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Gß dimers stimulate vascular L-type Ca²⁺ channels via phosphoinositide 3-kinase

^a Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 33076 Bordeaux, France; and

^b Institut für Pharmakologie, Freie Universität Berlin, D-14195 Berlin, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have previously reported that, in venous myocytes, Gß scavengers inhibit angiotensin AT_1A receptor-induced stimulation of L-type Ca²⁺ channels ⁽¹⁾ . Here, we demonstrate that intracellular infusion of purified Gß complexes stimulates the L-type Ca²⁺ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive G_o or of a peptide corresponding to the Gß binding region of the ß-adrenergic receptor kinase completely inhibited the Gß-induced stimulation of Ca²⁺ channel currents. The gating properties of the channel were not affected by intracellular application of Gß, suggesting that Gß increased the whole-cell calcium conductance. In addition, both the angiotensin AT_1A receptor- and the Gß-induced stimulation of L-type Ca²⁺ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gß-sensitive phosphoinositide 3-kinase, PI3K, mimicked Gß-induced stimulation of Ca²⁺ channels. Both Gß- and PI3K-induced stimulations of Ca²⁺ channel currents were reduced by protein kinase C inhibitors suggesting that the Gß/PI3K-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gß dimers stimulate the vascular L-type Ca²⁺ channels through a Gß-sensitive PI3K.—Viard, P., Exner, T., Maier, U., Mironneau, J., Nürnberg, B., Macrez, N. Gß dimers stimulate vascular L-type Ca²⁺ channels via phosphoinositide 3-kinase .

Key Words: G-protein • PI3K • protein kinase C • smooth muscle

	INTRODUCTION

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CALCIUM INFLUX THROUGH L-type Ca²⁺ channels is primarily regulated by the membrane potential, but is also subject to facilitatory and inhibitory modulations ⁽²⁾ . Modulatory processes include changes in the phosphorylation state of channel subunits ⁽³⁾ , membrane delimited or second messenger-driven regulation of Ca²⁺ channels by G-proteins ⁽⁴⁾ and voltage-dependent mechanisms ^{5, 6)} .

The understanding of protein kinase-mediated Ca²⁺ channel regulation has progressed by the assignment of specific phosphorylation sites for protein kinases C (PKC),¹ and cAMP-dependent protein kinases on channel subunits for selective aspects of modulation ^{7, 8)} . Moreover, receptor tyrosine kinases modulate Ca²⁺ channels presumably through phosphoinositide 3-kinase (PI3K) ⁽⁹⁾ . Only recently, the molecular mechanisms of voltage-dependent modulation and G-protein regulation of neuronal Ca²⁺ channels have begun to be elucidated ^10-13) . In neurons, presynaptic Ca²⁺ channels are generally inhibited by neurotransmitters via activation of G_o proteins ^{14, 15)} . Inhibition occurs by direct interaction between the Ca²⁺ channel complex and the ß subunit released from the activated G_o protein heterotrimer. The Gß complex binds to the cytoplasmic linker between transmembrane repeats I and II of the Ca²⁺ channel _1A, _1B, and _1E subunits ^{12, 13)} . This I-II linker region was also shown to bind the Ca²⁺ channel ß subunit that enhances Ca²⁺ channel currents. In addition, PKC-dependent phosphorylation of some amino acid residues in the I-II linker counteracts the effects of Gß ⁽¹³⁾ . Amino- and carboxyl-terminal regions of Ca²⁺ channel ₁ subunits are also predicted to be crucial for Gß binding ^{16, 17)} . The facilitation phenomenon observed in neuronal cells has been suggested to result from a voltage-dependent change in the Ca²⁺ channel protein conformation, thereby unmasking new phosphorylation sites and/or decreasing a tonic Gß-mediated inhibition of Ca²⁺ channels ⁽⁶⁾ . Direct binding of Gß to other ionic channels has been described, i.e., Gß binding to both the amino- and carboxyl-terminal domains of an inward rectifying K⁺ channel, thus regulating its activation ^{18, 19)} . Moreover, many effectors activated by Gß have been identified in the last few years, including Gß stimulation of class I phosphoinositide 3-kinases ^{20, 21)} . These new G-protein effector systems offer attractive hypotheses for Gß-mediated pathways.

The L-type Ca²⁺ channel current in smooth muscle cells may be enhanced by phosphorylation via PKC, probably of sites located on the carboxyl terminus of the ₁ subunit or on the ß subunit of the channels ⁽²²⁾ . Angiotensin II has been reported to stimulate L-type Ca²⁺ channels in vascular myocytes through a transduction pathway involving the AT_1A receptor and the ß complex of G₁₃ß₁₃ ^{1, 23)} . The purpose of the present study was to investigate the effects of Gß dimers on the vascular L-type Ca²⁺ channels. Here we show that intracellularly applied purified Gß activates the smooth muscle L-type Ca²⁺ channels by both PKC-dependent and PKC-independent mechanisms through stimulation of PI3K. These stimulatory effects of Gß on L-type Ca²⁺ channels contrast with the inhibitory effects of Gß on non-L-type Ca²⁺ channels and suggest a novel pathway of Ca²⁺ channel activation by G-protein-coupled receptors involving PI3K.

	MATERIALS AND METHODS

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Cell preparation
Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously ⁽²⁴⁾ . Cells were seeded at density of ~10³ cells/mm² on glass slides in physiological solution and used the same day.

Membrane current
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 2–4 M. Membrane potential and current records were stored and analyzed using a PC computer (P-clamp system, Axon Instruments, Foster City, Calif.). Cell capacitance was determined in each cell tested by imposing 10 mV hyperpolarizing steps from the holding potential (-40 mV) and analyzing the amplitude and the time course of the recorded currents. Charge density was expressed as the quantity of Ba²⁺ charges per capacitance unit (pC/pF). All experiments were performed at 30 ± 1°C.

Solutions
The physiological solution used to record Ba²⁺ currents contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl₂, 5 BaCl₂, 11 glucose, 10 HEPES, pH 7.4, with NaOH. The basic pipette solution contained (in mM): 130 CsCl, 10 EGTA, 5 ATPNa₂, 2 MgCl₂ 10 HEPES, pH 7.3, with CsOH. Bovine serum albumin (BSA) (0.1%) was added in the pipette solution to increase protein infusion and had no effect by itself on the current charge density. Gß proteins were stored in a solution containing 20 mM Tris, 1 mM EDTA, 11 mM CHAPS, and 20 mM ß-Mercaptoethanol. Depending on the concentration of Gß used in the experiments, the final concentration of detergent in the patch pipette solution was 55 to 220 µM CHAPS, which alone did not have any effect on Ba²⁺ charge density. PI3K was stored in a solution containing 50 mM Tris, 100 mM NaCl, 10 mM DTT, and 10 mM glutathione. This solution was diluted 50- to 200-fold in the final pipette solution and did not change the Ba²⁺ charge density. Angiotensin II was extracellularly applied to the recorded cell by pressure ejection from a glass pipette.

Recombinant PI3K
Construction of recombinant baculoviruses for expression of human p110 and of porcine GST-p101 fusion protein were described previously ⁽²⁵⁾ . For protein expression, cells were infected at a multiplicity of infection of 1 virus per cell. After 48–60 h of infection, cells were pelleted by centrifugation (1000 x g), washed with phosphate-buffered saline twice, and resuspended in ice-cold buffer A containing 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 50 mM Tris/HCl, pH 8.0, 10 mM DTT, 10 µg/ml each of aprotinin, benzamidin, leupeptin, and 0.2 mM Pefabloc (Boehringer Mannheim, Mannheim, Germany). Cells were disrupted by N₂ cavitation (30 min at 4°C, 25 bar) or by forcing the cell suspension through a 22-gauge needle (20 times) and then through a 26-gauge needle (10 times). Nuclei and debris were discarded. The cytosolic and membranous fractions were recovered by centrifugation at 100,000 x g for 50 min. Cytosol was incubated for 2 h with glutathione-Sepharose 4B beads (Pharmacia) prewashed with buffer A. The Sepharose-bound GST-fusion proteins could be stored at -20°C in buffer B containing 50% glycerol, 1 mM EDTA, 40 mM Tris/HCl, pH 8.0, 1 mM DTT, and 1.57 mg/ml benzamidin. For enzymatic assays, heterodimeric PI3K was freshly eluted with buffer C consisting of buffer A with 10 mM glutathione for 1 h at 4°C. Purified proteins were checked for stoichiometric expression of subunits and quantified by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with bovine serum albumin as the standard.

Lipid kinase assay
The assays were conducted in a final volume of 50 µl containing 0.1% BSA, 2 mM EGTA, 0.2 mM EDTA, 10 mM MgCl₂, 120 mM NaCl, 40 mM HEPES, pH 7.4, 1 mM DTT, and 1 mM ß-glycerophosphate as described previously ⁽²⁵⁾ with some modifications. Briefly, 30 µl of lipid vesicles (320 µM PtdEtn, 300 µM PtdSer, 140 µM PtdCho, 30 µM sphingomyelin, and 30 µM PI-4,5-P₂) were mixed with either purified Gß complexes or their vehicle and incubated on ice for 10 min. Thereafter, the enzyme fraction (1–10 ng) was added and the mixture was incubated for another 10 min at 4°C in a final volume of 40 µl. The assay was then started by adding 40 µM ATP (1 µCi [-³²P]ATP) in 10 µl of the above assay buffer (30°C). After 15 min, the reaction was stopped with ice-cold 150 µl of 1 N HCl and by placing tubes on ice. The lipids were extracted by vortexing samples with 450 µl of chloroform/methanol (1:1). After centrifugation and removal of the aqueous phase, the organic phase was washed twice with 200 µl of 1 N HCl. Subsequently, 40 µl of the organic phase were resolved on potassium oxalate-pretreated TLC plates (Whatman, Clifton, N.J.) with 35 ml of 2 N acetic acid and 65 ml of n-propanol as the mobile phase. Dried TLC plates were exposed to Fuji-Imaging plates, and autoradiographic signals were quantified with a BAS 1500 Fuji-Imager, that meets GLP requirements.

Preparation of G-proteins
For isolation of bovine brain Gß as well as G_o subunits, we used standard techniques with modifications. Bovine brain G_i/G_o protein subunits were purified to apparent homogeneity in the presence of aluminum fluoride ⁽²⁶⁾ . Isolation and final purification of G_o and Gß were achieved by using a Mono Q (Pharmacia) FPLC column. G-protein subunits were identified by their immunoreactivity. Concentrations of G subunits were determined by binding of ³⁵S-GTPS ⁽²⁶⁾ , amounts of G-protein ß subunits were determined by the method of Lowry et al. ⁽²⁷⁾ and by Coomassie blue staining after SDS-PAGE, with bovine serum albumin as the standard ⁽²⁸⁾ . Purified Gß complexes were free of G subunits as assessed by ³⁵S-GTPS binding and pertussis toxin-mediated ³²P-ADP-ribosylation. Purified proteins were stored at -70°C until use.

Gel electrophoresis, immunoblotting, and antibodies
Generation of the monoclonal antibody against p110 was detailed elsewhere ⁽²⁵⁾ . Polyclonal polypeptide p101-antiserum was generated in rabbits against a peptide corresponding to amino acids 398–411 (cYERPRRPGGHERRG) of porcine p101. The polyclonal anti-GST antibody was purchased from Santa Cruz (Heidelberg, Germany). For detection of GST fusion proteins, p110, or G-protein subunits, preparations were fractionated by SDS-PAGE transferred to nitrocellulose or PVDF-membranes (Millipore, Eschborn, Germany). Visualization of specific antisera was performed by using the ECL chemiluminescence system (Amersham, Braunschweig, Germany) or the CDP-Star chemiluminescence reagent (Tropix, Bedford, Pa.) according to the manufacturers' instructions.

Chemicals
Angiotensin II was from Neosystem Laboratories (Strasbourg, France). Peptides corresponding to the Gß binding domain of ß-adrenergic receptor kinase-1 (ßARK₁) (WKKELRDAYREAQQLVQRVPKMKNKPRS) or to a region outside the Gß binding site (AETDRLEARKKTKNKQLGHEEDY) were synthesized by Genosys (Cambridge, U.K.). Phorbol ester 12,13-dibutyrate and 4-phorbol 12,13-dibutyrate were from LC Laboratories (Woburn, Mass.). Phorbol 12-myristate 13-acetate was from RBI (Illkirch, France). The protein kinase C inhibitor GF109203X was a gift from Glaxo (Les Ulis, France). Protein kinase C inhibitor, 19–31 peptide, and the cAMP-dependent protein kinase inhibitor, H89, were from Calbiochem (Meudon, France). Wortmannin was from Sigma (Saint-Quentin Fallavier, France). 17-Hydroxywortmannin was a gift from Dr. A. Steinmeyer (Berlin, Germany).

	RESULTS

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Effects of Gß on L-type Ca²⁺ channels
Figure 1 A illustrates the time course of Ba²⁺ currents evoked by depolarizations from -40 to +10 mV in the presence or absence of purified bovine brain Gß complexes. Each current amplitude is expressed as a fraction of the first current obtained after breakthrough into the whole-cell recording mode. Under both conditions (with or without Gß), the recorded Ba²⁺ currents were completely blocked by 1 µM oxodipine or nifedipine (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of Gß on L-type Ca2+ channels in rat portal vein myocytes. A) Time course of Ba2+ current amplitude (expressed as a fraction of the first current obtained just after breakthrough into the whole-cell recording mode) in cells dialyzed with a control pipette solution () or with a pipette solution containing 400 nM Gß (). Inset: typical Ba2+ currents elicited by depolarization to +10 mV from a holding potential of -40 mV in control conditions and in the intracellular presence of 400 nM Gß. B) Ba2+ charge densities under resting conditions (control) and in the intracellular presence of 10 µM ßARK peptide or 400 nM Gß. Data are given as means ± SE, with the number of experiments in parentheses. *, values significantly different from those obtained in control conditions (P<0.05).

To study the effects of Gß on vascular L-type Ca²⁺ channels, we intracellularly infused a dilution (1:200 to 1:50) of a highly concentrated mixture of Gß purified from bovine brain G_i/G_o proteins, containing at least Gß₁ and G₃ isoforms ⁽²⁵⁾ . Under these experimental conditions, the protein buffer had no effect on Ca²⁺ channel currents; the controls shown in the figures have been obtained with a pipette solution containing the protein buffer. Intracellular applications of purified Gß (400 nM in the pipette solution) resulted in a time-dependent increase in Ba²⁺ currents when compared with control conditions (Fig. 1A ). Steady-state Ba²⁺ currents were obtained within 2–4 min after establishment of the whole-cell recording mode. Diffusion of molecules from the patch pipette into cells depends on molecular weight of the tested molecule, pipette size, access to the cell and finally, cell type, size, and volume. However, Pusch and Neher ⁽²⁹⁾ have proposed a simple method to approximate the theoretical diffusion time constant of a molecule in a given cell using the two following equations:

(1)

and

(2)

were is the time constant in seconds, R_A is the access resistance in M, and M the molecular mass of the diffusing molecule in daltons. Equation 2 is a volume scaling formula proposed by Pusch and Neher to relate the diffusion time constant of a given molecule in the cell of interest () to that of the same molecule in chromaffin cells (₀). The dimensions of venous myocytes (12–15 µM) are close to those of chromaffin cells, thus ₀. The proteins used in the present study have a molecular mass of ~40 kDa for G_o, 45 kDa for Gß, and 220 kDa for the dimeric PI3K. With an access resistance of 4–5 M, the diffusion time constants () for the different proteins were estimated to be 100 s for G_o, 105 s for Gß, and 180 s for PI3K. These values are in good agreement with the time course of Gß- (Fig. 1A ) and PI3K-induced increase in Ba²⁺ currents.

The mean control Ba²⁺ charge density was 1.37 ± 0.09 pC/pF (n=57). As shown in Fig. 1B, G ß (400 nM) stimulated Ba²⁺ currents up to a maximal value of 2.41 ± 0.15 pC/pF (n=30). Intracellular application of 10 µM of a Gß binding ßARK peptide (corresponding to the Gß binding region of ßARK₁) for 3–5 min decreased the control Ba²⁺ charge density to 0.92 ± 0.20 pC/pF (n=20, P<0.05; Fig. 1B ). This inhibitory effect of ßARK peptide was strongly pronounced in some cell batches displaying high-density Ba²⁺ currents under resting conditions. As a control, dialysis of 10 µM of a control peptide, which does not bind Gß, had no effect on the Ba²⁺ charge density (data not shown). One may speculate that the high-density Ba²⁺ currents observed in some cells may result from endogenous free Gß protein under resting conditions.

In Fig. 2 A, we show that Gß increased the Ba²⁺ current in a concentration-dependent manner. Since no significant increase of Ba²⁺ charge density was observed with heat-inactivated Gß (95°C for 30 min; Fig. 2B ), the stimulation of Ba²⁺ current was due specifically to the intact Gß proteins. In a second set of experiments, ßARK-peptide or an inactive control peptide was applied intracellularly together with Gß. In all cell batches, intracellular applications of Gß binding ßARK peptide (10 µM) suppressed the stimulatory effect of Gß, whereas the inactive control peptide (10 µM) had no significant effect (Fig. 2B ). In a third set of experiments, Gß was scavenged by preincubation with inactive GDP-bound G_o (400 nM in the pipette solution) 30 min before filling the patch pipette. Coinfusion of GDP-bound G subunits with a high affinity for Gß protein created conditions favoring the formation of inactive Gß heterotrimers, thereby suppressing Gß-induced increase in Ba²⁺ charge density (Fig. 2B ).

View larger version (31K): [in this window] [in a new window]   Figure 2. Specific effects of Gß on L-type Ba2+ currents. A) Concentration-dependent effects of Gß on Ba2+ currents elicited by depolarizations to +10 mV from -40 mV. Data are given as means ± SE, with the number of experiments in parentheses. *, values significantly different from those obtained in control conditions (P<0.05). B) Specificity of the Gß-induced stimulation of Ba2+ currents illustrated by the absence of a noticeable stimulation when 400 nM boiled Gß (95°C for 30 min) or when 400 nM Gß was dialyzed in the presence of either 10 µM ßARK peptide (corresponding to the Gß binding domain of ßARK1) or 400 nM inactive GDP-bound Go into the cells. Ba2+ charge densities obtained with 400 nM Gß were not affected in the intracellular presence of 10 µM control peptide (corresponding to a domain of ßARK1 not involved in Gß binding). Data are given as means ± SE, with the number of experiments in parentheses. *, values significantly different from those obtained in control conditions (P<0.05). Ba2+ currents were elicited by depolarizations to +10 mV from a holding potential of -40 mV and measured 4–5 min after breakthrough into the whole-cell recording mode.

In the absence and presence of 400 nM Gß, inward currents with similar kinetics but different amplitudes were evoked by increasing depolarizations from a holding potential of -40 mV. As illustrated by the current-voltage relationships (Fig. 3 A), the maximal Ba²⁺ current was increased without any change in the voltage threshold, the potential for the maximal current, and the extrapolated reversal potential. In addition, the steady-state inactivation of the L-type Ba²⁺ current was examined with a two-pulse protocol (Fig. 3B ). A test pulse to +10 mV (V₂) from a holding potential of -40 mV was preceded by a prepulse (V₁) of 20 s duration and of variable amplitude (-60 to -20 mV). For each prepulse, the amplitude of the test current was taken as an index of the remaining activatable channels. Relative availability was expressed by plotting the test current against the prepulse potential value. The amplitude of the test current was expressed as a fraction of the current obtained at the most negative prepulse. As shown in Fig. 3B , the curves obtained in the absence and presence of 400 nM of Gß were superimposed. Together, these results indicate that Gß stimulates L-type Ca²⁺ channels without affecting the gating properties of these channels.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of Gß on the current-voltage relationship and the steady-state inactivation curve of L-type Ba2+ currents. A) Current-voltage relationships obtained from a holding potential of -40 mV in cells dialyzed with a control pipette solution () or a pipette solution containing 400 nM Gß (). Currents are expressed as a fraction of the mean maximal current obtained with 400 nM Gß. Data are given as means ± SE for 3–5 cells. B) Steady-state inactivation curve obtained with the two-pulse protocol (inset). Currents are expressed as a fraction of maximal current (I/Imax) in each cell dialyzed with a control pipette solution () or with a pipette solution containing 400 nM Gß (). Data are fitted by curves of form 1/[1 + exp (Vm - Vh)/k], in which Vh is the potential at which half of the current is inactivated, Vm is the membrane potential, and k is the slope factor. Data are given as means ± SE for 5–7 cells. Ba2+ currents were measured 4–5 min after breakthrough into the whole-cell recording mode.

Phosphoinositide 3-kinase-mediated effect of Gß
The mechanism responsible for the stimulatory effect of Gß on L-type Ca²⁺ channel current was unlikely a direct effect on the channel since only N-, P-, and Q-type of channels have been shown to bind Gß ^{12, 16)} . In contrast, it has recently been speculated that L-type calcium channels might be activated by growth factor receptor-coupled PI3Ks ⁽⁹⁾ . Since a subset of class I PI3Ks is regulated by G-protein-coupled receptors ^{25, 30-33)} , we have checked the involvement of a Gß-sensitive PI3K on the stimulation of Ca²⁺ channels.

After pretreatment of the cells with the covalent membrane permeant-PI3K inhibitor wortmannin (100 nM for 1 h or 200 nM for 30 min), intracellular infusion of 400 nM Gß failed to induce any increase in Ba²⁺ current (Fig. 4 A, left panel). A shorter pretreatment with wortmannin (100 nM for 30 min) had a smaller inhibitory effect (~50%) on the Gß-induced stimulation of Ba²⁺ current (data not shown). Angiotensin II-induced stimulation of Ba²⁺ current (previously shown to be mediated by Gß) was also completely blocked by wortmannin (Fig. 4B ). 17-Hydroxywortmannin was even more effective since pretreatment of the cells with 50 nM of this 17-hydroxy derivative for 1 h inhibited the angiotensin II-induced Ca²⁺ responses (data not shown). However, no unspecific effect of wortmannin could be detected and the same cells pretreated with wortmannin were still responding to phorbol esters by an increase in Ba²⁺ current (Fig. 4B ). The cells were also dialyzed with a purified recombinant heterodimeric PI3K, which consists of a p110 catalytic subunit associated with a noncatalytic p101 subunit (Fig 4A , right panel). The function of the enzymatically inactive p101 subunit of PI3K is unclear. We have found that p101 subunit is not needed to convey Gß effects on p110 subunit activity ⁽²⁵⁾ , but rather protects the purified enzyme from rapid degradation [U. Maier, B. Nürnberg, unpublished data]. Intracellular infusion of enzymatically active recombinant heterodimeric PI3K (1 nM in the pipette solution) increased the charge density to the same extent as Gß (Fig 4A , right panel) whereas boiled PI3K or the protein buffer were without effect. In parallel, we measured the formation of the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5P₃) from phosphatidylinositol-4,5-bisphosphate by the purified heterodimeric PI3K (Fig 5 A) in the presence of various concentrations of Gß in vitro. Under these conditions, significant basal enzyme activity was seen that was stimulated by purified Gß up to 25-fold with a half-maximal concentration for Gß (EC₅₀) of ~10 nM (Fig. 5B ). In patch-clamp experiments, infusion of enzymatically active PI3K into the cell results in an increase of basal enzyme activity, thus mimicking the Gß-induced stimulation of PI3K and producing more PI-3,4,5P₃. Moreover, in cells pretreated with wortmannin, the stimulatory effect of 400 nM Gß was restored when a low concentration (0.1 nM) of exogenous PI3K was infused together with Gß (n=4, data not shown). Together, these data suggest that in venous myocytes, L-type Ba²⁺ currents are increased by Gß via stimulation of PI3K activity.

View larger version (27K): [in this window] [in a new window]   Figure 4. Role of PI3K in Gß-, AII-, and PMA-induced stimulation of L-type Ca2+ channels. A) Effects of Gß, PI3K, and PI3K inhibition on the stimulation of Ba2+ currents. Ba2+ charge densities in cells dialyzed with a control patch pipette solution (containing the protein buffer) or a pipette containing 400 nM Gß or 1 nM of either PI3K or boiled PI3K; some cells were also pretreated with 200 nM wortmannin (wort.) for 30 min. Data are given as means ± SE, with the number of experiments in parentheses. *, values significantly different from those obtained in control conditions. B) Effects of wortmannin pretreatment on angiotensin II (AII)- and phorbol ester-induced stimulation of L-type Ca2+ channels. Stimulation of the control Ba2+ current (cont.) by 10 nM AII and 0.3 µM PMA in control conditions (a, c) and after a pretreatment of the cells with wortmannin (200 nM for 30 min; b and d). Ba2+ currents were elicited by depolarization to +10 mV from a holding potential of -40 mV and measured 4–5 min after breakthrough into the whole-cell recording mode.

View larger version (32K): [in this window] [in a new window]   Figure 5. Gß stimulation of PI3K in vitro. A) Recombinant heterodimeric PI3K was expressed as a p101-GST-fusion protein and p110 catalytic subunit in Sf9 cells, and was purified from cytosol as detailed in Materials and Methods. Proteins were separated by SDS-PAGE and stained by Coomassie blue to check stoichiometric expression of subunits. PI3K subunits were identified by immunoblotting after SDS-PAGE with antisera against p110 or GST, as detailed in Materials and Methods. Apparent molecular masses of marker proteins are indicated. B) Representative concentration-response curve of purified recombinant PI3K activity by Gß purified from bovine brain. Enzyme activity was determined by measuring formation of radiolabeled PI-3,4,5P3 from PI-4,5P2 and [-32P]ATP using a phosphor imaging system. The inset shows the corresponding autoradiogram of 32P incorporation into PI-4,5P2. Basal PI3K activity in the absence of Gß corresponds to the left most PI-3P spot and was 4.3 nmol/min/mg protein.

PKC-dependent effect of Gß and PI3K
We have previously reported that PKC stimulates Ca²⁺ channels in vascular myocytes ^{24, 34)} . Here, we sought to assign the role of PKC in the Gß- and PI3K-induced stimulation of L-type Ca²⁺ channels. In the presence of PKC inhibitors, i.e., external application of GF109203X (1 µM) or infusion of 19–31 peptide (1 µM), phorbol ester-induced stimulation of Ba²⁺ currents was completely blocked (Fig. 6 These PKC inhibitors decreased the Gß- (Fig. 6B ) and PI3K-induced increase in Ba²⁺ charge density (Fig. 6C ) by ~50%. Increasing the concentration of GF109203X or 19–31 peptide up to 5 µM did not result in larger inhibition (n=6; data not shown). The PKC-independent effect of Gß was not mediated through a cAMP-dependent protein kinase, since exposure of the cells to the cAMP-dependent protein kinase inhibitor H89 (0.1 and 1 µM) in addition to infusion of 19–31 peptide did not increase the inhibition of the Gß-simulated Ba²⁺ current when compared with infusion of 19–31 peptide alone (n=5; data not shown). In addition, in cells pretreated with 10 µM of phorbol 12,13-dibutyrate (PDBu) for 24 h in order to desensitize PKC ⁽³⁵⁾ , the Gß- and PI3K-induced Ba²⁺ charge densities were also decreased by ~50%, whereas a similar pretreatment with 10 µM of the inactive derivative 4-PDBu had no significant effect (n=15; data not shown). Finally, the infusion of Gß (400 nM) in cells superfused with 0.3 µM phorbol 12-myristate 13-acetate (PMA) for 4 min evoked a Ba²⁺ charge density similar to that obtained in cells that were not treated with PMA (2.34 ± 0.25 pC/pF, n=9; data not shown). Together, these results suggest that the Gß/PI3K-induced stimulation Ca²⁺ channels is partly dependent on PKC.

View larger version (24K): [in this window] [in a new window]   Figure 6. Effects of protein kinase inhibitors on the phorbol ester-, Gß-, and PI3K-induced increases in L-type Ba2+ currents. A) Ba2+ charge densities in control cells and cells superfused with 0.3 µM PMA in the absence or presence of 1 µM GF109203X or 1 µM 19–31 peptide. B) Ba2+ charge densities obtained with intracellular infusion of 400 nM Gß in the absence or presence of 1 µM GF109203X or 1 µM 19–31 peptide compared with Ba2+ currents obtained in the absence of Gß in the pipette solution (control). C) Ba2+ charge densities obtained with intracellular infusion of 1 nM PI3K in the absence or presence of 1 µM GF109203X or 1 µM 10–31 peptide compared with Ba2+ currents obtained without internal PI3K (control). Data are given as means ± SE, with the number of experiments in parentheses. *, values significantly different from the control values (P<0.05). , values significantly different from the Gß-evoked response (B) and from the PI3K-evoked response (C) (P<0.05).

However, the maximal Ba²⁺ charge densities obtained after intracellular infusion of either Gß (400 nM; Fig. 6B ) or PI3K (1 nM; Fig. 6C ) always resulted in higher charge densities than those obtained with the application of PMA (0.3 µM; Fig 6A ). The higher charge densities in the presence of Gß or PI3K, together with the finding that PKC inhibitors are not able to completely inhibit Gß- and PI3K-induced increase in Ba²⁺ charge densities, suggest that Gß-activated PI3K also stimulate Ca²⁺ channels independently of PKC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our results indicate a novel means of Ca²⁺ channel modulation by G-protein-coupled receptors. Receptor-activated G-protein ß dimers stimulate vascular L-type Ca²⁺ channels through PI3K, and the Gß/PI3K-induced stimulation of Ca²⁺ channels is affected by PKC inhibitors. Our conclusions are based on several independent lines of evidence that distinguish the effects of Gß, PI3K, and phorbol esters.

First, we show that intracellular application of purified Gß complexes into vascular myocytes induced a concentration-dependent stimulation of L-type Ba²⁺ currents. The Gß-induced stimulation of L-type Ba²⁺ current was specific, as this effect was abolished when Gß had been inactivated by heating before infusion into the cells or in the presence of Gß scavengers, i.e., ßARK peptide and inactive GDP-bound G_o. As the voltage dependency of both current-voltage relationship and steady-state inactivation curve was not affected by Gß, we propose that Gß increases the whole-cell Ba²⁺ conductance through L-type Ca²⁺ channels. Under resting conditions, the existence of Ba²⁺ currents of high density in some cell batches may depend on a great number of phosphorylated Ca²⁺ channels or endogenous free Gß dimers. The latter hypothesis appears attractive in that recent data imply that cells contain a pool of free Gß ⁽³⁶⁾ . To suppress the effects of putative free Gß, we intracellularly applied Gß binding peptides derived from ßARK₁ ⁽³⁷⁾ , which significantly decreased the Ba²⁺ charge densities of unstimulated cells. Therefore, high-density Ba²⁺ currents may reflect a tonic stimulation of L-type Ca²⁺ channels by free Gß complexes present in the cell under resting conditions.

We show here, for the first time, that Gß stimulates the L-type Ca²⁺ channels. This is in contrast to the direct inhibitions of N-, P-, and Q-type Ca²⁺ channels by Gß that have been described recently ^{13, 16)} . The lack of direct inhibitory effects of Gß on vascular L-type Ca²⁺ channel is in agreement with 1) the fact that Gß cannot bind to either the I-II linker region of L-type _1C and _1S subunits ⁽¹²⁾ or the carboxyl terminus of _1C ⁽¹⁶⁾ , 2) the lack of effects of GDPßS on the double pulse-induced facilitation observed in CHO cells stably expressing the vascular class C_b Ca²⁺ channel ₁ subunit ⁽³⁸⁾ , and 3) the fact that such voltage-dependent Ca²⁺ channel current facilitation by double pulse is not observed in portal vein myocytes [P. Viard, N. Macrez, J. Mironneau, unpublished data].

Second, we show that the Gß-induced stimulation of L-type Ca²⁺ channels was mediated through a G-protein-sensitive PI3K. Wortmannin pretreatment of the cells at nanomolar concentrations specifically blocked the stimulatory effect of Gß on Ca²⁺ channels. At these concentrations, wortmannin acts specifically on PI3Ks ⁽³⁹⁾ . Accordingly, we showed that wortmannin pretreatment did not inhibit Ca²⁺ channel current or Ca²⁺ channel stimulation by direct activation of PKC by phorbol ester. These results indicate the presence of endogenously expressed G-protein-sensitive PI3Ks in these cells. Furthermore, intracellular infusion of purified PI3K dimers mimicked the Gß-induced stimulation of Ca²⁺ channels. By infusing enzymatically active PI3K into the cell, we increased the intracellular PI3K activity eliciting significant stimulation of Ca²⁺ channels. However, relatively high concentrations of PI3K (around 1 nM in the pipette solution) had to be infused in order to reproduce the Gß-stimulatory effect. In contrast, when Gß was infused, the efficiency of the Gß-PI3K system is greatly increased as illustrated by in vitro experiments showing half-maximal and maximal stimulation of recombinant purified PI3K dimer at ~10 nM and 100 nM Gß, respectively. Hence, the in vitro data are in good agreement with the fact that 200 to 400 nM purified Gß complexes present in the pipette solution stimulate L-type Ca²⁺ channels maximally through stimulation of endogenous PI3K activity. Together, our results show that, in vascular myocytes, the stimulation of Ca²⁺ channels via PI3K can be induced by Gß complexes. Despite a recent publication showing that a tyrosine kinase-activated PI3K stimulates L- and N-type Ca²⁺ channels ⁽⁹⁾ , the link between Gß/PI3K/Ca²⁺ channels was not evident since Gß has been shown to inhibit N-type Ca²⁺ channels ^{10, 13)} . In the present study, complementary experimental approaches show that the Gß-activated PI3K stimulates L-type Ca²⁺ channels. This may represent a common signaling pathway, providing a hypothesis to explain that, in vascular myocytes, L-type Ca²⁺ channels are convergent targets for stimulation by multiple G-protein-coupled receptors.

Third, we show that both Gß and PI3K stimulate L-type Ca²⁺ channels by two mechanisms. The involvement of a PKC-dependent mechanism is supported by the following observations: 1) in the presence of PKC inhibitors (GF109203X or 19–31 peptide) or after a PDBu pretreatment (which desensitizes PKC and inhibits PDBu-induced stimulation of Ba²⁺ current), the stimulation of L-type Ba²⁺ current either by Gß or PI3K is significantly decreased by ~50%, and 2) when PMA is applied on cells infused by Gß, the phorbol ester is not able to further stimulate the channels when compared with infusion of Gß alone. Since L-type Ca²⁺ channel activation by Gß was completely blocked by wortmannin and mimicked by PI3K infusion, this suggests that PKC may act downstream of PI3K. In agreement with this assumption are reports showing that PI-3,4,5-P₃ may bind to and activate some atypical PKC subtypes ^40-42) . PI-3,4,5-P₃ may also indirectly stimulate PKC through Rac activation ⁽⁴³⁾ ; the small G-protein may, in turn, activate a phospholipase D ^{44, 45)} , thus producing phosphatidic acid and further diacylglycerol. In addition, Parker's group ⁽⁴⁶⁾ recently reports that - and -subtypes of PKC are phosphorylated and activated by protein kinase D1 in a PI3K-dependent manner.

However, additional mechanisms for PI3K-induced L-type Ca²⁺ channel activation have to be considered. Indeed, PKC activation alone does not seem to be responsible for the entire effect of Gß and PI3K on L-type Ca²⁺ channels since, in the presence of PKC inhibitors (or after PKC desensitization), infusion of Gß or PI3K still produced a stimulatory effect on L-type Ca²⁺ channels. Moreover, the maximal charge densities obtained after a direct stimulation of PKC by phorbol esters were always lower than those obtained with either Gß or PI3K in the pipette solution on the same cell batches. One may speculate that PI-3,4,5-P₃ may directly act on Ca²⁺ channels, as discussed recently ⁽⁹⁾ . It should also be noted that voltage-independent Ca²⁺ channels have been suggested to be stimulated by the monomeric GTPase Rac ⁽⁴⁷⁾ , which is activated by Vav in a PI3K-dependent manner ⁽⁴²⁾ . The existence of a similar signaling pathway regulating voltage-operated Ca²⁺ channels should be clarified.

In conclusion, the present study shows for the first time that Gß complexes stimulate L-type Ca²⁺ channels in vascular myocytes and that this stimulatory effect is mediated by PI3K. The opposing effects of Gß on neuronal and on vascular Ca²⁺ channels may be of physiological relevance since the Gß-mediated inhibition of N, P, and Q types of neuronal Ca²⁺ channels results in a negative feedback controlling neuromediator release, whereas in vascular myocytes the Gß-induced stimulation of L-type Ca²⁺ channels may contribute to the contractile effects of neuromediators and hormones.

	ACKNOWLEDGMENTS

 
We are indebted to Dr. Günter Schultz and Dr. Tim Plant for critical reading of the manuscript and helpful suggestions. This work was supported by grants from Center National de la Recherche Scientifique, Center National des Etudes Spatiales, France, Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Insdustrie, Germany. We thank N. Biendon and J-L. Lavie for technical assistance.

	FOOTNOTES

 
^* Correspondence: N. Macrez, Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail:nathalie.macrez{at}esa5017.u-bordeaux2.fr or B. Nürnberg, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69/73, D-14195 Berlin, Germany. E-mail:bnue{at}zedat.fu-berlin.de

¹ Abbreviations: ßARK, ß-adrenergic receptor kinase; BSA, bovine serum albumin; PDBu, phorbol 12,13-dibutyrate; PI3K; phosphoinositide 3-kinase; PI-3,4,5P₃, phosphatidylinositol-3,4,5-trisphosphate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Received for publication October 5, 1998. Revision received November 21, 1998.

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