G Protein Heterotrimer Galpha 13beta 1gamma 3 Couples the Angiotensin AT1A Receptor to Increases in Cytoplasmic Ca2+ in Rat Portal Vein Myocytes*

(Received for publication, November 15, 1996, and in revised form, January 23, 1997)

Nathalie Macrez-Leprêtre Dagger §, Frank Kalkbrenner , Jean-Luc Morel Dagger , Günter Schultz and Jean Mironneau Dagger par

From the Dagger  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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

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 alpha 13, beta 1, and gamma 3 subunits. A correlating reduction in Galpha 13, Gbeta 1, and Ggamma 3 protein expression was confirmed by immunocytochemistry. In addition, anti-alpha 13 antibody and synthetic peptide corresponding to the carboxyl terminus of the Galpha 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 alpha 13/beta 1/gamma 3 subunits and that the carboxyl terminus of the Galpha 13 subunit interacts with the angiotensin AT1A receptor.


INTRODUCTION

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 Cgamma 1 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-Galpha 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 alpha  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 GTPgamma 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-alpha q/11 antibody (17). In rat portal vein myocytes, intracellular application of an anti-alpha 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. Galpha 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 Galpha 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, Galpha 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 Galpha 13, Gbeta 1, Ggamma 3, and angiotensin AT1A receptors. Anti-alpha 13 antibody and synthetic peptide corresponding to the carboxyl terminus of the Galpha 13 subunit also abrogated the angiotensin AT1A receptor-mediated Ca2+ response.


EXPERIMENTAL PROCEDURES

Microinjection of Oligonucleotides

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-alpha ocom, anti-alpha q, anti-alpha 11, anti-alpha 14, anti-beta 1, anti-beta 2, anti-beta 3, anti-beta 4, anti-gamma 1, anti-gamma 4, and anti-gamma 5 antisense oligonucleotides have been previously published (14, 28). The sequence of anti-alpha q/11com is ATGGACTCCAGAGT, corresponding to nucleotides 4-17 of alpha q cDNA (29); those of anti-alpha 12.1 and anti-alpha 12.2 are CTCCGGCCTCGGCCGGCAGCAAGC and CTAAGGGTCCGCACCACCCCGGACATG, respectively, corresponding to nucleotides 32-55 and nucleotides -1 to +25 of alpha 12 cDNA; those of anti-alpha 13.1 and anti-alpha 13.2 are TGGTCGAAGTCCTGGCCGTGG and CGACGGCAGGAAGTCCGCCATCTTG, respectively, corresponding to nucleotides 213-233 and nucleotides -4 to +21 of alpha 13 cDNA; that of nonsense alpha 13.2 is GTTCTACCGCCTGAAGGACGGCAGC, corresponding to nucleotides -4 to +21 of alpha 13 cDNA (18); that of anti-alpha 15 is CGTTATTGCTCAATCTCGGGTGGC, corresponding to nucleotides -177 to -156 of alpha 15 cDNA (30); that of anti-beta 5 is TGCCATCTTCGTCCGGATGCAGCC, corresponding to nucleotides -18 to +6 of beta 5 cDNA (31); that of anti-gamma 2 is TTCCTTGGCATGCGCTTCAC, corresponding to nucleotides 122-141 of gamma 2 cDNA (32); that of anti-gamma 3 is GTTCTCCGAAGTGGGCACAGGGGT, corresponding to nucleotides 165-188 of gamma 3 cDNA (33); that of anti-gamma 7 is CTGGGCGACGTTGTTAGTACCTGA, corresponding to nucleotides 7-30 of rat gamma 7 cDNA (34); that of anti-gamma 8 is GCGGGCCTCAGCGATCTTGGCCAT, corresponding to nucleotides 13-36 of gamma 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).

RNA Purification and Reverse Transcription-Polymerase Chain Reaction (PCR)

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 beta  and gamma  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 beta  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 gamma  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 beta 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).

Table I.

Sequence of primers used for DNA fragment amplifications of Gbeta and Ggamma subunits and of angiotensin AT1 receptors


Primer Primer sequence Amplified fragment (nucleotides) Ref. for cDNA sequences

 beta 1
  1 GCAGATGCAACTCTCTCTCAGAT
  2 GGCGTGGACCTTGTTGGTGGT 75 -275 38
  3 ATGTCCGGTAAACGTGGTCGTCT
 beta 2
  1 GGGGACTCAACACTGACCCAGAT
  2 GTTGGTGGTGTAGCTGTCCCAGA 18 -239 39
  3 TCGGCCCGCAGGTCGAAGAGG
 beta 3
  1 ATGGGGGAGATGGAGCCACTGCG
  2 AGCAGCTTAGAATCAGTGGCCCA 1 -211 40
  3 CCTCCTCAGTTCCAGATTTT
 beta 4
  1 AACGATGCCACGCTGGTTCAGAT
  2 CTCAGAGGGATGGCGTGCATCT 76 -287 41
  3 ATATCCCACAGCTTTGAGGAT
 beta 5
  1 GGCTGCATCCGGACGAAGATG
  2 CATGCACAGGACTTTGTTCCCG 18 -239 31
  3 CTTCTTCTTGGCGGCCATGTTC
 gamma 1
  1 AGATGCCAGTGATCAATATTGAGG
  2 CCAGCGTCACTTCTTTCTTGAGC 2 -145 42
  3 AATCACACAGCCTCCTTTGAGCT
 gamma 2
  1 ATGGCCAGCAACAACACCGCCA
  2 TTCCTTGGCATGCGCTTCACAGT 1 -141 32
  3 GATAGCACAGAAAAACTTCTTCTC
 gamma 3
  1 ATGAAAGGGGAGACCCCTGTGAACA
  2 GTTCTCGGAAGTGGGCACAGG 1 -186 43
  3 CAGAGGAGCGCACAGAAGAAC
 gamma 4
  1 GGAGCAGCTGAAGATGGAAGCCTG
  2 GTTTTCGGAGGCGGGCACTGGGA 130 43
  3 GAGGATGGTGCAGAAGAACTTC
 gamma 5
  1 ATGTCGGGTTCTTCTAGCGTCGC
  2 TGTACTTGAAGACACTCCAGTCAG 1 -168 44
  3 CTACAAAAAGGAGCAGACTTTCTG
 gamma 7
  1 GTCAGGTACTAACAACGTCGCC
  2 CAGAGGCTGGTACACCAACC 6 -195 34
  3 GAGAATTATGCAAGGCTTTTTGTC
 gamma 8
  1 GTCCAACAACATGGCCAAGATCG
  2 CATCCTTAGCGTGCGTTTCGC 139 35
  3 GAGCAGGGTGCAAAAGAGTCG
AT1A C terminus
    1 GTCATCCATGACTGTAAAATTT 808 -996 36
    2 CGTAGACAGGCTTGAGTGG
AT1A 3rd intracellular loop
    1 TAGGGCTGGGCCTTACCAAGAAT 578 -833 36
    2 TCAGAAATTTTACAGTCATGGATGAC
AT1B C terminus
    1 ATTATCCGTGACTGTGAAATTG 808 -996 37
    2 TGTTGACAAGCCTGCGTTGT
 beta 2 Microglobulin
    1 ATTCAGAAAACTCCCCAAATTCAAGT 61 -362 45
    2 GATTACATGTCTCGGTCCCAGGT

Measurements of Cytosolic Ca2+

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.

Immunocytochemistry

Three 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 Drugs

Medium 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 Galpha 13 peptide (LHDNLKQLMLQ; CN 371786), and carboxyl-terminal anti-alpha 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-alpha -nicotinoyl-Tyr-Lys-(N-alpha -benzyloxycarbonyl-Arg)-His-Pro-Ile-OH) were from Neosystem Laboratories (Strasbourg, France). The anti-alpha 12 antibody was a gift from K. Spicher (University of Berlin). Carboxyl-terminal Galpha q/11 peptide (LQLNLKEYNLV) was a gift from G. Guillon (INSERM U401, Montpellier, France). Anti-beta 1 (SC 379) and anti-gamma 3 (SC 375) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was from Immunotech (Marseille, France).


RESULTS

Identification of the Subunit Composition of the G Protein Coupling Angiotensin AT1 Receptors to Increases in [Ca2+]i

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 alpha , beta , and gamma  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 alpha 2- and beta -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-alpha 13.1 or anti-alpha 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 alpha 13.2 nonsense oligonucleotides (Fig. 2). As maximal inhibition of the AII-induced Ca2+ responses was obtained 3 days after injection of anti-alpha 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 alpha o1 and alpha o2 (anti-alpha ocom), alpha q (anti-alpha q), alpha 11 (anti-alpha 11), alpha q/11 (anti-alpha q/11com), alpha 12 (anti-alpha 12.1 and anti-alpha 12.2), alpha 14 (anti-alpha 14), and alpha 15 (anti-alpha 15) subunits showed no significant reduction in Ca2+ responses evoked by AII when compared with non-injected cells.


Fig. 1. Increase in [Ca2+]i evoked by AII, norepinephrine, and caffeine in rat portal vein myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs of Galpha q/11, Galpha 13, and Galpha 12 proteins. Ca2+ responses were obtained in the same cells with successive applications of 10 µM norepinephrine (NE), 10 nM AII, and 10 mM caffeine (Caf) (separated by a 3-min interval) in non-injected control cells (A) and in cells injected with 10 µM anti-alpha q/11 (B), anti-alpha 13.2 (C), and anti-alpha 12.2 (D) antisense oligonucleotides. Cells were used 3 days after nuclear injection of antisense oligonucleotides. Norepinephrine, AII, and caffeine were ejected from a glass pipette close to the cell for the periods indicated. Cells were loaded with fura-2/AM and not patch-clamped. CGP42112A (100 nM), rauwolscine (10 nM), and propranolol (1 µM) were present throughout the experiments.
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Fig. 2. Increase in [Ca2+]i evoked by 10 nM AII in myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs of alpha  subunits of G proteins. A, effects of anti-alpha q/11com, anti-alpha q, anti-alpha 11, anti-alpha 12.1, anti-alpha 12.2, anti-alpha 13.1, anti-alpha 13.2, anti-alpha 14, anti-alpha 15, and anti-alpha ocom antisense and of alpha 13.2 nonsense oligonucleotides on Ca2+ responses evoked by 10 nM AII. Bars show means ± S.E. in non-injected cells (open bars) and in oligonucleotide-injected cells (hatched bars). Numbers in parentheses indicate the number of cells tested. star , values significantly different from those obtained in non-injected cells (p < 0.01). Cells were loaded with fura-2/AM and not patch-clamped. CGP42112A (100 nM), rauwolscine (10 nM), and propranolol (1 µM) were present throughout the experiments. B, specific inhibition of Galpha 12 or Galpha 13 protein expression in myocytes injected with anti-alpha 12.2 or anti-alpha 13.2 antisense oligonucleotides, respectively. Myocytes were stained 3 days after injection with the anti-alpha 12 antibody (upper panels) and with the anti-alpha 13 antibody (lower panels). Visualization was obtained by staining with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200). Cells injected with anti-alpha 12.2 and anti-alpha 13.2 antisense oligonucleotides were compared with non-injected cells on the same glass slide.
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Antibodies directed against the carboxyl terminus of G protein alpha  subunits and synthetic peptides corresponding to specific regions of G protein alpha  subunits are useful tools for identifying the transduction pathways (13, 15). When the anti-alpha 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-alpha 13 antibody, the AII-induced Ca2+ response was decreased by ~60% (control: 115 ± 27 nM, n = 6; in the presence of the anti-alpha 13 antibody: 43 ± 17 nM, n = 6, p < 0.05). With 10 µg/ml anti-alpha 13 antibody, the AII-induced Ca2+ response was never observed (n = 12). In contrast, applications of 10 µg/ml boiled anti-alpha 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 alpha 13 subunit inhibited, in a concentration-dependent manner, the AII-induced increase in [Ca2+]i (Fig. 4A), whereas internal application of an alpha q/11 peptide (corresponding to the carboxyl terminus of the alpha q and alpha 11 subunits) had no effect on the AII-induced Ca2+ response. The concentration of alpha 13 peptide producing half-maximal inhibition was estimated to be 2.3 ± 0.4 ng/ml. In addition, intracellular application of 100 ng/ml alpha 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 alpha 13 peptide had no effect on the norepinephrine-induced Ca2+ response (control: 327 ± 25 nM, n = 5; in the presence of the alpha 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 alpha 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 alpha 13 subunit may specifically compete with endogenous Galpha 13 protein for interaction with angiotensin AT1 receptors.


Fig. 3. Effects of carboxyl-terminal anti-alpha 13 antibody on the AII-induced increase in [Ca2+]i in voltage-clamped single myocytes (at a holding potential of -50 mV). Ca2+ responses were evoked by 10 nM AII under control conditions (trace a) and in cells dialyzed for 7 min with a pipette solution containing 5 µg/ml anti-alpha 13 antibody (trace b), 10 µg/ml anti-alpha 13 antibody (trace c), or 10 µg/ml boiled anti-alpha 13 antibody (95 °C for 30 min; trace d). The pipette solution contained 50 µM Indo-1, and the external solution contained 100 nM CGP42112A.
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Fig. 4. Effects of carboxyl-terminal alpha 13 peptide on the AII-, norepinephrine- and caffeine-induced increases in [Ca2+]i. A, effects of carboxyl-terminal alpha 13 peptide (black-square) and carboxyl-terminal alpha q/11 peptide (bullet ) on the increase in [Ca2+]i evoked by 10 nM AII. The inhibition curve shows half-maximal and complete inhibition at 2.3 and 100 ng/ml alpha 13 peptide, respectively. Each point represents the mean ± S.E. for 6-12 cells. B and C, increases in [Ca2+]i evoked by 10 µM norepinephrine (NE) and 10 mM caffeine (Caf) under control conditions (trace a) and in cells dialyzed for 7 min with a pipette solution containing 100 ng/ml alpha 13 peptide (trace b). The pipette solution contained 50 µM Indo-1, and the external solution contained 100 nM CGP42112A.
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We also identified the beta  and gamma  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 beta 1 (anti-beta 1) and gamma 3 (anti-gamma 3) subunits significantly reduced the AII-induced Ca2+ response. Inhibition evoked by the oligonucleotides directed against the beta 1 and gamma 3 subunits (70 and 69%, respectively) was quantitatively similar to that induced by the oligonucleotides directed against the alpha 13 subunit, suggesting that the heterotrimeric G protein composed of alpha 13/beta 1/gamma 3 subunits may control the Ca2+ response evoked by AII application.


Fig. 5. Increase in [Ca2+]i evoked by 10 nM AII in myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs of beta  (A) and gamma  (B) subunits of G proteins. Cells were loaded with fura-2/AM 3 days after nuclear injection. Bars show means ± S.E. in non-injected cells (open bars) and in antisense oligonucleotide-injected cells (hatched bars). Numbers in parentheses indicate the number of cells tested. star , values significantly different from those obtained in non-injected cells (p < 0.01). CGP42112A (100 nM) was present throughout the experiments. Shown in C is a diagram illustrating the specific inhibition of Galpha 13 protein expression (immunostained with the anti-alpha 13 antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG) by injection of the anti-alpha 13.2 antisense oligonucleotide and the absence of effects of anti-beta 1 and anti-gamma 3 antisense oligonucleotides. Immunofluorescence is expressed in arbitrary units (AU). Open bars (non-injected cells) and hatched bars (antisense oligonucleotide-injected cells) show means ± S.E. Numbers in parentheses indicate the number of cells tested. star , values significantly different from those obtained in non-injected cells (p < 0.01).
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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 alpha 12- or alpha 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-alpha 12.2 antisense oligonucleotides, the immunofluorescence signal revealed by the anti-alpha 12 antibody was reduced by 80 ± 5% (n = 10), whereas it was only slightly affected in cells injected with anti-alpha 13.2 antisense oligonucleotides (12 ± 4%, n = 10) (Fig. 2B). Similarly, the immunofluorescence signal revealed by the anti-alpha 13 antibody was reduced by 85 ± 3% (n = 12) in cells injected with anti-alpha 13.2 antisense oligonucleotides, whereas it was little affected in cells injected with anti-alpha 12.2 antisense oligonucleotides (10 ± 2%, n = 12) (Fig. 2B). Then, we tested the effects of injection of anti-beta 1 and anti-gamma 3 antisense oligonucleotides on the expression of the alpha 13 subunit by staining with the anti-alpha 13 antibody (Fig. 5C). The immunofluorescence signal was not significantly reduced in the cells injected with anti-beta 1 or anti-gamma 3 antisense oligonucleotides, whereas it was largely inhibited in cells injected with anti-alpha 13.2 antisense oligonucleotides (Fig. 5C). Finally, we verified that, in cells stained with the anti-beta 1 or anti-gamma 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 alpha 1A-adrenoreceptors induces inositol 1,4,5-trisphosphate-induced Ca2+ release from the intracellular stores (26). In cells injected with either anti-alpha 13.1 or anti-alpha 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.

Expression of beta  and gamma  Subunit mRNAs

To determine which beta  and gamma  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 (beta 1-beta 5) and six (gamma 2-gamma 5, gamma 7, and gamma 8) subunits were found to be expressed (the beta 3 band is only weakly visible). The expression of the beta 2-microglobulin gene served as an internal positive control for the PCR.


Fig. 6. Amplification of G protein beta  and gamma  subunit DNA fragments from rat portal vein smooth muscle. Amplified cDNA fragments corresponding to beta  subunits (upper panel) and gamma  subunits (lower panel) and of the beta 2-microglobulin (beta 2M) were separated on a 2% agarose gel and visualized by staining with ethidium bromide. Numbers on the left indicate molecular size standards in base pairs (KB). For RNA purification and PCR conditions, see "Experimental Procedures."
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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.


Fig. 7. Expression of angiotensin AT1A receptor mRNA and effects of anti-AT1A and anti-AT1B antisense oligonucleotides in rat portal vein myocytes. A, amplified cDNA fragments corresponding to the carboxyl terminus (lane 1) and the third intracellular loop (lane 2) of the angiotensin AT1A receptor, the carboxyl terminus of the angiotensin AT1B receptor (lane 3), and part of the beta 2-microglobulin (lane 4) were separated on a 2% agarose gel and visualized by staining with ethidium bromide. Numbers on the left indicate molecular size standards in base pairs (KB). For RNA purification and PCR conditions, see "Experimental Procedures." B, effects of anti-AT1A and anti-AT1B antisense, scrambled anti-AT1A antisense, and AT1A sense oligonucleotides on Ca2+ responses evoked by 10 nM AII. Bars show means ± S.E. in non-injected cells (open bars) and in oligonucleotide-injected cells (hatched bars). Numbers in parentheses indicate the number of cells tested. star , values significantly different from those obtained in non-injected cells (p < 0.01).
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DISCUSSION

Our results show that, in rat portal vein myocytes, the G protein heterotrimer Galpha 13beta 1gamma 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 Galpha subunits to block interactions of G proteins with angiotensin AT1 receptors, and synthetic peptides corresponding to the carboxyl terminus of Galpha subunits to disrupt the angiotensin AT1 receptor-evoked activation of G proteins.

Antisense oligonucleotides have been used previously to demonstrate the involvement of both Galpha and Gbeta gamma 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-alpha 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-beta 1 or anti-gamma 3 antisense oligonucleotides. These inhibitions correlated well with reductions in the expression of the corresponding G protein subunits immunostained by anti-alpha 13, anti-beta 1, and anti-gamma 3 antibodies. The facts that the AII-induced increase in [Ca2+]i was selectively inhibited after blockade of Galpha 13beta 1gamma 3 protein expression and that anti-beta 1 and anti-gamma 3 oligonucleotides did not inhibit the expression of Galpha 13 protein support the concept that activation of G proteins by receptors requires the presence of all three alpha 13, beta 1, and gamma 3 subunits.

The specificity of beta 1gamma 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 beta gamma subunits expressed in portal vein smooth muscle) to associate with the alpha 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 beta 1gamma 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 beta 1gamma 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 beta 1gamma 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 alpha 13 subunit to couple the angiotensin AT1 receptor is supported by the observation that an anti-alpha 13 antibody, but not an anti-alpha q/11 antibody (raised against the carboxyl termini of the Galpha 13 and Galpha q/11 subunits) (5), selectively blocks the AII-induced increase in [Ca2+]i. Recently, peptides corresponding to various regions of Galpha 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 alpha 13 peptide inhibits, in a concentration-dependent manner, the AII-induced increase in [Ca2+]i, whereas the carboxyl-terminal alpha 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 alpha 13 subunit, in contrast to the alpha 1A-adrenoreceptor, which has been shown to bind to the carboxyl terminus of the alpha q/11 subunit (26). It is generally accepted that the angiotensin AT1 receptor couples phospholipase C-beta 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-gamma 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-alpha 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-alpha or C-delta (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 [alpha -32P]GTP azidoanilide into immunoprecipitated alpha 13 and alpha 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 alpha q/11 and alpha 13 subunit domains interacting with the angiotensin AT1 receptor seem to be independent of each other since the carboxyl-terminal alpha q/11 peptide does not interfere with the angiotensin AT1 receptor-induced activation of Galpha 13beta 1gamma 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 Galpha 13beta 1gamma 3 heterotrimer, leading to increases in [Ca2+]i, and that the interaction involves the extreme carboxyl terminus of the alpha 13 subunit.


FOOTNOTES

*   This work was supported in part by grants from CNRS (France) and from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (Germany).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.
§   Supported by a fellowship from the Fondation pour la Recherche Medicale (France).
par    To whom correspondence should be addressed. Tel.: 33-5-57-57-12-31; Fax: 33-5-57-57-12-26.
1   The abbreviations used are: AII, angiotensin II; [Ca2+]i, cytoplasmic Ca2+ concentration; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PCR, polymerase chain reaction.
2   R. Harhammer and F. Kalkbrenner, unpublished data.

ACKNOWLEDGEMENT

We thank E. Glass and N. Biendon for technical assistance.


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