Distinct Functions of Gq and G11 Proteins in Coupling alpha 1-Adrenoreceptors to Ca2+ Release and Ca2+ Entry in Rat Portal Vein Myocytes*

(Received for publication, October 22, 1996)

Nathalie Macrez-Leprêtre Dagger §, Frank Kalkbrenner , 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  Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69/71, D-14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In this study, we identified the subunit composition of Gq and G11 proteins coupling alpha 1-adrenoreceptors to increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes maintained in short-term primary culture. We used intranuclear antisense oligonucleotide injection to inhibit selectively the expression of subunits of G protein. Increases in [Ca2+]i were measured in response to activation of alpha 1-adrenoreceptors, angiotensin AT1 receptors, and caffeine. Antisense oligonucleotides directed against the mRNAs coding for alpha q, alpha 11, beta 1, beta 3, gamma 2, and gamma 3 subunits selectively inhibited the increase in [Ca2+]i activated by alpha 1-adrenoreceptors. A corresponding reduction of the expression of these G protein subunits was immunochemically confirmed. In experiments performed in Ca2+-free solution only cells injected with anti-alpha q antisense oligonucleotides displayed a reduction of the alpha 1-adrenoreceptor-induced Ca2+ release. In contrast, in Ca2+-containing solution, injection of anti-alpha 11 antisense oligonucleotides suppressed the alpha 1-adrenoreceptor-induced stimulation of the store-operated Ca2+ influx. Agents that specifically bound Gbeta gamma subunits (anti-beta com antibody and overexpression of a beta -adrenergic receptor kinase carboxyl-terminal fragment) had no effect on the alpha 1-adrenoreceptor-induced signal transduction. Taken together, these results suggest that alpha 1-adrenoreceptors utilize two different Galpha subunits to increase [Ca2+]i. Galpha q may activate phosphatidylinositol 4,5-bisphosphate hydrolysis and induce release of Ca2+ from intracellular stores. Galpha 11 may enhance the Ca2+-activated Ca2+ influx that replenishes intracellular Ca2+ stores.


INTRODUCTION

In vascular smooth muscle, activation of alpha 1-adrenoreceptors stimulates phospholipase C-beta which hydrolyzes phosphatidylinositol-4,5-bisphosphate to yield diacylglycerol and inositol 1,4,5-trisphosphate. In portal vein myocytes, the alpha 1A-adrenoreceptors are coupled to phospholipase C-beta through G proteins which have been identified to be Gq and/or G11, on the basis of intracellular applications of an anti-Galpha q/alpha 11 antibody. Inositol 1,4,5-trisphosphate subsequently releases Ca2+ from the intracellular store. Diacylglycerol in concert with cellular Ca2+ activates protein kinase C which, in turn, stimulates Ca2+ influx through voltage-dependent Ca2+ channels (1-2). In addition, depletion of the intracellular store by norepinephrine promotes a sustained Ca2+ entry through dihydropyridine-resistant Ca2+ channels by an unknown mechanism (3). Both norepinephrine-induced Ca2+ release and Ca2+ entry lead to a biphasic rise of the cytoplasmic Ca2+ concentration ([Ca2+]i).1 Although the G protein subtypes are currently defined by their alpha  subunits, of which 23 (including splice variants) are known, a functionally active heterotrimeric G protein includes an alpha , beta , and gamma  subunit. Up to now, 5 different beta  and 11 different gamma subunits have been identified (4). Thus, a great number of heterotrimers composed of specific alpha , beta , and gamma  subunits may exist and be involved in signal transduction pathways. In many cases, the coupling between receptor and G protein may appear unselective since one receptor may activate more than one G protein and thus initiate more than one signal-transduction pathway. However, there are many examples showing that different receptors activate the same heterotrimeric G protein to regulate the same effector system (5-6). The question remains whether in portal vein myocytes alpha 1-adrenoreceptors recognize a single heterotrimeric G protein (Gq or G11) to induce a rise of [Ca2+]i or whether different heterotrimers varying in the composition of alpha , beta , and gamma  subunits are required for this coupling.

Antisense oligonucleotides can be used for selective and transient knockout of cellular proteins (7). So far, microinjection is the only method available that allows for controlled intranuclear application of antisense oligonucleotides. Studies with this method in GH3 cells have revealed that the M4 muscarinic receptor in GH3 cells couples to the G protein trimer consisting of alpha o1beta 3gamma 4, the somatostatin receptor to the trimer alpha o2beta 1gamma 3, and the galanin receptor to the trimers alpha o1beta 2gamma 2 and alpha o1beta 3gamma 4 to inhibit voltage-dependent Ca2+ channels (8-11). In RBL-2H3-hm1 cells, G proteins composed of Galpha q/alpha 11·beta 1/beta 4·gamma 4 are required for effective coupling between the stably expressed human muscarinic m1 receptor and cellular increase in [Ca2+]i (12).

In the present study, we used the method of intranuclear microinjection of antisense oligonucleotides directed against individual G protein subunits and determined the composition of Gq and G11 proteins mediating the alpha 1-adrenoreceptor-induced increase in [Ca2+]i in short-term primary cultured rat portal vein myocytes. We show that alpha 1-adrenoreceptors utilize G proteins composed of alpha q, alpha 11, beta 1, beta 3, gamma 2, and gamma 3 subunits to increase [Ca2+]i and that the effector coupling is mediated by the alpha  subunits. Galpha q subunit may activate release of Ca2+ from intracellular stores and Galpha 11 subunit may modulate intracellular store-dependent Ca2+ entry.


EXPERIMENTAL PROCEDURES

Microinjection of Oligonucleotides

Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously (1). Cells were seeded at a density of about 103 cells per 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, 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, Germany). Oligonucleotides were from MWG-Biotech (Ebersberg, Germany) or synthesized in a DNA synthesizer (Milligen, model 8600); for synthesis of phosphorothioate oligonucleotides, the method described by Iyer et al. (13) was used. Injection of oligonucleotides was performed into the nucleus of myocytes by a manual injection system (Eppendorf, Hamburg, Germany). The injection solution contained 10 µM oligonucleotides in water; approximately 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 sense, scrambled, and 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-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 (11). The sequences of the anti-alpha q, anti-alpha 11, and anti-alpha 14 have been published (12). The sequence of anti-alpha q/11com is ATGGACTCCAGAGT and that of sense alpha q/11com is ACTCTGGAGTCCAT corresponding to nt 4-17 of alpha q cDNA (14), of scrambled anti-alpha q/11com is TACGGTCCAGAGTA corresponding to a scrambled sequence of nt 4-17 of alpha q cDNA, of anti-alpha 12 is CTCCGGCCTCGGCCGGCAGCAAGC corresponding to nt 32-55 of alpha 12 cDNA (15), of anti-beta 5 is TGCCATCTTCGTCCGGATGCAGCC corresponding to nt (-18)-(+6) of beta 5 cDNA (16), of anti-gamma 2 is TTCCTTGGCATGCGCTTCAC corresponding to nt 122-141 of gamma 2 cDNA (17), of anti-gamma 3 is GTTCTCCGAAGTGGGCACAGGGGT corresponding to nt 165-188 of gamma 3 cDNA (18), of anti-gamma 7 is CTGGGCGACGTTGTTAGTACCTGA corresponding to nt 7-30 of rat gamma 7 cDNA (19), of anti-gamma 8 is GCGGGCCTCAGCGAT CTTGGCCAT corresponding to nt 13-36 of gamma 8 cDNA (20).

Transfection

cDNAs encoding beta -adrenergic receptor kinase carboxyl-terminal fragment and the S65T green fluorescent protein were cloned into cytomegalovirus expression plasmids pRK5 and pcDNA3, respectively (Clontech, Palo Alto, CA). Plasmids were injected directly into the nucleus of vascular myocytes, as described for oligonucleotides. Briefly, cDNAs were diluted with water from stock solutions (0.5 µg/µl) to final concentrations of 0.1 µg/µl. The S65T green fluorescent protein was included to facilitate later identification of myocytes receiving a successful nuclear injection. Fluorescence produced by the S65T green fluorescent protein was observed 3 days after injection with a confocal microscope (Bio-Rad MRC 1000, Paris, France). The percentage of successful nuclear injection was estimated to be 20% (n = 185).

Measurements of Cytosolic Ca2+

Cells were loaded by incubation in physiological solution containing 1 µM fura-2-acetoxymethyl ester 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 (1). Briefly, fura-2-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). Single cells were alternately excited with UV light at 340 and 380 nm through a 10 × oil immersion objective, and emitted fluorescent light 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. In some experiments, cells were loaded through a patch-clamp pipette filled with a solution containing (in mM): 140 CsCl, 10 HEPES, 0.06 Fura-2, pH 7.3, as described previously (1). [Ca2+]i was calculated from mean ratios using a calibration for fura-2 determined in loaded cells. All measurements were made at 25 ± 1 °C.

The normal physiological solution contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, 10 HEPES, pH 7.4, with NaOH. Substances were applied to the cells by pressure ejection from a glass pipette for the period indicated on the records. 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 means of Student's t test. p values of < 0.05 were considered as significant.

Immunocytochemistry

Three days after injection, venous myocytes were washed with phosphate-buffered saline solution (PBS), fixed with 3% formaldehyde (v/v) for 30 min at room temperature, and permeabilized in PBS 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 or 1:1000 dilution overnight at 4 °C. Then, cells were washed in PBS containing 3% fetal calf serum and 0.01 (w/v) saponin (4 × 10 min) 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 PBS and mounted in Moviol (Hoechst, Frankfurt, Germany). Images of the stained cells were obtained with a confocal microscope (Bio-Rad MRC 1000). 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 MPL software (Bio-Rad).

Chemicals and Drugs

M199 medium was from Flow Laboratories (Puteaux, France). Fetal calf serum was from Flobio (Courbevoie, France). Streptomycin, penicillin, glutamate, and pyruvate were from Life Technologies, Inc. (Paisley, UK). Fura-2, Fura-2/AM, and anti-alpha q (CN 371752) antibody were from Calbiochem (Meudon, France). Norepinephrine, rauwolscine, and propranolol were from Sigma (St. Quentin Fallavier, France). Angiotensin II, CGP42112A (N-alpha -nicotinoyl-Tyr-Lys[N-alpha -CBZ-Arg]-His-Pro-Ile-OH) was from Neosystem Laboratories (Strasbourg, France). Caffeine was from Merck (Nogent sur Marne, France). Anti-alpha 11 (SC 394), anti-beta com (SC 378), anti-beta 1 (SC 379), and anti-gamma 3 (SC 375) were from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was from Immunotech (Marseille, France). Green fluorescent protein S65T expression construct was from Clontech (Palo Alto, CA). Oxodipine was a gift from Dr. Galiano (IQB, Madrid, Spain).


RESULTS

Identification of Subunit Composition of the G Proteins Coupling alpha 1-Adrenoreceptors to Increase in [Ca2+]i in Single Rat Portal Vein Myocytes

We previously showed that in portal vein myocytes, activation of alpha 1A-adrenoreceptors mediates both release of Ca2+ from intracellular stores and stimulation of voltage-dependent Ca2+ channels through a Gq/G11 protein that activates phospholipase C-beta (1). In order to identify the heterotrimeric G proteins involved in the alpha 1-adrenoreceptor-induced increase in [Ca2+]i, we injected phosphorothioate-modified antisense oligonucleotides directed against alpha , beta , and gamma  subunits into the nucleus of vascular myocytes. By measuring the norepinephrine-induced increases in [Ca2+]i after injection of an antisense oligonucleotide directed against both alpha q and alpha 11 subunits (anti-alpha q/11com), the highest inhibition (76 ± 12%, n = 7) was obtained 3 days after injection (data not shown). Therefore, all further measurements were performed 3 days after injection. 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 (2)), 10 mM caffeine and 10 nM angiotensin II (in the presence of 1 µM CGP42112A to inhibit angiotensin AT2 receptors (21)) on the same cells (Fig. 1). For each experiment, we compared the Ca2+ responses of antisense oligonucleotide-injected cells located within a marked area of the glass slide to sense or scrambled oligonucleotide-injected cells or non-injected cells outside this marked area. This procedure guaranteed that antisense oligonucleotide-injected cells were always compared with control cells that were otherwise grown, treated, and analyzed under identical conditions, i.e. culture, incubation, microinjection, and loading with fura-2AM. The increase in [Ca2+]i was measured for each cell, and mean values were calculated from all cells of each experiment. Myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs encoding for alpha q subunit (anti-alpha q) showed strongly reduced (75%) alpha 1-adrenoreceptor-induced Ca2+ responses, as compared with non-injected cells (Figs. 1B and 2A). Myocytes injected with 10 µM antisense oligonucleotides directed against the alpha 11 (anti-alpha 11) subunit showed reduced (40%) alpha 1-adrenoreceptor-induced Ca2+ responses as well (Figs. 1C and 2A). Interestingly, injection of both anti-alpha q and anti-alpha 11 oligonucleotides (anti-alpha q+11) did not induce a larger decrease of the alpha 1-adrenoreceptor-induced Ca2+ response than that evoked by anti-alpha q oligonucleotides alone (Fig. 2A). Myocytes injected with 10 µM antisense oligonucleotides directed against alpha o1 and alpha o2 (anti-alpha ocom), alpha 12 (anti-alpha 12), and alpha 14 (anti-alpha 14) subunits were comparable with non-injected cells. None of them showed a significant reduction in [Ca2+]i responses evoked by activation of alpha 1-adrenoreceptors (Figs. 1D and 2A). Furthermore, we used sense alpha q/11com and scrambled anti-alpha q/11com oligonucleotides which do not efficiently anneal to the target sequence of Galpha q/11 subunits. Ca2+ responses evoked by activation of alpha 1-adrenoreceptors were not significantly affected by injection of these oligonucleotides (non-injected cells = 418 ± 53 nM, n = 12; sense alpha q/11-injected cells = 379 ± 42 nM, n = 9; and scrambled anti-alpha q/11com-injected cells = 390 ± 32 nM, n = 13).


Fig. 1. Increase in [Ca2+]i evoked by norepinephrine (NE), caffeine (Caf), and angiotensin II (A II) in myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs of Galpha q, Galpha 11, and Galpha 14 proteins. Ca2+ responses were obtained for successive applications of 10 µM norepinephrine, 10 mM caffeine, and 10 nM angiotensin II in non-injected myocytes (A) and in myocytes injected with anti-alpha q (B), anti-alpha 11 (C), or anti-alpha 14 (D) antisense oligonucleotides. Myocytes were used 3 days after nuclear microinjection of oligonucleotides. Norepinephrine, caffeine, and angiotensin II were ejected from a glass pipette close to the cell for the period indicated on the records. External solution contained 10 nM rauwolscine, 1 µM propranolol, and 1 µM CGP42112A.
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Fig. 2. Peak increase in [Ca2+]i evoked by norepinephrine (A), caffeine (B), and angiotensin II (C) in myocytes injected with 20 µM anti-alpha q+11 antisense oligonucleotides and 10 µM anti-alpha q/11com, -alpha q, -alpha 11, -alpha ocom, -alpha 12, and -alpha 14 antisense oligonucleotides. Myocytes were used 3 days after injection. Norepinephrine (10 µM), caffeine (10 mM), and angiotensin II (10 nM) were ejected from a glass pipette close to the cell in the presence of 10 nM rauwolscine, 1 µM propranolol, and 1 µM CGP42112A. Data are given as mean ± S.E. with the number of experiments in parentheses, in non-injected cells (open bar), and in cells injected with antisense oligonucleotides (filled bar). star , values significantly different from those obtained under control conditions (p < 0.05).
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In order to identify the beta  subunits involved in the alpha 1-adrenoreceptor-induced Ca2+ response, we used antisense oligonucleotides directed against the mRNAs coding for beta 1, beta 2, beta 3, beta 4, and beta 5 subunits (Fig. 3A). Injection of 10 µM oligonucleotides directed against the beta 1 (anti-beta 1) and beta 3 (anti-beta 3) subunits significantly reduced the alpha 1-adrenoreceptor-induced Ca2+ responses. Inhibition of the alpha 1-adrenoreceptor-induced Ca2+ responses evoked by the oligonucleotides directed against the beta 1 and beta 3 subunits (80 and 40%, respectively) was quantitatively similar to those induced by the oligonucleotides directed against the alpha q and alpha 11 subunits. No significant reduction of the alpha 1-adrenoreceptor-induced Ca2+ response was seen in myocytes injected with oligonucleotides directed against beta 2 (anti-beta 2), beta 4 (anti-beta 4), and beta 5 (anti-beta 5) subunits. Injection of 10 µM antisense oligonucleotides against different gamma  subunits showed that anti-gamma 1, -gamma 4, -gamma 5, -gamma 7, and -gamma 8 oligonucleotides had no significant effect on the alpha 1-adrenoreceptor-induced Ca2+ responses (Fig. 3B). In contrast, injection of anti-gamma 3 and -gamma 2 oligonucleotides resulted in significant reduction of the alpha 1-adrenoreceptor-induced Ca2+ responses (73 and 40%, respectively). Amplification of cDNA fragments revealed that in portal vein smooth muscle five beta  (beta 1-beta 5) and six gamma  (gamma 2-gamma 8) subunits were expressed (data not shown). These results indicate that alpha 1-adrenoreceptors utilize G proteins composed of alpha q, alpha 11, beta 1, beta 3, gamma 2, and gamma 3 subunits to increase [Ca2+]i.


Fig. 3. Peak increase in [Ca2+]i evoked by norepinephrine in myocytes injected with 10 µM antisense oligonucleotides directed against the mRNAs of beta  (A) and gamma  (B) subunits of heterotrimeric G proteins. Ca2+ responses were obtained with 10 µM norepinephrine in myocytes maintained in primary culture for 3 days after injection. Norepinephrine was applied in the presence of 10 nM rauwolscine and 1 µM propranolol. Data are given as mean ± S.E. with the number of experiments in parentheses, in non-injected cells (open bar), and in cells injected with antisense oligonucleotides (filled bar). star , values significantly different from those obtained under control conditions (p < 0.05).
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Specificity of the Antisense Oligonucleotides

In order to verify that injection of antisense oligonucleotides directed against specific G protein subunits suppressed involvement of these subunits in the alpha 1-adrenoreceptor-activated transduction couplings, we performed two types of control experiments. First, we showed that injection of a specific antisense oligonucleotide inhibited only the immunofluorescence signal of the corresponding G protein subunit and did not affect the expression of other subunits. Cells were stained with either anti-alpha q or anti-alpha 11 specific antibodies, and the immunofluorescence was quantified by using the MPL software of the confocal microscope (Fig. 4A). Cells injected with either of the two different antisense oligonucleotides and non-injected cells located on the same glass slide were compared with each other, so that the staining procedure 3 days after injection of oligonucleotides was identical for the different cells. In cells injected with anti-alpha q oligonucleotides the immunofluorescence signal for the Galpha q subunit was reduced by 76% (n = 9), whereas that for the Galpha 11 subunit was only slightly affected (10%, n = 8). Similarly, in cells injected with anti-alpha 11 antisense oligonucleotides, the immunofluorescence signal for the Galpha 11 subunit was reduced by 70% (n = 7), whereas that for the Galpha q subunit was only slightly affected (12%, n = 12). Then we tested the effects of injection of anti-beta 1, anti-beta 3, anti-gamma 2, and anti-gamma 3 antisense oligonucleotides on the expression of Galpha q/alpha 11 subunits by staining with anti-alpha q/alpha 11 antibody (Fig. 4B). Although the immunofluorescence signal appeared to be slightly reduced in cells injected with beta  and gamma  antisense oligonucleotides (between 15 and 20%, n = 21), only the cells injected with the alpha q/alpha 11com antisense oligonucleotides showed a considerable inhibition of the immunofluorescence signal (85%, n = 7). Finally, we verified that in cells stained with an anti-beta 1 antibody, the immunofluorescence signal was inhibited in cells injected with anti-beta 1 antisense oligonucleotides (77%, n = 13), whereas it was slightly affected in cells injected with either anti-beta 3 and anti-gamma 2 antisense oligonucleotides (15%, n = 12). In cells stained with an anti-beta 3 antibody, the immunofluorescence signal was inhibited in cells injected with anti-beta 3 antisense oligonucleotides (81%, n = 12), whereas it was slightly affected in cells injected with either anti-beta 1 or anti-gamma 3 antisense oligonucleotides (22%, n = 12). In cells stained with an anti-gamma 3 antibody, the immunofluorescence signal was inhibited in cells injected with anti-gamma 3 antisense oligonucleotides (75%, n = 8), whereas it was slightly affected in cells injected with either anti-beta 3 or anti-gamma 2 antisense oligonucleotides (18%, n = 8). Taken together, these results indicate that each antisense oligonucleotide is selective for inhibiting the expression of the corresponding G protein subunit.


Fig. 4. Specific inhibition of Galpha q or Galpha 11 protein expression in single portal vein myocytes injected with anti-alpha , -beta , or -gamma antisense oligonucleotides. A, upper panel shows myocytes stained 3 days after injection with rabbit anti-alpha q antibody. Visualization was obtained by staining with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200). Cells injected with anti-alpha q and anti-alpha 11 antisense oligonucleotides were compared with control (non-injected cells) on the same glass slide. Lower panel shows myocytes stained with rabbit anti-alpha 11 antibody. Cells injected with anti-alpha 11 and anti-alpha q antisense oligonucleotides were compared with control (non-injected cells). B, diagram depicting Galpha q/11 protein expression in myocytes stained with anti-alpha q/11 antibody. Cells injected with anti-alpha q/11, anti-beta 1, anti-beta 3, anti-gamma 2, and anti-gamma 3 antisense oligonucleotides were compared with control (non-injected cells). Immunofluorescence was expressed in arbitrary units (AU). Open bars (non-injected cells) and filled bars (antisense oligonucleotide-injected cells) show means ± S.E. with the number of experiments in parentheses. star , values significantly different from those obtained under control conditions (p < 0.05).
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Second, we compared the effects of norepinephrine to those of angiotensin II (activating AT1 receptors, 22) and caffeine (releasing Ca2+ from the intracellular stores) in each cell studied (Fig. 1). We recently showed that activation of angiotensin AT1 receptors releases intracellularly stored Ca2+ without involving inositol 1,4,5-trisphosphate but through a Ca2+ release mechanism activated by Ca2+ influx through L-type Ca2+ channels (22-23). In the same cells injected with anti-alpha q/11com, -alpha q, and -alpha 11 antisense oligonucleotides, angiotensin II (in the presence of 1 µM CGP42112A) and caffeine evoked large Ca2+ responses, whereas alpha 1-adrenoreceptor-induced Ca2+ responses were inhibited (Fig. 2, A-C). We noted unspecific effects of phosphorothioate-modified antisense oligonucleotides only when oligonucleotides were injected at concentrations of 50 µM, i.e. 5 times higher than the concentration used in these experiments (n = 15). Taken together, these data indicate that suppression of alpha 1-adrenoreceptor-activated effects by antisense oligonucleotides does not interfere with other signaling pathways (e.g. that of angiotensin II) and with the intracellular Ca2+ stores of vascular myocytes.

Different G Proteins Are Involved in alpha 1-Adrenoreceptor-induced Ca2+ Release and Ca2+ Entry

We previously showed that norepinephrine activates Ca2+ entry even if the intracellular Ca2+ store is not completely emptied (3), possibly by involving a mechanism independent of Ca2+ store depletion. Therefore, experiments were performed in external Ca2+-free solution (containing 0.5 mM EGTA) on myocytes injected with anti-alpha q or anti-alpha 11 antisense oligonucleotides. As illustrated in Fig. 5, the alpha 1-adrenoreceptor-induced Ca2+ release was inhibited in cells injected with anti-alpha q oligonucleotides but was not affected in cells injected with anti-alpha 11 oligonucleotides. These results suggest different tasks for Gq and G11 proteins, i.e. induction of Ca2+ release from intracellular stores and induction of Ca2+ influx from extracellular medium, respectively.


Fig. 5. Effects of anti-alpha q and -alpha 11 antisense oligonucleotides on the norepinephrine-induced transient Ca2+ response in myocytes bathed in Ca2+-free solution (containing 0.5 mM EGTA) for 10 s. Ca2+ responses evoked by 10 µM norepinephrine in non-injected cells (open bars) and in cells injected with 10 µM anti-alpha q or anti-alpha 11 antisense oligonucleotides (filled bars). Norepinephrine was applied in the presence of 10 nM rauwolscine and 1 µM propranolol. Data are given as mean ± S.E. with the number of experiments in parentheses. star , values significantly different from those obtained under control conditions (p < 0.05).
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The sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin is commonly used to study the Ca2+ entry pathway activated after Ca2+ store depletion (24). Application of 1 µM thapsigargin depleted the intracellular Ca2+ stores (evidenced by the lack of caffeine-induced Ca2+ response) and increased the [Ca2+]i level to 118 ± 17 nM (n = 29; Fig. 6A). In the continuous presence of 10 µM oxodipine (a light-resistant dihydropyridine) to block voltage-dependent Ca2+ channels, activation of alpha 1-adrenoreceptors during the thapsigargin-induced Ca2+ plateau evoked a further rise in [Ca2+]i reaching 161 ± 15 nM (n = 5; Fig. 6A). This alpha 1-adrenoreceptor-induced Ca2+ response was never observed in Ca2+-free, 0.5 mM EGTA-containing solution (n = 15). In cells injected with anti-alpha 11 oligonucleotides, the alpha 1-adrenoreceptor-induced rise in [Ca2+]i was not observed (n = 7; Fig. 6B), although thapsigargin produced a progressive increase in [Ca2+]i reaching 123 ± 13 nM (n = 7). In contrast, in cells injected with anti-alpha q oligonucleotides, the amplitude of the alpha 1-adrenoreceptor-induced increase in [Ca2+]i (52 ± 12 mM, n = 6) was comparable with that obtained in non-injected control cells (48 ± 14 nM, n = 11; Fig. 6B).


Fig. 6. Effects of norepinephrine in the continuous presence of thapsigargin in cells injected with anti-alpha 11 and anti-alpha q antisense oligonucleotides. A, application of 10 µM norepinephrine (NE) during the sustained increase in [Ca2+]i induced by 1 µM thapsigargin (TG). B, amplitude of the norepinephrine-induced increase in [Ca2+]i, in the continuous presence of 1 µM thapsigargin, in non-injected cells (in 2 mM Ca2+-containing solution or in Ca2+-free solution with 0.5 mM EGTA, open bars), and in cells injected with anti-alpha q and anti-alpha 11 antisense oligonucleotides (in 2 mM Ca2+-containing solution, filled bars). All experiments were performed in the presence of 10 µM oxodipine, 10 nM rauwolscine, and 1 µM propranolol. Bars show mean ± S.E. with the number of experiments given in parentheses. star , values significantly different from those obtained under control conditions (p < 0.05).
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Depletion of caffeine-sensitive intracellular Ca2+ stores induced a Ca2+ response similar to that evoked by activation of alpha 1-adrenoreceptors. Fig. 7 displays representative traces of these experiments. In the continuous presence of 10 µM oxodipine, application of 10 mM caffeine for 50 s in the external solution (Fig. 7Aa) produced a large transient increase in [Ca2+]i (375 ± 20 nM, n = 16) and a sustained plateau of 70 ± 9 nM (n = 16). The rapid initial increase in [Ca2+]i was reduced in Ca2+-free solution (298 ± 25 nM, n = 10), and the subsequent sustained phase was absent (Fig. 7Ab). This indicates that in venous myocytes caffeine is able to induce a transient increase in [Ca2+]i due to Ca2+ release and a sustained phase representing Ca2+ entry into the cell from the extracellular space. As illustrated in Fig. 2B, the caffeine-induced Ca2+ responses were not affected by inhibition of the expression of any Galpha subunits, including Galpha q and Galpha 11 subunits. Activation of alpha 1-adrenoreceptors (in Ca2+-containing solution) during the second sustained phase of the caffeine-evoked Ca2+ response resulted in a 2-fold increase in [Ca2+]i which reached 134 ± 16 nM (n = 23; Fig. 7Ba). The alpha 1-adrenoreceptor-induced enhancement of [Ca2+]i during the second phase of the caffeine-induced Ca2+ response (64 ± 6 nM, n = 23) was not observed at all when norepinephrine was applied without Ca2+ and in the presence of 0.5 mM EGTA (n = 12; Fig. 7Bb), indicating that it corresponded to a Ca2+ entry from the extracellular medium. In myocytes injected with the anti-alpha q antisense oligonucleotides, the alpha 1-adrenoreceptor-induced Ca2+ entry in the continuous presence of caffeine (55 ± 9 nM, n = 6) was similar to that obtained in non-injected cells (61 ± 8 nM, n = 6; Fig. 7Bc). In contrast, in cells injected with anti-alpha 11 antisense oligonucleotides, no alpha 1-adrenoreceptor-induced Ca2+ entry in the continuous presence of caffeine was observed (n = 8; Fig. 7Bd). These results further support the idea that Galpha 11 subunit is involved in the modulation of store-operated Ca2+ entry by alpha 1-adrenoreceptors.


Fig. 7. Effects of norepinephrine in the continuous presence of caffeine in cells injected with anti-alpha 11 and anti-alpha q antisense oligonucleotides. A, Ca2+ responses evoked by external application of 10 mM caffeine (50 s) in 2 mM Ca2+-containing solution (a) and in Ca2+-free solution with 0.5 mM EGTA for 10 s (b). B, during the caffeine-induced sustained Ca2+ response, norepinephrine (10 µM) was ejected in Ca2+-containing solution (a, c, and d) or in Ca2+-free solution with 0.5 mM EGTA (b), in non-injected cells (a and b), or in cells injected with 10 µM anti-alpha q (c) or anti-alpha 11 (d) oligonucleotides. All the experiments were performed in the presence of 10 µM oxodipine, 10 nM rauwolscine, and 1 µM propranolol.
[View Larger Version of this Image (23K GIF file)]


Effector Coupling Is Dependent on alpha q and alpha 11 Subunits

The anti-alpha q/alpha 11 antibody and antisense oligonucleotide block of the alpha 1-adrenoreceptor-induced Ca2+ response cannot distinguish whether alpha  or beta gamma subunits are transducing the signal that activate Ca2+ release from the intracellular stores or Ca2+ entry. To determine which G protein subunits were involved in the alpha 1-adrenoreceptor-mediated effects, an anti-beta com antibody was dialyzed into the cell by the patch pipette for 3 min. Anti-beta com antibody (10 µg/ml in pipette solution) had no effect on the alpha 1-adrenoreceptor-induced Ca2+ response (Fig. 8A) since neither the transient peak (control = 305 ± 30 nM; in the presence of anti-beta com antibody = 295 ± 35 nM; n = 10) nor the sustained plateau (control = 55 ± 4 nM; in the presence of anti-beta com antibody = 53 ± 6 nM, n = 10) were significantly affected. In the same cells, the anti-beta com antibody inhibited the sustained angiotensin II-induced Ca2+ response in a concentration-dependent manner, with a maximal inhibition obtained at an antibody concentration of 10 µg/ml (n = 10).2 In a second set of experiments, we overexpressed a carboxyl-terminal fragment of beta ARK1 by intranuclear microinjection of expression plasmids containing cDNA inserts coding for beta ARK1. beta ARK has been used to bind beta gamma subunits and block activation of effectors (25-26). Overexpression of beta ARK1 had no effect on the alpha 1-adrenoreceptor-induced Ca2+ response (Fig. 8B) since neither the transient peak (control = 290 ± 25 nM; in the presence of beta ARK1 = 285 ± 20 nM; n = 12) nor the sustained plateau (control = 65 ± 5 nM; in the presence of beta ARK1 = 60 ± 8 nM; n = 12) were significantly affected. In contrast, the angiotensin II-induced Ca2+ response was inhibited when beta ARK1 was overexpressed in the same cells (n = 12).2 Taken together, our results indicate that application of anti-beta com antibody and beta ARK1, both able to bind free beta gamma subunits, had no effects on both Ca2+ release and Ca2+ entry induced by activation of alpha 1-adrenoreceptors.


Fig. 8. Effects of anti-beta com antibody and overexpression of beta ARK1 on the norepinephrine-induced Ca2+ response. A, norepinephrine-induced Ca2+ responses in cells dialyzed for 3 min with a pipette solution containing 60 µM Fura-2 in the absence (a) or in the presence of 10 µg/ml anti-beta com antibody (b). B, transient norepinephrine-induced Ca2+ response in control conditions (a) and after overexpression of a carboxyl-terminal fragment of beta ARK1 (b, obtained by nuclear injection of beta ARK1 mini-gene construct, 3 days before the experiments). Norepinephrine (NE) was applied in the presence of 10 nM rauwolscine and 1 µM propranolol.
[View Larger Version of this Image (18K GIF file)]



DISCUSSION

G Protein Subunits Mediating alpha 1-Adrenoreceptor-induced [Ca2+]i Increase

Here we show that the alpha 1-adrenoreceptor-induced increase in [Ca2+]i in rat portal vein myocytes involves both Gq and G11 proteins. Using nuclear injection of antisense oligonucleotides corresponding to the mRNA sequences coding for G protein alpha , beta , and gamma  subunits, we identified G protein heterotrimers composed of alpha q, alpha 11, beta 1, beta 3, gamma 2, and gamma 3 involved in the coupling of alpha 1-adrenoreceptors to Ca2+ release and intracellular store-dependent Ca2+ entry.

We proved the specificity of the injected oligonucleotides by studying the increase in [Ca2+]i induced by application of two hormonal stimuli (norepinephrine and angiotensin II) and caffeine. All three substances release Ca2+ from the same intracellular store via different pathways. Caffeine is known to release Ca2+ by acting on the ryanodine-sensitive Ca2+ channels of the sarcoplasmic reticulum. In portal vein myocytes, we recently showed that activation of angiotensin AT1 receptors evoked an increase in [Ca2+]i which depended on both activation of L-type Ca2+ channels and opening of ryanodine-sensitive Ca2+ channels of the sarcoplasmic reticulum, without involving inositol 1,4,5-trisphosphate generation (22); this effect is mediated by G proteins different from Gq/11 protein, as shown by the absence of effect of intracellular application of anti-alpha q/alpha 11 antibodies (27). Therefore, we used caffeine as a control for the availability of the Ca2+ stores in control and oligonucleotide-injected cells, whereas the angiotensin AT1 receptor-induced response was used as a control for the specificity of antisense oligonucleotide effects on G protein subunits. Antisense oligonucleotides against Galpha subunits showed no effect on the caffeine-induced Ca2+ responses excluding non-antisense effects on intracellular Ca2+ stores (see Fig. 2). Such non-antisense but sequence-specific effects have been described for oligonucleotides directed against c-myb and p53 (28-29). To have a second control for the specificity of the antisense oligonucleotides, we compared injected cells to non-injected control cells located on the same glass slide in each experiment. We show that the value of the increase in [Ca2+]i induced by norepinephrine is not significantly different comparing non-injected cells to cells injected with alpha q/11 sense or scrambled antisense oligonucleotides and antisense oligonucleotides directed against G protein subunits which are not involved in the alpha 1-adrenoreceptor-induced increase in [Ca2+]i (see Figs. 1 and 2). Furthermore, injection of antisense oligonucleotides directed against Galpha subunits involved in alpha 1-adrenoreceptor-mediated effects did not change the increase in [Ca2+]i achieved by angiotensin II or caffeine (see Fig. 2, B-C). To demonstrate the extent of the antisense knockout effects, we studied protein depletion by immunocytochemistry of Galpha subunits. The results of these experiments revealed that the time course by which anti-alpha q or anti-alpha 11 antisense oligonucleotides were effective in suppressing functional receptor-mediated effects paralleled suppression of the Galpha q or Galpha 11 protein level which decreased maximally within 3 days after injection. Similar results were recently obtained using alpha o-, alpha i-, and alpha q/alpha 11-antisense oligonucleotides in different cells (8, 12, 30). In addition, we demonstrated that injection of antisense oligonucleotides directed against a given G protein subunit did not modify significantly the expression of other G protein subunits (see Fig. 4). Therefore, the fact that anti-beta 1, -beta 3, -gamma 2, and -gamma 3 oligonucleotides inhibit the alpha 1-adrenoreceptor-induced [Ca2+]i increase cannot be related to an inhibition of the Galpha subunit expression and means that these beta  and gamma  subunits are necessary for activation of the Gq and G11 proteins by alpha 1-adrenoreceptors. Since anti-alpha q, -beta 1, and -gamma 3 oligonucleotides largely inhibited the alpha 1-adrenoreceptor-induced increase in [Ca2+]i whereas anti-alpha 11, -beta 3 and -gamma 2 oligonucleotides produced a limited inhibition, one may speculate that the composition of G protein heterotrimers required for the two phases of the alpha 1-adrenoreceptor-induced Ca2+ response is alpha q/beta 1/gamma 3 and alpha 11/beta 3/gamma 2.

Distinct Functions of Gq and G11 Proteins

The experiments presented in this work suggest that two different heterotrimeric G proteins mediate Ca2+ release from intracellular stores and Ca2+ entry in response to stimulation of alpha 1-adrenoreceptors. Evidence supporting this proposal are the following. 1) Cells injected with a mixture of anti-alpha q and anti-alpha 11 oligonucleotides (anti-alpha q+11) showed no further reduction of alpha 1-adrenoreceptor-induced Ca2+ response compared with cells injected with either anti-alpha q or anti-alpha q/alpha 11 oligonucleotides. 2) In Ca2+-free solution, anti-alpha q oligonucleotides strongly reduced the alpha 1-adrenoreceptor-induced Ca2+ release, whereas anti-alpha 11 oligonucleotides were without effect, suggesting that Galpha 11 protein-mediated modulation of oxodipine-resistant Ca2+ entry required a preceding Ca2+ release mediated by Galpha q protein. 3) The oxodipine-resistant Ca2+ entry evoked by activation of alpha 1-adrenoreceptors in the presence of thapsigargin or caffeine was selectively suppressed by anti-alpha 11 oligonucleotides. Therefore, we propose that the Galpha 11 subunit may enhance the alpha 1-adrenoreceptor-induced Ca2+ entry activated by a previous release of Ca2+ from intracellular stores. Several types of Ca2+ entry mechanisms have been described in various cellular systems (31). In smooth muscle cells, activation of Ca2+ entry by application of mediators (histamine, endothelin, vasopressin) has been reported (32-34). We show that caffeine also induces intracellular store-operated Ca2+ entry in rat portal vein myocytes (see Fig. 6), and we used caffeine and thapsigargin pretreatment to study the modulation of the store-operated Ca2+ entry by activation of alpha 1-adrenoreceptors. This dihydropyridine-resistant Ca2+ entry may be mediated by cation channels. Interestingly, a nonselective cation channel, the Drosophila trpl channel, has been shown to be stimulated in a membrane-confined way by Galpha 11 protein (35), and a similar nonspecific cation channel permeable for Ca2+ ions has been previously identified in portal vein myocytes (36). Furthermore, experiments performed in an epithelial cell line have shown that overexpression of Galpha q protein increases Ca2+ release-activated Ca2+ influx (37). Thus, the ubiquitously expressed Galpha q family may have a general role in modulating Ca2+ entry through Ca2+-permeable nonselective cation channels which may be controlled by both the filling state of Ca2+ stores (38) and, as shown here, more directly by G protein. Recently, the composition of G proteins coupling the stably expressed human muscarinic m1 receptor in the rat basophilic leukemia cell line (RBL-2H3-hm1) to increase in [Ca2+]i has been determined by using the same method and the same antisense oligonucleotides (12). In these cells, the authors have proposed that a complex of G protein subunits, i.e. Galpha q/alpha 11·beta 1/beta 4·gamma 4, is activated by m1 receptors. As [Ca2+]i measurements were performed in Ca2+-containing solution, a differential coupling of Galpha q and Galpha 11 subunits to Ca2+ release and Ca2+ entry, respectively, could not have been detected. Finally, transient expression of a carboxyl-terminal fragment of beta ARK1 that scavenged Gbeta gamma subunits after their dissociation from the receptor-activated heterotrimers had no effect on alpha 1-adrenoreceptor-induced Ca2+ responses suggesting that beta gamma subunits did not display direct interactions with the effectors, i.e. phospholipase C and Ca2+-permeable nonselective cation channels. This conclusion is supported by the results showing that intracellular application of anti-beta com antibody did not modify significantly the alpha 1-adrenoreceptor-mediated Ca2+ release and Ca2+ entry. The possibility that Gbeta gamma subunits that are dissociated from both Galpha q and Galpha 11 subunits after activation of alpha 1-adrenoreceptors may activate other cellular effectors remains to be investigated.

In conclusion, we show that in rat venous myocytes, the Gq proteins may couple by their alpha  subunits endogenous alpha 1-adrenoreceptors to phospholipase C, whereas the Galpha 11 proteins, activated at the same time by the same receptors, may couple to Ca2+ entry. These results point out distinct functions of Gq and G11 in receptor-activated [Ca2+]i increase.


FOOTNOTES

*   This work was supported by grants from Centre National de la Recherche Scientifique (France) and from the Deutsche Forschungsgemeinschaft and 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: Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Faculté de Pharmacie, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France. Tel.: 33-5-57-57-12-31; Fax: 33-5-57-57-12-26.
1    The abbreviations used are: [Ca2+]i, cytoplasmic Ca2+ concentration; PBS, phosphate-buffered saline solution; nt, nucleotide(s); beta ARK, beta -adrenergic receptor kinase.
2    N. Macrez-Leprêtre, F. Kalkbrenner, J. L. Morel, G. Schultz, and J. Mironneau, manuscript in preparation.

Acknowledgments

We thank Dr. R. J. Lefkowitz for generously donating beta ARK1 minigene; Drs. K. Spicher and B. Nürnberg for anti-alpha q/11, anti-beta 3, and anti-gamma 2 antibodies; Dr. A. Lückhoff for helpful discussions, and N. Biendon for secretarial assistance.


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