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(Circulation Research. 2001;89:692.)
© 2001 American Heart Association, Inc.

Cellular Biology

Phosphoinositide 3-Kinase Isoforms Selectively Couple Receptors to Vascular L-Type Ca²⁺ Channels

Nathalie Macrez, Chantal Mironneau, Valérie Carricaburu, Jean-François Quignard, Aleksei Babich, Cornelia Czupalla, Bernd Nürnberg, Jean Mironneau

From Laboratoire de Signalisation et Interactions Cellulaires (N.M., C.M., V.C., J.-F.Q., J.M.), Université de Bordeaux II, France, and Abteilung Pharmakologie und Toxikologie (A.B., C.C., B.N.), Universität Ulm, Germany.

Correspondence to N. Macrez, Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail nathalie.macrez{at}umr5017.u-bordeaux2.fr

	Abstract

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Abstract—— Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca²⁺ channels by various mediators. In this study, we bring evidences that vascular L-type Ca²⁺ channels can be modulated by both tyrosine kinase–regulated class Ia and G protein–regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca²⁺ channel current density when applied intracellularly. Furthermore, PI3K-, ß-, and -mediated stimulations of Ca²⁺ channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3K- and ß-mediated effects were increased by Gß. In freshly isolated and cultured vascular myocytes, angiotensin II and Gß stimulated L-type Ca²⁺ channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca²⁺ channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3K and PI3K. Interestingly, when endogenous PI3Kß expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca²⁺ channels through PI3Kß, as revealed by the inhibitory effect of an anti-PI3Kß antibody. These results suggest that endogenous PI3Kß but not PI3K is specifically involved in PDGF receptor–induced stimulation of Ca²⁺ channels and that different isoforms of PI3K regulate physiological increases of Ca²⁺ influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Key Words: phosphoinositide 3-kinase isoforms • L-type Ca²⁺ channel • smooth muscle • platelet-derived growth factor

	Introduction

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In striated and smooth muscles, voltage-gated L-type Ca²⁺ channels represent the major pathway for Ca²⁺ entry and play a crucial role in excitation-contraction coupling. The regulatory role of L-type channels is enhanced by the modulation of this Ca²⁺ current by a large variety of hormones and mediators mainly through protein kinase activation.^1–4 Angiotensin II activates Ca²⁺ entry by stimulating L-type Ca²⁺ channels through Gß-sensitive phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) in venous myocytes^5,6 and through activation of tyrosine kinase and PI3K in A7r5 smooth muscle cells.⁷ Neuronal L-type Ca²⁺ channels are also stimulated in a PI3K-dependent mechanism activated on stimulation of the tyrosine kinase IGF-1 receptor.⁸ Platelet-derived growth factor (PDGF), also acting on a receptor tyrosine kinase (RTK), has been reported to stimulate vascular voltage-gated Ca²⁺ channels.⁹ Moreover, both Ca²⁺ entry and PI3K activation in vascular cells have been reported to be necessary for PDGF-induced and angiotensin II–induced DNA synthesis.^10–13

PI3Ks form an ubiquitously expressed enzyme family that phosphorylates membrane inositol lipids in the D3 position of the inositol ring. Class I PI3Ks are capable of phosphorylating in vitro phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate, whereas in vivo phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] and its metabolites seem the predominant products.^14,15 PI3Ks also possess a protein kinase activity, which is assumed to regulate their own lipid kinase activity for PI3K and or to regulate the mitogen-activated protein kinase signaling pathways as reported for PI3K.¹⁶ Many cell-surface receptors, including RTKs or G protein–coupled receptors (GPCRs), activate class I PI3Ks, and a large variety of cellular functions are regulated by the lipid products generated by these enzymes.

Four class I enzymes have been identified in humans and other mammals. These are divided into two subclasses (Ia and Ib) on the basis of their noncatalytic subunits. The Ia subgroup consists of the classical p110 and two additional, closely related enzymes, p110ß and p110. The p110 and p110ß isoforms both display a broad tissue distribution in adults, whereas p110 is mainly expressed in hematopoietic cells. All class Ia enzymes are associated with a p85 adapter subunit to form a heterodimeric complex. All three isoforms of this class are activated by the binding of specific phosphotyrosyl motifs to the two SH2 domains of the regulatory subunits. In addition, activation of PI3Kß by direct interaction of Gß with p110ß has been reported.^17–19

The sole member of class Ib, p110, is activated by ß subunits of heterotrimeric G proteins, which are released on activation of 7-transmembrane domain receptors. The p101 subunit, which bears no resemblance to any other known proteins, modulates the sensitivity of the kinase toward Gß.^18,20 PI3K has been shown to be expressed in hematopoietic cells as well as in exocrine glands, heart, kidney, and bovine aortic endothelial cells.^20–22

Because there is increasing evidence that different isoforms of the enzyme have distinct functions,^23–25 the goal of the present study was two-fold. First, we determined which of the four class I PI3K isoforms are able to stimulate vascular L-type Ca²⁺ channels by comparing the Ca²⁺ channel current densities in vascular myocytes infused with purified recombinant dimeric PI3Ks through the patch pipette. Second, we studied the recruitment of endogenous class Ia and class Ib PI3Ks by RTK and GPCR. We showed that efficiency of endogenous class Ia PI3Ks in transducing stimulation of Ca²⁺ channels depends on the expression of the ß isoform, which increases in cultured myocytes.

	Materials and Methods

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Cell Preparation
Isolated myocytes were obtained from Wistar rat portal vein by enzymatic dispersion, as described previously.²⁶ Cells were seeded on glass slides in physiological solution and used on the same day or maintained 4 days in primary culture in M199 medium containing 5% FCS, 2 mmol/L glutamine, 1 mmol/L pyruvate, 20 U/mL penicillin, and 20 µg/mL streptomycin.

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). Whole-cell recordings were performed with patch pipettes having a resistance of 2 to 4 M. Membrane potential and current records were stored and analyzed using P-clamp system (Axon Instruments). L-type Ba²⁺ currents were elicited by depolarizations to +10 mV from a holding potential of -40 mV and measured 4 to 5 minutes after breakthrough into the whole-cell recording mode and were digitally corrected for leakage current. Cell capacitance was determined in each cell tested by imposing 10-mV hyperpolarizing steps from the holding potential (-40 mV). Peak current density was expressed as the maximal amplitude of the Ba²⁺ current per capacitance unit (pA/pF). All experiments were performed at 30±1°C.

Solutions
The physiological solution used to record Ba²⁺ currents contained (in mmol/L) NaCl 130, KCl 5.6, MgCl₂ 1, BaCl₂ 5, glucose 11, and HEPES 10, pH 7.4 with NaOH. The basic pipette solution contained (in mmol/L) CsCl 130, EGTA 10, ATPNa₂ 5, MgCl₂ 2, and HEPES 10, pH 7.3 with CsOH. 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 (in mmol/L) Tris 20, EDTA 1, CHAPS 11, and ß-mercaptoethanol 20. PI3Ks were stored in a solution containing (in mmol/L) Tris 50, NaCl 100, and DTT 10 for the GST-tagged proteins and an additional glutathione 10 for the His-tagged PI3Kß. These solutions were diluted 50- to 200-fold in the final pipette solution and did not change either the Ba²⁺ charge densities or the peak Ba²⁺ current densities.

Purified Proteins
Construction of recombinant baculoviruses for expression of PI3K subunits was described previously.^21,27,28 For protein expression, cells were incubated at a multiplicity of infection of 1 virus per cell. Subunits of heterodimeric PI3Ks were coexpressed at equal multiplicity of infection numbers in Sf9 cells and purified as previously described.¹⁸ For functional studies, we used GST-p110 and -p110 fusion proteins and hexa-His–tagged p110ß coexpressed with p85; the p110 protein was coexpressed with GST-p101 fusion protein. Gß-proteins were purified from bovine brain as previously described.²⁹

Western Blot Analysis
Microsomal proteins were prepared from rat portal vein media homogenates and treated with Laemmli sample buffer containing 5% ß-mercaptoethanol, boiled for 10 minutes, and separated by SDS-PAGE (10% separating gel with 4% stacking gel). The resolved proteins were transferred to polyvinylidene difluoride (Bio-Rad) membrane (1 hour at 100 V). Polyvinylidene difluoride membrane was then blocked for 1 hour with 3% BSA in PBS complemented with 0.1% Tween 20 (PBS-T) and then incubated overnight with the primary anti-PI3K isoform antibodies at 2 µg/mL. After extensive washes in PBS-T, membranes were incubated for 2 hours with a 0.4-µg/mL dilution of peroxidase-coupled anti-rabbit or anti-goat IgG in PBS-T complemented with 3% BSA. Specific antigen detection was performed using the Bio-Rad Opti 4CN Substrate Kit. Gels were analyzed with KDS1D 2.0 software (Kodak Digital Science). Immunoblot analysis of purified proteins was detailed elsewhere.²⁸

Immunocytochemistry
Myocytes were washed with PBS, fixed with 4% (vol/vol) formaldehyde for 30 minutes at 20°C, and permeabilized in PBS containing 5% FCS and 0.1% (wt/vol) saponin for 30 minutes. Cells were incubated overnight at 4°C plus 1 hour at 20°C with the same solution containing the specific rabbit anti-PI3K isoform antibody at 2 µg/mL. Then cells were washed (4x10 minutes) in PBS containing 5% FCS and 0.1% (wt/vol) saponin and incubated with goat anti-rabbit or donkey anti-goat IgG conjugated to fluorescein isothiocyanate (diluted 1:500) in the same solution for 2 hours at 20°C. After extensive washes (4x10 minutes) in PBS, cells were mounted in Vectashield (Biosys). Images of the stained cells were obtained with a confocal microscope (MRC 1000, Bio-Rad). Cells were compared with each other by keeping acquisition parameters (such as gray values, exposure time, and aperture) constant.

Chemicals
PDGF-BB was from Sigma-RBI. Angiotensin II was from Neosystem Laboratories. The doubly tyrosine-phosphorylated peptide used in this study (CGGY(P)MDMSKDESVDY(P)VPMLDM) was based on that of the human platelet-derived growth factor receptor³⁰ and was kindly donated by Dr Andreas Steimeyer (Schering AG, Berlin, Germany). A nonphosphorylated peptide from the same supplier was used as a control and had no effect. Rabbit and goat anti-p110 isoform antibodies (sc-1331 and sc-7174 for p110, sc-603 and sc-7175 for p110ß, sc-7176 for p110, and sc-7177 for p110) and rabbit anti-p85 antibody (sc-423) were from Santa Cruz Biotechnology (Santa Cruz, Calif). The fluorescein isothiocyanate–conjugated goat anti-rabbit IgG and donkey anti-goat IgG were from Interchim-Jackson Immunoresearch Laboratories (Montluçon, France).

Data Analysis
All values are given as mean±SEM. A Student’s t test was performed to estimate the significance of the differences between mean values. A value of P<0.05 was considered significant.

	Results

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Effects of PI3K Isoforms on L-Type Ca²⁺ Channels
We investigated the capability of all four PI3K isoforms to stimulate vascular L-type Ca²⁺ channels by intracellular infusion of purified recombinant heterodimeric enzymes that have been checked for their enzymatic activities, as reported in a previous study.¹⁸

First, we used relatively high concentrations (1 nmol/L) of PI3K to stimulate the L-type Ca²⁺ channel current in freshly isolated portal vein myocytes. As illustrated in Figure 1, intracellular infusion of all four heterodimeric isoforms through the patch pipette resulted in an increase (about 2-fold) of the maximal peak current density measured 4 to 5 minutes after breakthrough by the whole-cell recording mode. This effect was not observed when the PI3Ks were boiled (95°C, 30 minutes) before intracellular application, thus showing that the stimulation of the current was strictly related to the integrity of the protein (Figure 1). These control experiments together with the measurements of current densities in cells infused with the protein buffers alone (data not shown) attested to the absence of any nonspecific effect. The peak Ca²⁺ channel current densities were 5.2±0.8 pA/pF (n=5) in control cells, 4.8±0.9 pA/pF (n=5) in cells infused with the buffer of the GST-tagged proteins (PI3K, PI3K, and PI3K), and 5.1±0.6 pA/pF (n=5) in cells infused with the buffer of His-tagged proteins (PI3Kß).

View larger version (30K): [in this window] [in a new window]   Figure 1. Effects of PI3K isoforms on L-type Ca2+ channels in freshly isolated rat portal vein myocytes. A, Typical Ba2+ currents elicited by depolarizations to +10 mV from a holding potential of -40 mV in control conditions (a) and in the intracellular presence of 1 nmol/L PI3K (b). Cell capacitance values were 36 pF and 34 pF for a and b, respectively. B, Peak Ba2+ current densities under resting conditions (control) and in the intracellular presence of 1 nmol/L of intact or boiled PI3K as indicated for each isoform. Data are given as mean±SEM, with the number of experiments in parentheses. Values significantly different from those obtained in control conditions (P<0.05).

Although a dose-response of PI3K-induced stimulation of Ca²⁺ channels was not fully achieved because of the difficulty to control the exact amount of PI3K in the cytoplasm of the patch-clamped cells, we noted a clear difference between 0.1 nmol/L and 1 nmol/L of PI3Ks in the pipette solution, because 0.1 nmol/L PI3Ks failed to increase the Ca²⁺ channel current densities (Figure 2).

View larger version (33K): [in this window] [in a new window]   Figure 2. Phosphotyrosyl-mediated regulation of the effect of PI3Ks on L-type Ba2+ currents in freshly isolated myocytes. Compiled data illustrating peak Ba2+ current densities under resting conditions and in the intracellular presence of Ptyr alone (A) or coapplied with 0.1 nmol/L PI3K (B). Effects of intracellular 1 nmol/L PI3K serve as positive controls, and Tyr is the control unphosphorylated peptide. Data are mean±SEM, with the number of experiments in parentheses. Values significantly different from those obtained under resting conditions (P<0.05).

Gß- or Phosphotyrosyl-Activated Effects of PI3K Isoforms
Class I PI3Ks have been shown to be activated in vitro by Gß or phosphotyrosyl peptides (Ptyr) resembling an intracellular domain of the PDGF receptor. We have previously shown that 400 nmol/L Gß maximally stimulates the Ca²⁺ channel current in vascular myocytes.⁶ In this study, we show that in freshly isolated vascular myocytes, Ptyr was unable to stimulate Ca²⁺ channel currents (Figure 2A). In contrast, when this peptide was coinfused with a low concentration (0.1 nmol/L) of any of the p85-associated p110 subunits, it did increase the Ca²⁺ channel peak current density (Figure 2B). As controls, the unphosphorylated peptide (Tyr) failed to stimulate any PI3K-mediated effect on L-type Ca²⁺ channel, and when the Ptyr was coinfused with low concentrations of PI3K, the mixture did not affect the Ca²⁺ channel peak current density (Figure 2B).

The four PI3K heterodimers were then assayed for their Gß sensitivity. As previously described, Gß stimulated L-type Ca²⁺ channel activity in a concentration-dependent manner,⁶ and even a low concentration (50 nmol/L) of Gß slightly increased the current in some cells, although the mean increase in Ca²⁺ channel current density was not significant (Figure 3A). Therefore, the study of the effect of exogenous Gß associated with various exogenous PI3Ks on Ca²⁺ channel currents was performed after pretreatment of the cells with 100 nmol/L wortmannin for 1 hour to inhibit Gß-activated endogenous PI3Ks⁶ (Figure 3A). In these conditions and when associated with PI3Kß or PI3K, 50 nmol/L Gß maximally stimulated Ca²⁺ channel currents, whereas it was ineffective when associated with PI3K or PI3K (Figure 3B). Because PI3Kß was also activated by Ptyr, we examined the combined effects of Gß and Ptyr but we did not note additional stimulation of Ca²⁺ channels when compared with the effects of one stimulus or the other (Figure 3B).

View larger version (34K): [in this window] [in a new window]   Figure 3. Gß-mediated regulation of the effect of PI3Ks on L-type Ba2+ currents in freshly isolated myocytes. Compiled data illustrating peak Ba2+ current densities under resting conditions and in the intracellular presence of Gß alone (A) or coapplied with 0.1 nmol/L of PI3Ks (B). Effects of intracellular 1 nmol/L PI3Ks serve as positive controls on each cell batches. Most experiments have been performed after a pretreatment of the cells with 0.1 µmol/L wortmannin for 1 hour to inhibit activation of endogenous PI3Ks. Data are mean±SEM, with the number of experiments in parentheses. Values significantly different from those obtained under resting conditions (P<0.05).

Effect of Cell Culture on PI3K-Induced Stimulation of L-Type Ca²⁺ Channels
Because the role of PI3Ks in transducing proliferative signals has been well-documented, we compared the Gß- and Ptyr-mediated stimulation of Ca²⁺ channels in freshly isolated and cultured vascular myocytes. As mentioned above, in freshly isolated myocytes (day 0), intracellular infusion of 400 nmol/L Gß increased the peak Ca²⁺ channel current densities to the same extent as 1 nmol/L of heterodimeric PI3K, whereas 1 µmol/L Ptyr was without effect (Figure 4, left). In contrast, in myocytes cultured for 4 days (day 4), Ca²⁺ channel current densities were similarly increased by Ptyr, Gß, and PI3K (Figure 4, right), whereas the unphosphorylated peptide (Tyr) was ineffective. Moreover, pretreatment of the cells with wortmannin (100 nmol/L for 1 hour) before patch-clamp experiments abolished Gß- as well as Ptyr-mediated stimulations of Ca²⁺ channels (n=12; data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 4. Stimulation of L-type Ca2+ channels through activation of endogenous PI3Ks by Gß and Ptyr in freshly isolated myocytes. Compiled data illustrating Ba2+ current densities under resting conditions (control) and in the intracellular presence of Gß or PTyr alone in freshly isolated myocytes (day 0) and in myocytes cultured for 4 days (day 4). At day 4, the culture medium was depleted from its FCS 1 hour before the experiments. Effects of intracellular 1 nmol/L PI3K serve as positive controls on each cell batch. Data are mean±SEM, with the number of experiments in parentheses. Values significantly different from those obtained in control conditions (P<0.05).

In agreement with these effects of Gß and Ptyr peptide, we have previously shown that in freshly isolated and cultured myocytes, angiotensin II stimulates Ca²⁺ channel current via Gß and PI3K.^5,6 In this study, we show that extracellular application of PDGF-BB, a RTK ligand, failed to increase the Ca²⁺ channel current in freshly isolated myocytes (Figure 5), whereas it did significantly increase peak current density in cultured myocytes by 39±4.2% (n=7; Figure 5). The ineffectiveness of PDGF-BB in freshly isolated myocytes was not related to the absence of PDGF receptors on the membrane of the cells after enzymatic dissociation, because when the cells were infused with low concentrations (0.1 nmol/L) of either PI3K or PI3Kß, external application of PDGF-BB produced a significant increase in peak Ca²⁺ channel current density (5.6±1.8 pA/pF in control cells versus 10.1±2.3 pA/pF in PDGF-BB–stimulated cells, n=12; data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 5. Effects of PDGF-BB and angiotensin II on L-type Ca2+ channels in freshly isolated and cultured myocytes. Time courses of the effect of PDGF-BB (0.1 µg/mL) on the Ca2+ channel current at day 0 on freshly isolated myocytes (A) or, at day 4, on myocytes cultured for 4 days (B). C, Compiled data showing the effects of PDGF (0.1 µg/mL) and angiotensin II (100 nmol/L) on freshly isolated myocytes (day 0) and on myocytes cultured for 4 days (day 4). The culture medium was deprived of FCS for 1 hour before performing the experiments. Ba2+ currents were expressed as a fraction of the current amplitude measured on the same cell before extracellular application of the agonist (I/Ic).

Effect of Cell Culture on PI3K Isoform Expression
Different patterns of expression of PI3K isoforms could provide an explanation for the absence of effect of PDGF-BB and Ptyr in freshly isolated myocytes. Therefore, we performed immunostaining experiments with antibodies specifically targeting each p110 catalytic subunit on freshly isolated and cultured portal vein myocytes.

First, we verified the specificity of the anti-p110 subunit antibodies by Western blot analysis, showing that each antibody specifically recognizes only one of the four recombinant p110 catalytic subunits (Figure 6A). The same antibodies have then been used to determine the p110 subunits expressed in rat portal vein myocytes. Western blot performed on portal vein media homogenates revealed that only p110 and p110 were expressed (Figure 6B). The absence of p110ß was confirmed in Western blot by using a second anti-p110ß antibody (sc-603; data not shown) and by immunostaining. As illustrated in Figure 7, freshly isolated myocytes expressed only two isoforms of PI3Ks, namely p110 and p110 subunits, whereas p110ß subunit was not detected by FITC-labeled immunostainings. When cells were cultured for 4 days, the p110ß subunit was clearly immunostained by two different antibodies (sc-7175, Figure 7 and sc-603, data not shown) in addition to p110 and p110. We did not use the antibody targeting p110 in immunostaining, because Western blot analysis revealed that this antibody weakly recognized several bands but no p110 in rat portal vein homogenates (Figure 6B) and because expression of PI3K has been reported to be restricted to hematopoietic cells.²⁷

View larger version (38K): [in this window] [in a new window]   Figure 6. Specificity of anti-PI3K antibodies and expression of PI3K isoforms in portal vein microsomes. A, Recombinant PI3Ks (100 ng/vial) were electrophoresed and analyzed by Western blot with anti-PI3K antibodies selectively recognizing p110, p110ß, p110, and p110, respectively. B, Microsomes of rat portal vein media (50 µg of protein/vial) were separated on SDS-PAGE and analyzed by Western blot with the same antibodies as in panel A. Molecular weight standards were electrophoresed on the same gels.

View larger version (66K): [in this window] [in a new window]   Figure 7. Cellular expression of PI3K subunit in freshly isolated and cultured rat portal vein myocytes. Immunostaining of p110, p110ß, p110, and p85 subunits in freshly isolated myocytes (day 0) and in myocytes cultured for 4 days (day 4) with the specific antibodies used in Figure 6. FITC-conjugated secondary antibodies revealed the expression of p110, p110, and p85 subunits in freshly isolated and cultured myocytes. The p110ß subunit was expressed only in cultured myocytes.

Because one member of the class Ia PI3K (p110) was expressed in freshly isolated myocytes that did not display PTyr-induced Ca²⁺ channel stimulation, we verified that a class Ia regulatory subunit was present in these cells. Immunostainings performed with an antibody targeting the regulatory subunits (anti-p85) revealed that at least one of the regulatory subunits was expressed at day 0 as well as day 4 (Figure 7), suggesting that a lack of regulatory subunit could unlikely explain the lack of PDGF-BB–mediated effect on freshly isolated myocytes.

To determine causality between the appearance of PI3Kß in cultured cells and the ability of PDGF-BB to stimulate L-type Ca²⁺ channel in these cells, we performed experiments with anti-PI3K antibodies. When the anti-PI3Kß antibody was infused into the intracellular medium through the patch pipette, PDGF-BB was not able to stimulate Ca²⁺ channels (Figures 8A and 8B). In contrast, infusion of anti-PI3K (Figure 8B) or boiled anti-PI3Kß antibody (not shown) did not affect the stimulation of Ca²⁺ channel by PDGF-BB. The efficiency of anti-PI3K antibody to inhibit recombinant PI3K-mediated stimulation of Ca²⁺ channels was shown in Figure 8C.

View larger version (23K): [in this window] [in a new window]   Figure 8. Effects of anti-PI3K antibodies on PDGF-BB–induced stimulation of L-type Ca2+ channels in cultured myocytes. A, Typical Ba2+ currents elicited by depolarizations to +10 mV from a holding potential of -40 mV before (a) and during (b) application of 0.1 µg/mL PDGF-BB in a control cell (capacitance 32 pF) and in the intracellular presence of 10 µg/mL anti-PI3Kß antibody (capacitance 30 pF). B, Compiled data showing the effects of anti-PI3K and anti-PI3Kß on the stimulation of Ca2+ channel by PDGF-BB in myocytes cultured for 4 days. The culture medium was deprived of FCS for 1 hour before performing the experiments. Ba2+ currents were expressed as a fraction of the current amplitude measured on the same cell before extracellular application of the agonist (I/Ic). C, Compiled data showing the efficacy of anti-PI3K in inhibiting PI3K-induced stimulation of peak Ba2+ current density. Peak current density significantly different from that obtained in control conditions (P<0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results show L-type Ca²⁺ channel regulation by different PI3Ks depending on the agonist and on the stage of cell culture. The main findings are that (1) all four recombinant class I PI3Ks are potentially able to stimulate vascular L-type Ca²⁺ channels; (2) the endogenously expressed PI3K does not transduce L-type channels stimulation; and (3) expression of PI3Kß in cultured myocytes is responsible of Ptyr-mediated and PDGF-BB–mediated regulation of L-type channels by PI3Ks.

The first finding is supported by a series of experiments where recombinant heterodimeric isoforms of PI3K were assayed for their ability to increase Ca²⁺ channel current density. Intracellular infusion of either a high concentration of PI3K or a low concentration of Gß- or PTyr-stimulated PI3K led to a maximal increase in peak Ca²⁺ channel current density. These results show that all four isoforms are potentially able to stimulate Ca²⁺ channels. However, because the four class I PI3K isoforms possess both lipid kinase and protein kinase activities,³¹ the present study does not help to discriminate whether PI3Ks stimulate Ca²⁺ channel through a protein phosphorylation mechanism or through the second messenger PtdIns(3,4,5)P₃.

Receptor-induced regulation of PI3Ks has been classically assigned to the noncatalytic subunit of the enzymes. In the present study, we confirm that the Ca²⁺ channel stimulation by p85-associated p110 subunits is increased by PTyr whereas the p101-associated p110 subunit–mediated effect is increased by Gß. However, because low concentrations of p85/p110ß are fully able to stimulate L-type Ca²⁺ channels once activated by Gß, this suggests that the p101 subunit usually associated with p110 is not required to confer the Gß sensitivity of the p110 catalytic subunits. Our results are in agreement with studies showing that the p101 subunit is not required for p110 activity in vitro²⁸ and that p110ß or p110 activation by Gß occurs regardless of the noncatalytic subunit it is associated with.¹⁸ In our cellular system, Gß-activated exogenous PI3Kß and PI3K stimulate L-type Ca²⁺ channel activity to the same extent, suggesting that PI3Kß may represent the GPCR-activated PI3K counterpart in cells that do not express PI3K.

The lack of additivity of Gß- and PTyr-activated effects in increasing PI3Kß-induced stimulation of Ca²⁺ channels contrasts with previous in vitro studies, showing a synergistic activation of lipid kinase activity when both stimuli were coapplied.^17,18 This difference might be attributable to an intrinsic limitation of Ca²⁺ channel facilitation or to a limitation of the endogenous phosphatidylinositol-4,5-bisphophate that serves as a substrate for the production of PtdIns(3,4,5)P₃ by PI3K.

The second main conclusion of the present study points out that the stimulatory mechanism of Ca²⁺ channels by PI3K can be differently regulated by hormones and mediators in function of the culturing stage of the cells. This statement is supported by results showing that in freshly isolated myocytes, PTyr and PDGF-BB were unable to stimulate Ca²⁺ channels except when low concentrations of exogenous PI3Ks were infused into the cytoplasm. In contrast, Gß and angiotensin II stimulate Ca²⁺ channels in the same cell batches without needing exogenous PI3Ks.⁶ Although we showed that in addition to PI3K, PI3Kß can fully activate L-type Ca²⁺ channels when activated by Gß only, the PI3K isoform involved in the Gß-activated and angiotensin II–activated effects in native myocytes is believed to be PI3K. Arguments supporting this proposal are that the ß isoform is not expressed in freshly isolated myocytes and crude portal vein media as revealed by immunostaining and Western blot analysis and that an anti-PI3K antibody specifically inhibits the Gß-induced and angiotensin II–induced stimulation of Ca²⁺ channels.³²

In this study, we show that PTyr and PDGF-BB failed to stimulate L-type channels in freshly isolated vascular myocytes, although the PTyr-activatable class Ia PI3K is expressed as revealed by immunocytochemistry and by Western blot analysis. This could be attributable to a negative regulation of p110 either by Ruk-like adaptor proteins that have been shown to inhibit p85-associated p110 lipid kinase activity³³ or by some isoforms of the regulatory subunit, ie, p85ß, p55, or p55, which are recognized by the same anti-p85 antibody as p85 but transduce very weak, if any, activation of PI3Ks.³⁴ An alternative possibility would be that enzymatic dissociation alters PDGF receptor coupling efficiency, but this is unlikely, because PTyr is also not able to stimulate Ca²⁺ channels through PI3K in freshly isolated myocytes. Finally, one may speculate that the efficiency and subcellular compartmentalization of endogenous PI3K do not allow stimulation of Ca²⁺ channels.

Because we noticed that p110ß subunit expression differs in tissue extract and freshly isolated vascular myocytes versus cultured myocytes, we hypothesized that the appearance of endogenous PI3Kß might be responsible for the PTyr-mediated and PDGF-BB–mediated effects on L-type Ca²⁺ channels observed in cultured cells. Damage of PI3Kß during the cell isolation process is unlikely, because Western Blot analysis performed on tissue extract led to the same result as immunostaining in freshly isolated myocytes, ie, the absence of p110ß subunit. Our hypothesis has been confirmed by using the anti-PI3K antibodies in functional experiments, showing that anti-PI3Kß, but not anti-PI3K, inhibits PDGF-BB–induced stimulation of Ca²⁺ channels. During intracellular infusion of exogenous PI3K, the loss of specificity of PI3Kß versus PI3K in transducing RTK- and PTyr-mediated stimulation of L-type Ca²⁺ channels argues in favor of specific subcellular localization of endogenous PI3Ks. Referring to recent studies where PI3K and ß isoforms have been reported to have different roles in RTK-mediated signaling,^24,35 it is likely that the PI3K expressed in vascular cells may be involved in other cellular responses than Ca²⁺ channel stimulation.

In conclusion, we show that Ca²⁺ channel activity can potentially be enhanced by all four types of class I PI3K but that, physiologically, this cellular response involves specific isoforms depending on the agonist acting on the cells and the culture stage of the cells. Our results also point out a specificity of endogenous PI3Kß versus endogenous PI3K in transducing stimulation of Ca²⁺ channels in cultured cells.

	Acknowledgments

 
This work was supported by grants from Center National de la Recherche Scientifique and Center National des Etudes Spatiales, France and Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Insdustrie, Germany.

Received June 6, 2001; revision received August 7, 2001; accepted August 17, 2001.

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