst, Frankfurt, Germany). Images of the stained cells were obtained with a confocal microscope (MRC 1000, Bio-Rad, Paris). Only cells on the same glass slide were compared with each other by keeping acquisition parameters (gray values, exposure time, aperture, etc.) constant. Immunostaining fluorescence was estimated by gray level analysis using the macro-programming language (MPL) software (Bio-Rad).
Chemicals and DrugsMedium M199 was from Flow Laboratories
(Puteaux, France). Fetal calf serum was from Flobio (Courbevoie,
France). Streptomycin, penicillin, glutamate, and pyruvate were from
Gibco (Paisley, United Kingdom). Fura-2/AM, Indo-1, carboxyl-terminal
G13 peptide (LHDNLKQLMLQ; CN 371786), and
carboxyl-terminal anti-
13 antibody (CN 371784) were from
Calbiochem (Meudon, France). Norepinephrine, rauwolscine, and
propranolol were from Sigma (St.-Quentin Fallavier, France). Caffeine was from Merck (Nogent-sur-Marne, France).
Angiotensin II and CGP42112A
(N-
-nicotinoyl-Tyr-Lys-(N-
-benzyloxycarbonyl-Arg)-His-Pro-Ile-OH) were from Neosystem Laboratories (Strasbourg, France). The
anti-
12 antibody was a gift from K. Spicher (University
of Berlin). Carboxyl-terminal G
q/11 peptide
(LQLNLKEYNLV) was a gift from G. Guillon (INSERM U401, Montpellier,
France). Anti-
1 (SC 379) and anti-
3 (SC
375) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was from
Immunotech (Marseille, France).
We have previously shown that,
in rat portal vein myocytes, AII stimulates L-type Ca2+
channels by binding to angiotensin AT1 receptors through a
transduction pathway involving both pertussis toxin- and cholera
toxin-insensitive G protein, phosphatidylcholine-specific phospholipase
C, and protein kinase C (5). L-type Ca2+ channel activation
leads to a slow elevation of [Ca2+]i, which
triggers a subsequent Ca2+ release from intracellular
stores (6, 46). To identify the heterotrimeric G protein that
transduces the AT1 receptor signal, we injected completely
phosphorothioate-modified antisense oligonucleotides directed against
the ,
, and
subunits of G proteins into the nucleus of
vascular myocytes. We measured the increase in
[Ca2+]i induced by successive applications of 10 µM norepinephrine (in the presence of 10 nM
rauwolscine and 1 µM propranolol to inhibit both
2- and
-adrenoreceptors), 10 nM AII (in
the presence of 100 nM CGP42112A to inhibit angiotensin
AT2 receptors), and 10 mM caffeine on the same
cells 3 days after oligonucleotide injection (Fig. 1).
For each experiment, we compared the Ca2+ responses of
antisense or nonsense oligonucleotide-injected cells located within a
marked area on the glass slide with those of non-injected cells outside
this marked area. This procedure ensures that oligonucleotide-injected
cells were always compared with control cells that were grown, treated
(i.e. incubation and loading with fura-2/AM), and analyzed
under identical conditions. The increases in
[Ca2+]i were measured for each cell, and mean
values were calculated from all cells of each experiment. Myocytes
injected with anti-
13.1 or anti-
13.2
antisense oligonucleotides showed a strong inhibition (70-75%) of the
AII-induced Ca2+ response when compared with non-injected
cells or cells injected with
13.2 nonsense
oligonucleotides (Fig. 2). As maximal inhibition of the
AII-induced Ca2+ responses was obtained 3 days after
injection of anti-
13.2 oligonucleotides, all further
measurements were performed under similar conditions. As illustrated in
Figs. 1 and 2, cells injected with 10 µM antisense oligonucleotides directed against
o1 and
o2 (anti-
ocom),
q (anti-
q),
11 (anti-
11),
q/11 (anti-
q/11com),
12
(anti-
12.1 and anti-
12.2),
14 (anti-
14), and
15
(anti-
15) subunits showed no significant reduction in
Ca2+ responses evoked by AII when compared with
non-injected cells.
Antibodies directed against the carboxyl terminus of G protein subunits and synthetic peptides corresponding to specific regions of G
protein
subunits are useful tools for identifying the transduction
pathways (13, 15). When the anti-
13 antibody was added
to the basic pipette solution for 7 min, the AII-induced increase in
[Ca2+]i was concentration-dependently
inhibited (Fig. 3). With 5 µg/ml intracellular
anti-
13 antibody, the AII-induced Ca2+
response was decreased by ~60% (control: 115 ± 27 nM, n = 6; in the presence of the
anti-
13 antibody: 43 ± 17 nM,
n = 6, p < 0.05). With 10 µg/ml
anti-
13 antibody, the AII-induced Ca2+
response was never observed (n = 12). In contrast,
applications of 10 µg/ml boiled anti-
13 antibody
(95 °C for 30 min) did not alter the AII-induced Ca2+
response (107 ± 31 nM, n = 8) (Fig.
3). Similarly, intracellular application of a synthetic peptide
corresponding to the carboxyl terminus of the
13 subunit
inhibited, in a concentration-dependent manner, the
AII-induced increase in [Ca2+]i (Fig.
4A), whereas internal application of an
q/11 peptide (corresponding to the carboxyl terminus of
the
q and
11 subunits) had no effect on
the AII-induced Ca2+ response. The concentration of
13 peptide producing half-maximal inhibition was
estimated to be 2.3 ± 0.4 ng/ml. In addition, intracellular application of 100 ng/ml
13 peptide had no effect on the
Ca2+ channel current evoked by a depolarizing pulse to 0 mV
from a holding potential of
50 mV (n = 14) (data not
shown). As additional control experiments, we verified that internal
application of 100 ng/ml
13 peptide had no effect on the
norepinephrine-induced Ca2+ response (control: 327 ± 25 nM, n = 5; in the presence of the
13 peptide: 321 ± 17 nM,
n = 5, p > 0.05) (Fig. 4B)
and the caffeine-induced Ca2+ response (control: 345 ± 31 nM, n = 7; in the presence of the
13 peptide: 329 ± 33 nM,
n = 7, p > 0.05) (Fig. 4C).
These results suggest that the peptide corresponding to the carboxyl
terminus of the
13 subunit may specifically compete with
endogenous G
13 protein for interaction with angiotensin
AT1 receptors.
We also identified the and
subunits that compose the G protein
heterotrimer activated by AT1 receptors by using injection of antisense oligonucleotides. As illustrated in Fig. 5
(A and B), only the oligonucleotides directed
against the
1 (anti-
1) and
3 (anti-
3) subunits significantly reduced
the AII-induced Ca2+ response. Inhibition evoked by the
oligonucleotides directed against the
1 and
3 subunits (70 and 69%, respectively) was quantitatively similar to that induced by the oligonucleotides directed
against the
13 subunit, suggesting that the
heterotrimeric G protein composed of
13/
1/
3 subunits may
control the Ca2+ response evoked by AII application.
Specificity of Antisense Oligonucleotides
To verify that
injection of antisense oligonucleotides directed against a G protein
subunit specifically suppresses involvement of this subunit in the
AII-activated transduction coupling, we performed two types of control
experiments. First, the cells were stained with either
12- or
13-specific antibodies and
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, and
immunofluorescence was quantified by using the MPL software of the
confocal microscope (Fig. 2B). In cells injected with
anti-
12.2 antisense oligonucleotides, the
immunofluorescence signal revealed by the anti-
12
antibody was reduced by 80 ± 5% (n = 10),
whereas it was only slightly affected in cells injected with
anti-
13.2 antisense oligonucleotides (12 ± 4%,
n = 10) (Fig. 2B). Similarly, the
immunofluorescence signal revealed by the anti-
13
antibody was reduced by 85 ± 3% (n = 12) in
cells injected with anti-
13.2 antisense
oligonucleotides, whereas it was little affected in cells injected with
anti-
12.2 antisense oligonucleotides (10 ± 2%,
n = 12) (Fig. 2B). Then, we tested the
effects of injection of anti-
1 and anti-
3
antisense oligonucleotides on the expression of the
13
subunit by staining with the anti-
13 antibody (Fig.
5C). The immunofluorescence signal was not significantly
reduced in the cells injected with anti-
1 or
anti-
3 antisense oligonucleotides, whereas it was
largely inhibited in cells injected with anti-
13.2
antisense oligonucleotides (Fig. 5C). Finally, we verified
that, in cells stained with the anti-
1 or
anti-
3 antibody, the immunofluorescence signal was inhibited only in cells injected with the corresponding antisense oligonucleotides (n = 9-19) (data not shown).
Second, we compared the effects of AII with those of norepinephrine and
caffeine in each cell studied. We have recently shown that activation
of 1A-adrenoreceptors induces inositol
1,4,5-trisphosphate-induced Ca2+ release from the
intracellular stores (26). In cells injected with either
anti-
13.1 or anti-
13.2 antisense
oligonucleotides, increases in [Ca2+]i evoked by
norepinephrine and caffeine were unchanged when compared with control
measurements (n = 44) (Fig. 1). We noted unspecific
effects of antisense oligonucleotides only at concentrations of 50 µM, i.e. 5 times higher than the concentration used in these experiments (n = 15). Taken together,
these results indicate that suppression of AII-mediated effects by
antisense oligonucleotides does not interfere with another signaling
pathway (i.e. that of norepinephrine) and with the
intracellular Ca2+ stores.
To determine
which and
subunits are expressed in rat portal vein smooth
muscle, mRNA was purified and reversibly transcribed into cDNA,
and a fragment of cDNA of each subunit was amplified using
subtype-specific primers for the PCR (Fig. 6). Five
(
1-
5) and six
(
2-
5,
7, and
8)
subunits were found to be expressed (the
3 band is only
weakly visible). The expression of the
2-microglobulin gene served as an internal positive control for the PCR.
Expression of Angiotensin AT1 Receptor mRNAs and Effects of Oligonucleotides Directed against Angiotensin AT1A and AT1B Receptors
The expression of
angiotensin AT1 receptor subtypes was examined in rat
portal vein smooth muscle by using subtype-specific primers designed to
amplify cDNA fragments of AT1A or AT1B
receptors (Fig. 7A). Only the
AT1A receptor was found to be expressed. The third
intracellular loop and carboxyl-terminal fragments of angiotensin AT1 receptors have been shown to be involved in G protein
coupling (11, 47). Primers designed to amplify these two regions
produced amplificates displaying size (Fig. 7A) and sequence
(data not shown) corresponding to those of the cloned rat angiotensin
AT1A receptor (43). As illustrated in Fig. 7B,
cells injected with 10 µM antisense oligonucleotides
directed against angiotensin AT1A receptors
(anti-AT1A) showed a high level of inhibition (70%) of the
AII-induced increase in [Ca2+]i when compared
with non-injected cells and cells injected with anti-AT1B
antisense, scrambled anti-AT1A antisense, or nonsense AT1A oligonucleotides.
Our results show that, in rat portal vein myocytes, the G protein
heterotrimer G13
1
3 is
coupled to angiotensin AT1A receptors and activates a
transduction pathway leading to increases in
[Ca2+]i. This conclusion is based on experiments
using antisense oligonucleotides to block expression of G protein
subunits, antibodies raised against the carboxyl terminus of G
subunits to block interactions of G proteins with angiotensin
AT1 receptors, and synthetic peptides corresponding to the
carboxyl terminus of G
subunits to disrupt the angiotensin
AT1 receptor-evoked activation of G proteins.
Antisense oligonucleotides have been used previously to demonstrate the
involvement of both G and G
subunits in the selectivity of
receptor-mediated inhibition of voltage-dependent
Ca2+ channels and Ca2+ release from
intracellular stores (14, 28, 48-50). Our results indicate that
anti-
13 oligonucleotide injection into the nucleus of
myocytes selectively inhibited the AII-induced increase in [Ca2+]i by ~75%, a value similar to that
obtained with injection of anti-
1 or
anti-
3 antisense oligonucleotides. These inhibitions correlated well with reductions in the expression of the corresponding G protein subunits immunostained by anti-
13,
anti-
1, and anti-
3 antibodies. The facts
that the AII-induced increase in [Ca2+]i was
selectively inhibited after blockade of
G
13
1
3 protein expression
and that anti-
1 and anti-
3
oligonucleotides did not inhibit the expression of G
13
protein support the concept that activation of G proteins by receptors
requires the presence of all three
13,
1,
and
3 subunits.
The specificity of 1
3 subunits in
G13 protein-mediated AII-induced increases in
[Ca2+]i may depend on a high affinity or efficacy
of this dimer (compared with other
subunits expressed in portal
vein smooth muscle) to associate with the
13 subunit and
angiotensin AT1 receptors in a functional protein complex.
An alternative cause of specificity may be the generation of a high
concentration of
1
3 dimer in a restricted
plasma membrane area. Clustered distributions of G protein associated
with cytoskeletal proteins or localized in membrane domains
corresponding to caveolae have been previously reported (51-53).
Finally, it is also possible that
1
3
regulates the activity of specific effector proteins transducing the
AII-induced signal (for review, see Refs. 54-56). Further experiments
are in progress to identify the role of
1
3 subunits in AII-mediated transmembrane
signaling.
Antisense oligonucleotide blockade alone cannot distinguish which part
of the G protein is required for interaction with the angiotensin
AT1 receptor. The ability of the carboxyl terminus of the
13 subunit to couple the angiotensin AT1
receptor is supported by the observation that an anti-
13
antibody, but not an anti-
q/11 antibody (raised against
the carboxyl termini of the G
13 and G
q/11
subunits) (5), selectively blocks the AII-induced increase in
[Ca2+]i. Recently, peptides corresponding to
various regions of G
subunits have been tested for their ability to
block receptor-mediated activation of effectors or to bind directly to
receptors and to stabilize them in a high affinity conformation state
for agonists (15, 57, 58). Thus, these peptides act as competitive
agonists at the receptor/G protein interface (59) and may be used to block receptor-activated transduction pathways. Intracellular application of the carboxyl-terminal
13 peptide
inhibits, in a concentration-dependent manner, the
AII-induced increase in [Ca2+]i, whereas the
carboxyl-terminal
q/11 peptide is ineffective. Taken
together, these results show that, in portal vein myocytes, the
angiotensin AT1 receptor binds to the extreme carboxyl
terminus of the
13 subunit, in contrast to the
1A-adrenoreceptor, which has been shown to bind to the
carboxyl terminus of the
q/11 subunit (26). It is
generally accepted that the angiotensin AT1 receptor couples phospholipase C-
via a Gq/11 protein and
stimulates phosphatidylinositol breakdown, leading to the production of
inositol phosphates and diacylglycerol (60). This seems not to be the
case in all vascular smooth muscles since a phospholipase
C-
1 has been reported to mediate the AII-induced
inositol phosphate accumulation in rat aorta (7). Furthermore, in
membranes derived from NG 108-15 cells and rat liver, the AII-induced
stimulation of phosphatidylinositol hydrolysis is reduced by 30-60%,
but is not completely suppressed, in the presence of an
anti-
q/11 antibody, suggesting the possible involvement
of G proteins different from Gq/11 protein in the AII-mediated transduction coupling (17). This Gq/11
protein-independent inositol phosphate production evoked by AII could
be the result of activation of Ca2+-modulated phospholipase
C-
or C-
(7, 61). Finally, we showed that activation of the
angiotensin AT1 receptors in venous cell line membranes
(RVF-SMC) led to increased incorporation of the photoreactive GTP
analogue [
-32P]GTP azidoanilide into
immunoprecipitated
13 and
q/11 subunits, indicating that angiotensin AT1 receptors may potentially
couple to both G13 and Gq/11
proteins.2 Nevertheless, in portal vein
myocytes, the
q/11 and
13 subunit domains
interacting with the angiotensin AT1 receptor seem to be
independent of each other since the carboxyl-terminal
q/11 peptide does not interfere with the angiotensin
AT1 receptor-induced activation of
G
13
1
3 protein. The fact
that the AII-induced increase in [Ca2+]i is only
transduced by G13 protein could be related to the existence
of a particular subtype of angiotensin AT1 receptor. This
possibility can be discarded since the reverse transcription-PCR analysis shows that portal vein smooth muscle expresses only the angiotensin AT1A receptor. The sequences of the receptor
domains known to interact with the G protein show 100% identity to
those of the cloned angiotensin AT1A receptor. Moreover,
inhibition of angiotensin AT1A receptor expression largely
inhibited the AII-induced Ca2+ responses. These
observations suggest that the specificity of the angiotensin
AT1A receptor/G13 protein interaction in portal vein myocytes depends probably on both membrane environment and structural constraints achieved by the receptor-G protein-effector complex (62).
In conclusion, we show that, in portal vein myocytes, the angiotensin
AT1A receptor binds to a specific
G13
1
3 heterotrimer, leading to increases in [Ca2+]i, and that the
interaction involves the extreme carboxyl terminus of the
13 subunit.
We thank E. Glass and N. Biendon for technical assistance.