Distinct functions of Gq and G11 proteins in coupling alpha1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes.

In this study, we identified the subunit composition of Gq and G11 proteins coupling α1-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 α1-adrenoreceptors, angiotensin AT1 receptors, and caffeine. Antisense oligonucleotides directed against the mRNAs coding for αq, α11, β1, β3, γ2, and γ3 subunits selectively inhibited the increase in [Ca2+]i activated by α1-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-αq antisense oligonucleotides displayed a reduction of the α1-adrenoreceptor-induced Ca2+ release. In contrast, in Ca2+-containing solution, injection of anti-α11 antisense oligonucleotides suppressed the α1-adrenoreceptor-induced stimulation of the store-operated Ca2+ influx. Agents that specifically bound Gβγ subunits (anti-βcom antibody and overexpression of a β-adrenergic receptor kinase carboxyl-terminal fragment) had no effect on the α1-adrenoreceptor-induced signal transduction. Taken together, these results suggest that α1-adrenoreceptors utilize two different Gα subunits to increase [Ca2+]i. Gαq may activate phosphatidylinositol 4,5-bisphosphate hydrolysis and induce release of Ca2+ from intracellular stores. Gα11 may enhance the Ca2+-activated Ca2+ influx that replenishes intracellular Ca2+ stores.

In vascular smooth muscle, activation of ␣ 1 -adrenoreceptors stimulates phospholipase C-␤ which hydrolyzes phosphatidylinositol-4,5-bisphosphate to yield diacylglycerol and inositol 1,4,5-trisphosphate. In portal vein myocytes, the ␣ 1A -adrenoreceptors are coupled to phospholipase C-␤ through G proteins which have been identified to be G q and/or G 11 , on the basis of intracellular applications of an anti-G␣ q /␣ 11 antibody. Inositol 1,4,5-trisphosphate subsequently releases Ca 2ϩ from the intracellular store. Diacylglycerol in concert with cellular Ca 2ϩ activates protein kinase C which, in turn, stimulates Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (1)(2). In addition, depletion of the intracellular store by norepinephrine promotes a sustained Ca 2ϩ entry through dihydropyridine-resistant Ca 2ϩ channels by an unknown mechanism (3). Both norepinephrine-induced Ca 2ϩ release and Ca 2ϩ entry lead to a biphasic rise of the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ). 1 Although the G protein subtypes are currently defined by their ␣ subunits, of which 23 (including splice variants) are known, a functionally active heterotrimeric G protein includes an ␣, ␤, and ␥ subunit. Up to now, 5 different ␤ and 11 different ␥ subunits have been identified (4). Thus, a great number of heterotrimers composed of specific ␣, ␤, and ␥ 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 ␣ 1 -adrenoreceptors recognize a single heterotrimeric G protein (G q or G 11 ) to induce a rise of [Ca 2ϩ ] i or whether different heterotrimers varying in the composition of ␣, ␤, and ␥ 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 GH 3 cells have revealed that the M 4 muscarinic receptor in GH 3 cells couples to the G protein trimer consisting of ␣ o1 ␤ 3 ␥ 4 , the somatostatin receptor to the trimer ␣ o2 ␤ 1 ␥ 3 , and the galanin receptor to the trimers ␣ o1 ␤ 2 ␥ 2 and ␣ o1 ␤ 3 ␥ 4 to inhibit voltage-dependent Ca 2ϩ channels (8 -11). In RBL-2H3-hm1 cells, G proteins composed of G␣ q /␣ 11 ⅐␤ 1 /␤ 4 ⅐␥ 4 are required for effective coupling between the stably expressed human muscarinic m 1 receptor and cellular increase in [Ca 2ϩ ] 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 G q and G 11 proteins mediating the ␣ 1 -adrenoreceptor-induced increase in [Ca 2ϩ ] i in short-term primary cultured rat portal vein myocytes. We show that ␣ 1 -adrenoreceptors utilize G proteins composed of ␣ q , ␣ 11 , ␤ 1 , ␤ 3 , ␥ 2 , and ␥ 3 subunits to * 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. This article must therefore be hereby marked "advertisement" in accordance with 18  increase [Ca 2ϩ ] i and that the effector coupling is mediated by the ␣ subunits. G␣ q subunit may activate release of Ca 2ϩ from intracellular stores and G␣ 11 subunit may modulate intracellular store-dependent Ca 2ϩ 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 10 3 cells per mm 2 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 g/ml streptomycin; they were kept in an incubator gassed with 95% air, 5% CO 2 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 M oligonucleotides in water; approximately 10 fl were injected with commercially available microcapillaries (Femtotips, Eppendorf) with an outlet diameter of 0.5 m. 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 Ca 2ϩ measurements. The sequences of anti-␣ ocom , anti-␤ 1 , anti-␤ 2 , anti-␤ 3 , anti-␤ 4 , anti-␥ 1 , anti-␥ 4 , and anti-␥ 5 antisense oligonucleotides have been previously published (11). The sequences of the anti-␣ q , anti-␣ 11 , and anti-␣ 14 have been published (12). The sequence of anti-␣ q/11com is ATGGACTCCAGAGT and that of sense ␣ q/11com is ACTCTGGAGTCCAT corresponding to nt 4 -17 of ␣ q cDNA (14), of scrambled anti-␣ q/11com is TACGGTCCAGAGTA corresponding to a scrambled sequence of nt 4 -17 of ␣ q cDNA, of anti-␣ 12 is CTCCGGC-CTCGGCCGGCAGCAAGC corresponding to nt 32-55 of ␣ 12 cDNA (15), of anti-␤ 5 is TGCCATCTTCGTCCGGATGCAGCC corresponding to nt (Ϫ18)-(ϩ6) of ␤ 5 cDNA (16), of anti-␥ 2 is TTCCTTGGCATGCGCTTCAC corresponding to nt 122-141 of ␥ 2 cDNA (17), of anti-␥ 3 is GTTCTC-CGAAGTGGGCACAGGGGT corresponding to nt 165-188 of ␥ 3 cDNA (18), of anti-␥ 7 is CTGGGCGACGTTGTTAGTACCTGA corresponding to nt 7-30 of rat ␥ 7 cDNA (19), of anti-␥ 8 is GCGGGCCTCAGCGAT CTTGGCCAT corresponding to nt 13-36 of ␥ 8 cDNA (20).
Transfection-cDNAs encoding ␤-adrenergic receptor kinase carboxyl-terminal fragment and the S65T green fluorescent protein were cloned into cytomegalovirus expression plasmids pRK 5 and pcDNA 3 , 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 g/l) to final concentrations of 0.1 g/l. 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 ϭ 185).
Measurements of Cytosolic Ca 2ϩ -Cells were loaded by incubation in physiological solution containing 1 M 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 Ca 2ϩ 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 ϫ oil immersion objective, and emitted fluorescent light from the Ca 2ϩ -sensitive dye was collected through a 510-nm-long pass filter with a chargecoupled 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). [Ca 2ϩ ] i was calculated from mean ratios using a calibration for fura-2 determined in loaded cells. All measurements were made at 25 Ϯ 1°C.
The normal physiological solution contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl 2 , 2 CaCl 2 , 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 Ϯ S.E. Significance was tested by means of Student's t test. p values of Ͻ 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 ϫ 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 ϫ 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).

Identification of Subunit Composition of the G Proteins Coupling ␣ 1 -Adrenoreceptors to Increase in [Ca 2ϩ ] i in Single Rat
Portal Vein Myocytes-We previously showed that in portal vein myocytes, activation of ␣ 1A -adrenoreceptors mediates both release of Ca 2ϩ from intracellular stores and stimulation of voltage-dependent Ca 2ϩ channels through a G q /G 11 protein that activates phospholipase C-␤ (1). In order to identify the heterotrimeric G proteins involved in the ␣ 1 -adrenoreceptorinduced increase in [Ca 2ϩ ] i , we injected phosphorothioate-modified antisense oligonucleotides directed against ␣, ␤, and ␥ subunits into the nucleus of vascular myocytes. By measuring the norepinephrine-induced increases in [Ca 2ϩ ] i after injection of an antisense oligonucleotide directed against both ␣ q and ␣ 11 subunits (anti-␣ q/11com ), the highest inhibition (76 Ϯ 12%, n ϭ 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 [Ca 2ϩ ] i induced by successive applications of 10 M norepinephrine (in the presence of 10 nM rauwolscine and 1 M propranolol to inhibit both ␣ 2 -and ␤-adrenoreceptors (2)), 10 mM caffeine and 10 nM angiotensin II (in the presence of 1 M CGP42112A to inhibit angiotensin AT 2 receptors (21)) on the same cells (Fig. 1). For each experiment, we compared the Ca 2ϩ 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 [Ca 2ϩ ] i was measured for each cell, and mean values were calculated from all cells of each experiment. Myocytes injected with 10 M antisense oligonucleotides directed against the mRNAs encoding for ␣ q subunit (anti-␣ q ) showed strongly reduced (75%) ␣ 1 -adrenoreceptor-induced Ca 2ϩ responses, as compared with non-injected cells (Figs. 1B and 2A). Myocytes injected with 10 M antisense oligonucleotides directed against the ␣ 11 (anti-␣ 11 ) subunit showed reduced (40%) ␣ 1 -adrenoreceptor-induced Ca 2ϩ responses as well (Figs. 1C and 2A). Interestingly, injection of both anti-␣ q and anti-␣ 11 oligonucleotides (anti-␣ qϩ11 ) did not induce a larger decrease of the ␣ 1 -adrenoreceptor-induced Ca 2ϩ response than that evoked by anti-␣ q oligonucleotides alone ( Fig. 2A). Myocytes injected with 10 M antisense oligonucleotides directed against ␣ o1 and ␣ o2 (anti-␣ ocom ), ␣ 12 (anti-␣ 12 ), and ␣ 14 (anti-␣ 14 ) subunits were comparable with non-injected cells. None of them showed a significant reduction in [Ca 2ϩ ] i responses evoked by activation of ␣ 1 -adrenoreceptors (Figs. 1D and 2A). Furthermore, we used sense ␣ q/11com and scrambled anti-␣ q/ 11com oligonucleotides which do not efficiently anneal to the target sequence of G␣ q/11 subunits. Ca 2ϩ responses evoked by activation of ␣ 1 -adrenoreceptors were not significantly affected by injection of these oligonucleotides (non-injected cells ϭ 418 Ϯ 53 nM, n ϭ 12; sense ␣ q/11 -injected cells ϭ 379 Ϯ 42 nM, n ϭ 9; and scrambled anti-␣ q/11com -injected cells ϭ 390 Ϯ 32 nM, n ϭ 13).
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 ␣ 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-␣ q or anti-␣ 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-␣ q oligonucleotides the immunofluorescence signal for the G␣ q subunit was reduced by 76% (n ϭ 9), whereas that for the G␣ 11 subunit was only slightly affected (10%, n ϭ 8). Similarly, in cells injected with anti-␣ 11 antisense oligonucleotides, the immunofluorescence signal for the G␣ 11 subunit was reduced by 70% (n ϭ 7), whereas that for the G␣ q subunit was only slightly affected (12%, n ϭ 12). Then we tested the effects of injection of anti-␤ 1 , anti-␤ 3 , anti-␥ 2 , and anti-␥ 3 antisense oligonucleotides on the expression of G␣ q /␣ 11 subunits by staining with anti-␣ q /␣ 11 antibody (Fig. 4B). Although the immunofluorescence signal appeared to be slightly reduced in cells injected with ␤ and ␥ antisense oligonucleotides (between 15 and 20%, n ϭ 21), only the cells injected with the ␣ q /␣ 11com antisense oligonucleotides showed a considerable inhibition of the immunofluorescence signal (85%, n ϭ 7). Finally, we verified that in cells stained with an anti-␤ 1 antibody, the immunofluorescence signal was inhibited in cells injected with anti-␤ 1 antisense oligonucleotides (77%, n ϭ 13), whereas it was slightly affected in cells injected with either anti-␤ 3 and anti-␥ 2 antisense oligonucleotides (15%, n ϭ 12). In cells stained with an anti-␤ 3 antibody, the immunofluorescence signal was inhibited in cells injected with anti-␤ 3 antisense oligonucleotides (81%, n ϭ 12), whereas it was slightly affected in cells injected with either anti-␤ 1 or anti-␥ 3 antisense oligonucleotides (22%, n ϭ 12). In cells stained with an anti-␥ 3 antibody, the immunofluorescence signal was inhibited in cells injected with anti-␥ 3 antisense oligonucleotides (75%, n ϭ 8), whereas it was slightly affected in cells injected with either anti-␤ 3 or anti-␥ 2 antisense oligonucleotides (18%, n ϭ 8). Taken together, these results indicate that each antisense oligonucleotide is selective for inhibiting the expression of the corresponding G protein subunit.
Second, we compared the effects of norepinephrine to those of angiotensin II (activating AT 1 receptors, 22) and caffeine (releasing Ca 2ϩ from the intracellular stores) in each cell studied (Fig. 1). We recently showed that activation of angiotensin AT 1 receptors releases intracellularly stored Ca 2ϩ without involving inositol 1,4,5-trisphosphate but through a Ca 2ϩ release mechanism activated by Ca 2ϩ influx through L-type Ca 2ϩ channels (22)(23). In the same cells injected with anti-␣ q/11com , -␣ q , and -␣ 11 antisense oligonucleotides, angiotensin II (in the presence of 1 M CGP42112A) and caffeine evoked large Ca 2ϩ responses, whereas ␣ 1 -adrenoreceptor-induced Ca 2ϩ 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 M, i.e. 5 times higher than the concentration used in these experiments (n ϭ 15). Taken together, these data indicate that suppression of ␣ 1 -adrenoreceptor-activated effects by antisense oligonucleotides does not interfere with other signaling pathways (e.g. that of angiotensin II) and with the intracellular Ca 2ϩ stores of vascular myocytes.
Different G Proteins Are Involved in ␣ 1 -Adrenoreceptor-induced Ca 2ϩ Release and Ca 2ϩ Entry-We previously showed that norepinephrine activates Ca 2ϩ entry even if the intracel- lular Ca 2ϩ store is not completely emptied (3), possibly by involving a mechanism independent of Ca 2ϩ store depletion. Therefore, experiments were performed in external Ca 2ϩ -free solution (containing 0.5 mM EGTA) on myocytes injected with anti-␣ q or anti-␣ 11 antisense oligonucleotides. As illustrated in Fig. 5, the ␣ 1 -adrenoreceptor-induced Ca 2ϩ release was inhibited in cells injected with anti-␣ q oligonucleotides but was not affected in cells injected with anti-␣ 11 oligonucleotides. These results suggest different tasks for G q and G 11 proteins, i.e. induction of Ca 2ϩ release from intracellular stores and induction of Ca 2ϩ influx from extracellular medium, respectively.
Depletion of caffeine-sensitive intracellular Ca 2ϩ stores induced a Ca 2ϩ response similar to that evoked by activation of ␣ 1 -adrenoreceptors. Fig. 7  these experiments. In the continuous presence of 10 M oxodipine, application of 10 mM caffeine for 50 s in the external solution (Fig. 7Aa) produced a large transient increase in [Ca 2ϩ ] i (375 Ϯ 20 nM, n ϭ 16) and a sustained plateau of 70 Ϯ 9 nM (n ϭ 16). The rapid initial increase in [Ca 2ϩ ] i was reduced in Ca 2ϩ -free solution (298 Ϯ 25 nM, n ϭ 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 [Ca 2ϩ ] i due to Ca 2ϩ release and a sustained phase representing Ca 2ϩ entry into the cell from the extracellular space. As illustrated in Fig. 2B, the caffeine-induced Ca 2ϩ responses were not affected by inhibition of the expression of any G␣ subunits, including G␣ q and G␣ 11 subunits. Activation of ␣ 1adrenoreceptors (in Ca 2ϩ -containing solution) during the second sustained phase of the caffeine-evoked Ca 2ϩ response resulted in a 2-fold increase in [Ca 2ϩ ] i which reached 134 Ϯ 16 nM (n ϭ 23; Fig. 7Ba). The ␣ 1 -adrenoreceptor-induced enhancement of [Ca 2ϩ ] i during the second phase of the caffeine-induced Ca 2ϩ response (64 Ϯ 6 nM, n ϭ 23) was not observed at all when norepinephrine was applied without Ca 2ϩ and in the presence of 0.5 mM EGTA (n ϭ 12; Fig. 7Bb), indicating that it corresponded to a Ca 2ϩ entry from the extracellular medium. In myocytes injected with the anti-␣ q antisense oligonucleotides, the ␣ 1 -adrenoreceptor-induced Ca 2ϩ entry in the continuous presence of caffeine (55 Ϯ 9 nM, n ϭ 6) was similar to that obtained in non-injected cells (61 Ϯ 8 nM, n ϭ 6; Fig. 7Bc). In contrast, in cells injected with anti-␣ 11 antisense oligonucleotides, no ␣ 1 -adrenoreceptor-induced Ca 2ϩ entry in the continuous presence of caffeine was observed (n ϭ 8; Fig. 7Bd). These results further support the idea that G␣ 11 subunit is involved in the modulation of store-operated Ca 2ϩ entry by ␣ 1 -adrenoreceptors.
Effector Coupling Is Dependent on ␣ q and ␣ 11 Subunits-The anti-␣ q /␣ 11 antibody and antisense oligonucleotide block of the ␣ 1 -adrenoreceptor-induced Ca 2ϩ response cannot distinguish whether ␣ or ␤␥ subunits are transducing the signal that activate Ca 2ϩ release from the intracellular stores or Ca 2ϩ entry. To determine which G protein subunits were involved in the ␣ 1 -adrenoreceptor-mediated effects, an anti-␤ com antibody was dialyzed into the cell by the patch pipette for 3 min. Anti-␤ com antibody (10 g/ml in pipette solution) had no effect on the ␣ 1 -adrenoreceptor-induced Ca 2ϩ response (Fig. 8A) since neither the transient peak (control ϭ 305 Ϯ 30 nM; in the presence of anti-␤ com antibody ϭ 295 Ϯ 35 nM; n ϭ 10) nor the sustained plateau (control ϭ 55 Ϯ 4 nM; in the presence of anti-␤ com antibody ϭ 53 Ϯ 6 nM, n ϭ 10) were significantly affected. In the same cells, the anti-␤ com antibody inhibited the sustained angiotensin II-induced Ca 2ϩ response in a concentration-dependent manner, with a maximal inhibition obtained at an antibody concentration of 10 g/ml (n ϭ 10). 2 In a second set of experiments, we overexpressed a carboxyl-terminal fragment of ␤ARK 1 by intranuclear microinjection of expression plasmids containing cDNA inserts coding for ␤ARK 1 . ␤ARK has been used to bind ␤␥ subunits and block activation of effectors (25)(26). Overexpression of ␤ARK 1 had no effect on the ␣ 1 -adrenoreceptor-induced Ca 2ϩ response (Fig. 8B) since neither the transient peak (control ϭ 290 Ϯ 25 nM; in the presence of ␤ARK 1 ϭ 285 Ϯ 20 nM; n ϭ 12) nor the sustained plateau (control ϭ 65 Ϯ 5 nM; in the presence of ␤ARK 1 ϭ 60 Ϯ 8 nM; n ϭ 12) were significantly affected. In contrast, the angiotensin II-induced Ca 2ϩ response was inhibited when ␤ARK 1 was overexpressed in the same cells (n ϭ 12). 2 Taken together, our results indicate that application of anti-␤ com antibody and ␤ARK 1 , both able to bind free ␤␥ subunits, had no effects on both Ca 2ϩ release and Ca 2ϩ entry induced by activation of ␣ 1 -adrenoreceptors. other cellular effectors remains to be investigated.
In conclusion, we show that in rat venous myocytes, the G q proteins may couple by their ␣ subunits endogenous ␣ 1 -adrenoreceptors to phospholipase C, whereas the G␣ 11 proteins, activated at the same time by the same receptors, may couple to Ca 2ϩ entry. These results point out distinct functions of G q and G 11 in receptor-activated [Ca 2ϩ ] i increase.