INTRODUCTION

Specific heterotrimeric G proteins composed of different , , and  subunits transmit signals from membrane receptors to intracellular effectors (1-2). When a receptor is activated by an agonist, it catalyzes the exchange of GDP for GTP on the subunit of G proteins, resulting in dissociation of  subunits from the dimers. It is now well documented that both the G subunit and the G complex are able to transmit signals to effector molecules (3-4). After the initial observation that G could activate K⁺ channels (5), it was found that G can regulate certain isoforms of adenylyl cyclase (6) and phospholipase C- (7), activate the mitogen-activated protein kinase (8) and c-Jun N-terminal kinase (9) pathways, and mediate the translocation of the -adrenergic receptor kinase (ARK)¹ (10).

In portal vein myocytes, the G protein heterotrimer that couples angiotensin AT_1A receptors to increase [Ca²⁺]_i has been identified using an antisense oligonucleotide strategy. The G protein is composed of ₁₃, ₁, and ₃ subunits, all three being required for activation of the transduction pathway (11). Angiotensin II (AII)-induced increase in [Ca²⁺]_i is initiated by activation of L-type Ca²⁺ channels, producing a slow elevation of [Ca²⁺]_i (12) that, in turn, activates a Ca²⁺-induced Ca²⁺ release from the intracellular store (13). It has been shown that AII produces Ca²⁺ release from the intracellular store by opening of ryanodine-sensitive Ca²⁺ release channels, as evidenced by the increase in Ca²⁺ spark frequency (14).

The purpose of the present study was to identify which G protein subunits transduce the signal for activation of Ca²⁺ channels after stimulation of the angiotensin AT₁ receptor in rat portal vein myocytes. A specific G₁₃ function-blocking antibody and G₁₃ peptide (corresponding to the last 11 amino acids of the carboxyl terminus) abrogated AII-induced stimulation of Ca²⁺ channel current when dialyzed into the cells through the patch pipette. Intracellular infusion of specific G binding agents, i.e. anti-_com antibody and ARK peptide, also blocked the AII-induced stimulation of Ca²⁺ channel current. Finally, overexpression of a ARK₁ fragment and of G scavengers, i.e. wild type of G_o1 and G₁₂ subunits, largely inhibited the AII-induced increase in [Ca²⁺]_i. We conclude that the angiotensin AT₁ receptor uses the dimers of G₁₃ to transduce the signal leading to activation of Ca²⁺ channels.

EXPERIMENTAL PROCEDURES

Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously (15). Cells were seeded at a density of about 10³ cells/mm² on glass slides imprinted with squares for localization of injected cells and maintained in short-term primary culture in M199 medium containing 2% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 200 units/ml penicillin, and 200 µg/ml streptomycin; they were kept in an incubator gassed with 95% air, 5% CO₂ at 37 °C and used within 72 h.

Voltage clamp and membrane current recordings were made with a standard patch-clamp technique using a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed with patch pipettes having resistances of 1-3 megaohms. Membrane potential and current records were stored and analyzed using an IBM PC computer (P-clamp system, Axon Instruments, Inc., Foster City, CA). Simultaneous measurements of intracellular Ca²⁺ concentration were carried out in some experiments. Briefly, 60 µM fura-2 was added to the pipette solution and entered cells after establishment of the whole-cell recording mode. [Ca²⁺]_i was estimated from the 340/380-nm fluorescence ratio using a calibration determined within cells (15). All measurements were made at 25 ± 1 °C.

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 dual-wavelength fluorescence method, as described previously (15). 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 alternatively 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. [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.

cDNA encoding -adrenergic receptor kinase was cloned into expression plasmids pRK₅ (8). cDNAs encoding for G_o and G₁₂ subunits were cloned into pECE (16). cDNA encoding for S65T green fluorescent protein was cloned into pcDNA₃ (CLONTECH, Palo Alto, CA). Briefly, plasmids were diluted with water from stock solutions (0.5 µg/µl) to final concentrations of 0.1 µg/µl and injected directly into the nucleus of vascular myocytes. The S65T green fluorescent protein (GFP) was included to facilitate later identification of myocytes having a successful nuclear injection and plasmid expression. Fluorescence produced by the S65T GFP was observed 3 days after injection with a confocal microscope (Bio-Rad MRC 1000, Paris, France). From injected cells (n = 280), about 20% showed a detectable fluorescence signal.

The normal physiological solution contained 130 mM NaCl, 5.6 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 11 mM glucose, 10 mM HEPES, pH 7.4, with NaOH. The basic pipette solution contained 130 mM CsCl, 10 mM HEPES, pH 7.4, with CsOH. Ca²⁺-free external solution was prepared by omitting CaCl₂ and adding 0.5 mM EGTA. For the recording of Ca²⁺ channel current, 5 mM BaCl₂ was substituted for CaCl₂ in the reference solution, and CsCl was used in the pipette and external solutions to block outward potassium currents. In addition, 10 mM EGTA, 5 mM Na₂ATP, and 1 mM MgCl₂ were added to the basic pipette solution. Substances were applied to the cells by pressure ejection from a glass pipette for the period indicated on the records.

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/AM, carboxyl-terminal G₁₃ peptide (LHDNLKQLMLQ) and anti-₁₃ antibody were from Calbiochem (Meudon, France). Norepinephrine, rauwolscine, and propranolol were from Sigma. Angiotensin II and CGP 42112A (N--nicotinoyl-Tyr-Lys[N--CBZ-Arg]-His-Pro-Ile-OH) was from Neosystem Laboratories (Strasbourg, France). Anti-₁₂ and anti-₁₃ antisera were a gift from B. Nürnberg (University of Berlin). Anti-_com (raised to the carboxyl-terminal amino acids, TDDGMAVATGSWDSFLKIWN, of G₁ subunit) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-_q/11 antibody was a gift from G. Guillon (INSERM U401 Montpellier, France). Carboxyl-terminal G_q/11 peptide (LQLNLKEYNLV) and peptides corresponding to the G binding domain of ARK₁ (peptide G, WKKELRDAYREAQQLVQRVPKMKNKPRS) or to a region outside the G binding site (peptide A, AETDRLEARKKTKNKQLGHEEDY) were synthesized by Genosys (Cambridge, UK).

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.

RESULTS

To identify the G proteins activated by the angiotensin AT₁ receptor, we used antibodies raised against the carboxyl terminus of  subunits to block interactions of G proteins with angiotensin AT₁ receptors and synthetic peptides corresponding to the carboxyl terminus of  subunits to disrupt the angiotensin AT₁ receptor-evoked activation of G proteins. In the continuous presence of 100 nM CGP 42112A (to block angiotensin AT₂ receptors; Ref. 17), 10 nM AII increased the Ba²⁺ current by about 40% (Fig. 1A). The stimulatory effect of AII reached a steady state within 1-2 min and was progressively reversed within 5-10 min. When anti-₁₂ antiserum (at 1:100 or 1:50) was added to the pipette solution for 7-10 min, the AII-induced stimulation of the Ba²⁺ current was not significantly affected (Fig. 1B). Similarly, intracellular applications of the anti-_q/11 antibody (15 µg/ml) for 7-8 min were without effect (Fig. 1B). In contrast, intracellular applications of anti-₁₃ antiserum (1:100) or anti-₁₃ purified antibody (10 µg/ml) blocked the AII-induced stimulation of the Ba²⁺ current (Fig. 1, A and B). The specificity of the anti-₁₃ antibody is documented in Fig. 1, A and B, since intracellular application of 10 µg/ml boiled anti-₁₃ antibody (95 °C for 30 min) did not alter the AII-induced stimulation of the Ba²⁺ current. Fig. 2 illustrates the effects of synthetic peptides corresponding to the carboxyl terminus of _q/11 and ₁₃ subunits. Intracellular applications of the ₁₃ peptide for 7-8 min inhibited the AII-induced stimulation of the Ba²⁺ current in a concentration-dependent manner (Fig. 2, A and B). The concentration of ₁₃ peptide producing half-maximal inhibition was estimated to be 4 ng/ml. Complete inhibition was obtained with 0.1 µg/ml ₁₃ peptide. It has to be noted that the ₁₃ peptide by itself had no effect on the Ba²⁺ current, as the current density, normalized by the cell capacitance, was not significantly modified (control: 12.5 ± 2.5 microampere/microfarad, n = 10; in the presence of 1 µg/ml ₁₃ peptide: 13.2 ± 3.5 microampere/microfarad, n = 8). Intracellular applications of _q/11 peptide (0.1-1 µg/ml) had no effect on the AII-induced stimulation of the Ba²⁺ current (control: 46 ± 5%, n = 10; in the presence of 1 µg/ml _q/11 peptide: 44 ± 4%, n = 6). Taken together, these results suggest that G₁₃, but not G₁₂ and G_q/11, functionally couples the angiotensin AT₁ receptors to stimulation of L-type Ca²⁺ Channels.

Effects of anti-_q/11, anti-₁₃ antibodies and anti-₁₂, anti-₁₃ antisera on the AII-induced stimulation of the Ca²⁺ channel current.

Effects of carboxyl-terminal G_q/11 and G₁₃ peptides on the AII-induced stimulation of Ca²⁺ channel current.

G Is Required for AII-induced Stimulation of Ca²⁺ Channels

Anti-G antibody and G subunit peptide block of the response alone cannot distinguish which G protein subunit (G or G) transduces the signal to Ca²⁺ channels. To determine which G protein subunit was involved in effector activation, an anti-_com antibody (18), raised to the carboxyl terminus of G₁ subunit, was dialyzed into the cell for 7-8 min. As shown in Fig. 3A, intracellular applications of 10 µg/ml anti-_com antibody blocked the AII-induced stimulation of the Ba²⁺ current. In contrast, application of the same concentration of boiled anti-_com antibody (95 °C for 30 min) had no significant effect on the AII-induced stimulation of the Ba²⁺ current (Fig. 3A).

Effects of anti-_com antibody and ARK₁ peptides on the AII-induced stimulation of Ca²⁺ channel current.

In a second set of experiments, we dialyzed peptides corresponding to fragments of ARK₁ (19) into the cells for 5-6 min. Carboxyl-terminal fragments of ARK₁ have been used to bind G subunits and to block activation of effectors (19-21). Intracellular applications of peptide G (corresponding to the G binding domain of ARK₁) inhibited the AII-induced stimulation of the Ba²⁺ current in a concentration-dependent manner (Fig. 3B). The concentration of peptide G producing half-maximal inhibition was estimated to be 65 nM. Complete inhibition was obtained with 1 µM peptide G. In contrast, intracellular applications of 10-100 µM peptide A (corresponding to a domain of ARK₁ not involved in G binding) had no effect on the AII-induced stimulation of the Ba²⁺ current (Fig. 3B). These results suggest that the angiotensin AT₁ receptors transduce their signal to Ca²⁺ channels through G.

G Transduces AII-evoked Increase in [Ca²⁺]_i

We have previously reported that the AII-evoked increase in [Ca²⁺]_i is dependent on L-type Ca²⁺ channel activation, leading to a slow elevation of [Ca²⁺]_i that triggers, in turn, a subsequent Ca²⁺ release from the intracellular store through activation of ryanodine-sensitive Ca²⁺ release channels (13). As shown in Fig. 4A, the AII-evoked increase in [Ca²⁺]_i was selectively inhibited by intracellular applications of 10 µg/ml anti-_com antibody for 7-8 min. The same concentration of boiled anti-_com antibody was without effect. Furthermore, intracellular dialysis of 10 µM peptide G also inhibited the AII-induced increase in [Ca²⁺]_i, whereas 10 µM peptide A was ineffective. It has to be noted that complete inhibition of the AII-induced increase in [Ca²⁺]_i was not obtained with increasing concentrations (100 µM) of peptide G (n = 8). This is in contrast to the block of the AII-induced stimulation of the Ba²⁺ current obtained with 1 µM peptide G. Inhibition of the AII-dependent Ca²⁺ signaling by binding of G could be related to two distinct mechanisms. (i) The inhibitory proteins (ARK₁ peptide, anti-_com antibody) prevented the AII-induced dissociation of G₁₃ and G subunits, thus inhibiting the transduction process or (ii) they bind to G after its release from G and, therefore, inhibit the G-effector coupling. To distinguish between these possibilities, the cells were stimulated by 10 µM norepinephrine to produce an increase in [Ca²⁺]_i dependent on Ca²⁺ release from the intracellular store. This transduction pathway involves G_q, which activates a phospholipase C-, leading to InsP₃ production and the subsequent activation of InsP₃-gated channels (15). As illustrated in Fig. 4B, intracellular applications of 10-100 µM peptide G to scavenge free G had no effect on the norepinephrine-induced Ca²⁺ release that was mediated by the _q subunit. Moreover, intracellular applications of both peptide G (100 µM) and _q/11 peptide (10 µg/ml) inhibited the norepinephrine-induced Ca²⁺ release (Fig. 4B). These results show that, although G is bound to the peptide G, G_q on its own can transduce and support the Ca²⁺ response evoked by norepinephrine.

Effects of ARK₁ peptides and G_q/11 peptide on the increase in [Ca²⁺]_i evoked by AII and norepinephrine.

In a second set of experiments, we overexpressed a carboxyl-terminal fragment of ARK₁ by intranuclear microinjection of expression plasmids containing cDNA inserts coding for ARK₁ fragment and S65T GFP. Overexpression of the ARK₁ fragment was followed by cytoplasmic detection of S65T GFP, 3 days after injection. As illustrated in Fig. 5A, the AII-induced increase in [Ca²⁺]_i was inhibited by about 75% in cells overexpressing the ARK₁ fragment (control: 103 ± 13 nM, n = 20; after ARK₁ fragment overexpression: 25 ± 9 nM, n = 12). It has to be noted that in all the cells injected with expression plasmids, the basal [Ca²⁺]_i level was significantly increased (control: 47 ± 5 nM, n = 50; after plasmid injection: 88 ± 5 nM, n = 56). However, in cells injected with pRK₅ plasmids alone, AII evoked a Ca²⁺ response whose amplitude (98 ± 9 nM, n = 12) was not significantly different from that measured in noninjected cells (Fig. 5B).

Effects of overexpression of a ARK₁ fragment and G subunits on AII-induced increase in [Ca²⁺]_i.

p

p

p

Another strategy to confirm the role of G in AII-induced increase in [Ca²⁺]_i was to increase the normal G:G ratio by overexpressing G subunits. Excess of GDP-bound G subunits with a high affinity for G subunits should create conditions favoring heterotrimeric formation. Injection of expression plasmids containing cDNA inserts coding for wild type (WT) G_o1 (which is not endogenously expressed in vascular myocytes; Ref. 22) or WT G₁₂ inhibited the AII-induced increase in [Ca²⁺]_i by about 75% (Fig. 5B). In contrast, overexpression of the constitutively active Q229L G₁₂ did not significantly affect the AII-induced increase in [Ca²⁺]_i. Taken together, these results are consistent with the idea that G controls the transduction pathway, leading to stimulation of Ca²⁺ channels and increase in [Ca²⁺]_i in response to activation of angiotensin AT₁ receptors.

DISCUSSION

Our results show that, in rat portal vein myocytes, G₁₃ couples angiotensin AT₁ receptors to stimulation of L-type Ca²⁺ channels. The G₁₃₁₃ heterotrimer provides the specificity for the coupling with AT₁ receptors (11), but the signal to Ca²⁺ channel is transduced by the G dimer. This conclusion is based on experiments using antibodies raised against the carboxyl-terminal G or G subunits, synthetic peptides corresponding to the carboxyl terminus of G subunits or to ARK₁ fragments, and overexpression of a ARK₁ fragment and of G subunits to disrupt the angiotensin AT₁ receptor-evoked activation of Ca²⁺ channels.

Involvement of G₁₃ in the Ca²⁺ responses evoked by AII is demonstrated by intracellular perfusion of an anti-₁₃ antibody that selectively blocks the AII-induced stimulation of Ca²⁺ channels and the increases in [Ca²⁺]_i. These results support the idea that activation of Ca²⁺ channels via G₁₃ is the initial step leading to an increase in [Ca²⁺]_i. To confirm these results, we used synthetic peptides corresponding to the carboxyl terminus of G subunits. These peptides have been shown to bind to receptors and to stabilize them in a high affinity conformational state (23-25). Thus, synthetic peptides may act as competitive agonists at the receptor/G protein interface and block receptor-mediated activation of effectors (26). As carboxyl-terminal G₁₃ peptide selectively abrogates the AII-induced stimulation of Ca²⁺ channels whereas carboxyl-terminal G_q/11 peptide is ineffective, our results suggest that the activated angiotensin AT₁ receptors specifically interact with the extreme carboxyl terminus of G₁₃ to promote dissociation of the heterotrimer involved in the regulation of [Ca²⁺]_i.

Involvement of G in Ca²⁺ channel stimulation is supported by the following results. (i) The anti-_com antibody inhibited the AII-induced stimulation of Ca²⁺ channels and increase in [Ca²⁺]_i; (ii) the ARK₁ peptide G (corresponding to the G binding domain of ARK₁) specifically abrogated the AII-mediated responses; (iii) transient overexpression of a ARK₁ fragment and of G_o and G₁₂ inhibited the AII-induced Ca²⁺ response. These experiments clearly show that G is necessary to mediate the AII-induced increase in [Ca²⁺]_i. The fact that the inhibitory proteins interacting either with G or G inhibit the AII-induced Ca²⁺ signaling may indicate that they bind to the undissociated heterotrimer and prevent the dissociation of the G protein into its subunits. Another possibility is that the inhibitory proteins bind to the dissociated G protein subunits and suppress their action on the effectors. This latter possibility is supported by our observations that overexpression of G_o and G₁₂ (which cannot bind to heterotrimeric G proteins) inhibited the AII-induced Ca²⁺ responses. In addition, intracellular applications of the ARK₁ peptide G did not inhibit the norepinephrine-activated Ca²⁺ response as would be expected if this peptide prevented the G protein heterotrimer dissociation. Although G is bound to the peptide G, G_q on its own can transduce and support Ca²⁺ release from the intracellular store in response to activation of ₁-adrenoreceptors (27). Taken together, these observations indicate that these inhibitory proteins interact with the free G protein subunits and, thus, suppress their actions on the effectors.

Coupling of G proteins with Ca²⁺ channels has been previously reported and generally promotes an inhibition of neuronal Ca²⁺ currents (28-30). This inhibition can result from a direct interaction between G and the pore-forming ₁ subunits of N- and P/Q-type Ca²⁺ channels but not of L-type Ca²⁺ channels (31). In contrast, L-type Ca²⁺ currents in cardiac and vascular myocytes can be enhanced by phosphorylation by protein kinases A and C, probably on phosphorylation sites located on the carboxyl terminus of the ₁ subunit or on the  subunit of Ca²⁺ channels (32, 33). Protein kinase C has been reported to increase Ca²⁺ channel activity through the modulation of the mean open time, a voltage-independent property, whereas protein kinase A enhances channel activity through the modification of the voltage-dependence of activation and inactivation (34). In portal vein myocytes, AII increases the Ca²⁺ channel current at all potentials but does not shift the current-voltage relationship. However, the relative increase in Ca²⁺ current induced by AII is more pronounced at negative potentials, i.e. between 40 and 20 mV, than at 0 mV,² supporting our previous data that the AII-induced stimulation of Ca²⁺ channels involves activation of protein kinase C (12).

G-induced Ca²⁺ signaling may also involve various effector systems leading to second messenger production. It has been reported in several cell types that G-mediated activation of phospholipase C-₂ or -₃ is associated with a relatively low increase in InsP₃ (35). Moreover, G may increase the apparent affinity of the InsP₃-gated Ca²⁺ channels to InsP₃, so that Ca²⁺ release may occur at low InsP₃ concentrations (36). Recently, it has been shown that G is involved in the regulation of the activity of the small GTP-binding proteins Rho and Rac (37). Thus, G may mediate the association of activated Rho and Rac to the membrane and activate, in turn, various enzymes such as phospholipase D or phosphoinositide 3-kinase (38-40), leading to generation of various second messengers. The specificity of the G-activated coupling could depend on localized distribution of certain subsets of receptors, G proteins, and effectors within distinct membrane domains that may have access to each other (41). Our results suggest that G activates L-type Ca²⁺ channels by a transduction pathway that does not involve InsP₃-gated Ca²⁺ release channels, since the AII-induced increase in [Ca²⁺]_i is insensitive to both heparin (an inhibitor of the InsP₃ receptor) and anti-phosphatidylinositol antibody (13). In addition, biochemical and pharmacological approaches have proposed that AII may induce phosphatidylcholine hydrolysis by phospholipases D (42) or C (12, 43), leading to diacylglycerol formation. Release of diacylglycerol from phosphatidylcholine hydrolysis is a slow phenomenon (42), supporting the observation that the AII-induced stimulation of Ca²⁺ channel current reaches a maximum value within 1-2 min. Activation of protein kinase C by diacylglycerol may be responsible for phosphorylation of L-type Ca²⁺ channels and the subsequent increase in Ca²⁺ channel activity (34). In vascular myocytes, openings of L-type Ca²⁺ channels at the resting potential (50 mV) have been previously proposed to serve as a pathway for Ca²⁺ influx in response to receptor activation (44, 45).

In conclusion, we show that in rat portal vein myocytes, activation of angiotensin AT₁ receptors requires G₁₃ heterotrimer for specificity and G dimer for transducing the signal to L-type Ca²⁺ channels and increase in [Ca²⁺]_i.