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Regulation of Vascular L-type Ca2+ Channels by Phosphatidylinositol 3,4,5-Trisphosphate

Originally publishedhttps://doi.org/10.1161/01.RES.0000138017.76125.8bCirculation Research. 2004;95:300–307

Abstract

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gβγ-sensitive PI3Kγ in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3Kγ transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3Kγ in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.

Class I phosphoinositide 3-kinases (PI3Ks) have been implicated in an increasing number of signal transduction pathways linking virtually every class of extracellular stimulus to intracellular activation of kinase.1–3 For several years, PI3K has been shown to be involved in the regulation of Ca2+ signals. PI3K has been proposed to indirectly modulate inositol 1,4,5-trisphosphate (IP3) receptors and Ca2+ release-activated Ca2+ channels [through PI(3,4,5)P3-dependent IP3 production] in nonexcitable cells.4 A noncapacitative Ca2+ influx is also regulated by PI(3,4,5)P3 in RBL-2H3 cells.5 In excitable cells, PI3K stimulates voltage-gated Ca2+ channels.6–8 This mechanism seems to underlie the increased vascular spontaneous tone observed in hypertensive rats.9 However, how PI3K is able to regulate Ca2+ channels activity remains to be elucidated.10

Class I PI3Ks are enzymes that selectively phosphorylate the 3′-OH position of the PI(4,5)P2 inositol ring in vivo to generate PI(3,4,5)P3, further metabolized by inositol lipid phosphatases to PI(3,4)P2. PI(3,4)P2 and PI(3,4,5)P3 are absent in resting cells, increase on class I PI3K activation during cellular stimulation, and interact with pleckstrin homology (PH) domains of cellular proteins to transduce signal. Class I PI3Ks have been subclassified according to their structure and mode of activation by cell surface receptors. Class IA PI3Ks are heterodimers composed of a catalytic subunit (the ubiquitous p110α or more tissue-restricted p110β, or p110δ) tightly complexed to a regulatory adapter subunit (p85α, p85β, p55, or their splice variants). These regulatory subunits dock the holoenzyme to the membrane through interactions with specific phosphotyrosyl-containing sequences within receptor tyrosine kinases or other membrane-associated proteins. Class IB PI3K is composed of the p110γ catalytic subunit associated with a p101 regulatory protein. PI3Kγ is specifically stimulated by Gβγ dimers liberated on G protein-coupled receptor (GPCR) activation. The p110γ catalytic subunit contains all the structural elements necessary for Gβγ-induced stimulation. The p101 noncatalytic regulatory subunit, which is able to bind lipid substrates, increase p110γ activity.11 PI3Kβ is synergistically activated by GPCRs and receptor tyrosine kinases. Like for other class IA enzymes, phosphotyrosyl peptide interaction with the p85 regulatory subunit leads to activation of PI3Kβ. However, p110β catalytic subunits can be directly activated by membrane-bound βγ dimers.12

In addition to their function as lipid-kinases, PI3Ks have intrinsic serine-kinase activity. This protein-kinase activity is assumed to regulate the lipid-kinase activity for PI3Kα, PI3Kβ, and PI3Kδ. For example, p110α phosphorylates itself and its associated p85 regulatory subunit, which results in decreased lipid-kinase activity for the complex. p110α can also phosphorylate the insulin receptor substrate-1.1 The protein-kinase activity of PI3Kγ autophosphorylates its p110γ subunit without changing its own lipid-kinase activity13 but regulates the MAP kinase signaling pathway in Cos-7 cells.14 Inhibitors such as wortmannin and LY294002 cannot be used to discriminate PI3K lipid-kinase and protein-kinase activity because they interfere with both activities. However, PI3K-mediated signals can be identified with a PI3Kγ mutant engineered to be selectively defective in lipid-kinase activity. Studies with this mutant indicate that the protein-kinase activity of PI3Kγ is sufficient to activate the ERK cascade, whereas in contrast, 3′-phosphorylated lipids are required for PI3K/Akt activation.14

To date, the mechanism for calcium channel activation by PI3K isoforms remains unknown. Several nonexclusive mechanisms are possible, including direct effects of PI(3,4,5)P3 to modulate channel gating, PI(3,4,5)P3-dependent activation of PDK1 and phosphorylation/activation of a PKC isoform or Akt. In addition, the protein-kinase activity of PI3K might regulate calcium channel activity by phosphorylation of either the channel itself or a protein associated to the channel.

The present study was performed to answer the question of whether PI(3,4,5)P3 and/or protein-kinase activity of PI3K are responsible for the stimulation of L-type Ca2+ channels in vascular myocytes. Therefore, we designed experiments challenging the ability of various PIs to stimulate Ca2+ channel currents. We have also used anti-PI(3,4,5)P3 antibody and various PI3Kγ mutants to discriminate between the 2 enzymatic activities of PI3Ks responsible for angiotensin II (Ang II)-activated Ca2+ responses. These experiments provide evidences supporting the essential role of PI(3,4,5)P3 to stimulate Ca2+ channels, whereas serine-kinase activity of PI3K is not required for Ca2+ channel stimulation.

Materials and Methods

Cell Preparation

Isolated myocytes from Wistar rat portal vein were obtained by enzymatic dispersion, as described previously.15 Cells were seeded at density of ≈103 cells/mm2 on glass slides in physiological solution and used on the same day or maintained in short primary culture in M199 medium complemented with 5% fetal calf serum for 24 hours.

Plasmid Constructs and Transfection

Engineering of chimeric mutants of PI3Kγ (accession number X83368) with modified substrate specificity was described in detail previously.14 In brief, a short region within the conserved catalytic domain of PI3Kγ was replaced by synthetic oligonucleotides corresponding to the sequences of PI3Ks of class IV (FKBP12-rapamycin-associated protein [FRAP], a lipid kinase-inactive member of the target-of-rapamycin family). The protein kinase activity was unaffected in this hybrid protein. The point mutation K832R yielded a kinase-deficient PI3Kγ without protein or lipid kinase activities. For permanent membrane attachment, PI3Kγ was extended by a C-terminal isoprenylation signal of K-Ras (CAAX box). GFP constructs were from Clontech (BD Biosciences, Le Pont de Claix, France). GFP and PI3Kγ plasmids were diluted to 0.1 μg/μL and 0.4 μg/μL, respectively, in phosphate-buffered solution and electroporated in freshly isolated rat portal vein myocytes with an electrical field of 0.5 kV/cm for 10 ms generated by the BTX Electrosquare porator (Qbiogene, Illkirch, France). The myocytes were then rapidly resuspended in warmed M199 plus 5% fetal calf serum, seeded on glass slides, and kept in an incubator gassed with 5% CO2 and 95% air for 24 hours.

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 to 4 Mohms. Membrane potential and current records were stored and analyzed using P-clamp system (Axon Instruments). Cell capacitance was determined in each cell tested by imposing 10 mV hyperpolarizing steps from the holding potential (−40 mV). Current density was expressed as the maximum Ba2+ current per cell capacitance unit (pA/pF). All experiments were performed at 30°C±1°C.

Measurements of Cytosolic Ca2+

Cells were loaded by incubation in physiological solution containing 1 μmol/L fura-2/acetoxymethylester for 30 minutes at room temperature. These cells were washed and allowed to cleave the dye to the active fura-2 compound for at least 30 minutes. Fura-2 loading was usually uniform over the cytoplasm. Measurement of cytosolic Ca2+ concentration was performed by the dual-wavelength fluorescence method as described previously.16 Briefly, fura-2-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope IX71 (Olympus). Single cells were alternately excited with ultraviolet light at 340 and 380 nm through a 100× oil immersion objective, and fluorescent light emitted from the Ca2+-sensitive dye was collected through a 510-nm-long pass filter with a charge-coupled device camera CoolSnap HQ (Photometrics). The signal was processed by correcting each fluorescence image for background fluorescence and calculating 340/380-nm fluorescence ratios on a pixel-to-pixel basis by Metafluor software (Roper Scientifique). Averaged frames were usually collected at each wavelength every 0.5 seconds. In some experiments, cells were loaded through a patch-clamp pipette filled with a solution containing 140 mmol/L CsCl, 10 mmol/L HEPES, and 50 μmol/L Fura-2 (pH 7.3). Measurements were made at 25°C±1°C for fura-2/AM-loaded cells and at 30°C±1°C for cells loaded with fura-2 through the patch pipette.

Solutions

The physiological solution used to record Ba2+ currents contained (in mmol/L): 130 NaCl, 5.6 KCl, 1 MgCl2, 5 BaCl2, 11 glucose, and 10 HEPES, and pH 7.4 with NaOH. Ca2+ ions were substituted to Ba2+ for intracellular calcium measurements by omitting BaCl2 and adding 2 mmol/L CaCl2 in the physiological solution. The basic pipette solution used to record Ba2+ currents contained (in mmol/L) : 130 CsCl, 10 EGTA, 5 ATPNa2, 2 MgCl2 and 10 HEPES, and pH 7.3 with CsOH. Chaps (100 μmol/L) was added in the pipette solution to increase phospholipid infusion and had no effect by itself on the current densities (4.7±0.8 pA/pF, n=7 in control conditions versus 4.9±0.7 pA/pF, n=7 in Chaps-containing solution). Phospholipid stock solutions were prepared in 50% H2O/50% DMSO and kept at −20°C in glass containers; they were then diluted into Chaps-containing pipette solution just before use. Ang II was diluted in physiological solution and extracellularly applied to the recorded cell by pressure ejection from a glass pipette.

Chemicals

Angiotensin II was from Neosystem Laboratories (Strasbourg, France). Monoclonal anti-PIP3 antibody (clone RC6F8) was from Molecular Probes (Montluçon, France). PI(3,4,5)P3, PI(3,4)P2, PI(4,5)P2, and PI(3,5)P2 were purchased from Echelon (Salt lake City, Utah). Wortmannin and Neurogranin (W-NG 28-43) were from Sigma (Saint-Quentin Fallavier, France).

Results

Effects of Various PIs on L-type Ca2+ Channels and Intracellular Calcium Concentration

Considering our previous results showing PI3K-mediated L-type Ca2+ channel stimulation, we decided to shunt the enzyme and directly observe the effects of the lipid products to investigate the role of the lipid-kinase activity of PI3K on L-type channel. We measured Ca2+ channel current density in the presence of various lipid products infused through the patch pipette in freshly isolated portal vein myocytes.

As illustrated in Figure 1, intracellular infusion of PI(3,4,5)P3 through the patch pipette resulted in a concentration-dependent increase of the maximal peak Ba2+ current density measured 3 to 4 minutes after breakthrough into the whole-cell recording mode. The same effect was observed with the primary metabolite of PI(3,4,5)P3, ie, PI(3,4)P2. Both PI-derived second messengers were maximally effective in stimulating L-type Ca2+ channel currents at concentrations of 1 to 2 μmol/L. In contrast, PI(3,5)P2, which is produced by a completely independent pathway, was not effective at all in stimulating L-type Ca2+ channel current density as illustrated in Figure 1C. PI(4,5)P2 was also not able to significantly stimulate L-type Ca2+ channel current density (Figure 1C). We noticed nonspecific effects of PIs for concentration higher than 5 to 10 μmol/L, which resulted in a dramatic decrease of Ba2+ current density (data not shown). As already published for Ang II-induced, PI3Kγ-induced, or PKC-induced stimulation of L-type channel current, 7,17–18 PI(3,4,5)P3-induced increase in current density results in an augmentation of current amplitude without altering the current-voltage relationship (data not shown).

Figure 1. Stimulation of Ba2+ current by PI(3,4)P2 et PI(3,4,5)P3 in rat portal vein myocytes. A, Typical Ba2+ currents elicited by membrane depolarizations from −40 mV to +10 mV. Membrane capacitances were 30 pF and 31 pF for the control cell and the cell infused with 1 μmol/L PI(3,4,5)P3 respectively. B, Bar graph illustrating the mean Ba2+ current densities obtained in control conditions or in the intracellular presence of various concentrations of PI(3,4,5)P3 or PI(3,4)P2 as indicated. C, Bar graph illustrating the mean Ba2+ current densities obtained in control conditions or in the intracellular presence of 1 μmol/L PI(4,5)P2 or PI(3,5)P2 as indicated. Data are given as means±SEM with the number of experiments in parentheses. ★, Values significantly different from those obtained in control conditions (P<0.05).

This L-type Ca2+ channel-stimulating effect of PI(3,4,5)P3 prompted us to examine its ability to increase intracellular Ca2+ concentration ([Ca2+]i). Figure 2 illustrates the effects of PI(3,4,5)P3 or PI(3,5)P2 intracellularly applied together with Fura-2 through the patch pipette into myocytes. Figure 2A shows that after breakthrough into the whole-cell recording mode, the fluorescence ratio stabilized within 30 to 40 seconds, corresponding to the delay required for diffusion of the Ca2+ dye Fura-2 from the pipette to the cytoplasm. This ratio was almost stable over the next 5 minutes, although the amount of fluorescence for each wavelength continues to increase during the first 3 minutes. We compared Fura-2 ratios obtained in cells patched with a pipette containing a control solution or a solution containing 1 μmol/L of PI(3,4,5)P3, PI(3,4)P2, or PI(3,5)P2. The results illustrated in Figure 2B and 2C show that PI(3,4,5)P3 and PI(3,4)P2 increase Fura-2 ratio, measured 3 minutes after breakthrough into the whole-cell recording mode, whereas PI(3,5)P2 does not modify the ratio when compared with the one obtained with the control pipette solution.

Figure 2. Effects of PIs on basal calcium levels. A and B, Typical traces of Fura-2 ratio obtained in voltage-clamped (holding potential −50 mV) portal vein myocytes after breakthrough into the whole-cell recording mode with a control pipette solution (A) or a pipette solution containing 1 μmol/L of either PI(3,4,5)P3 or PI(3,5)P2 (B). Triangles at the beginning of the traces show the time-point of seal break, allowing the pipette solution to enter the cytoplasm of the cell. C, Bar graph showing the mean Fura-2 ratios measured 3 minutes after breakthrough into the whole-cell recording mode with or without PIs in the pipette solution. Data are given as means±SEM, with the number of experiments in parentheses. ★, Values significantly different from those obtained in control conditions (P<0.05).

Effects of Anti-PIP3 Antibody on Ang II-Induced Stimulation of Ba2+ Currents and Calcium Responses

We have previously shown that Ang II-induced stimulation of Ca2+ channel and increase in [Ca2+]i were blocked by wortmannin and anti-PI3Kγ antibody.7,17 Moreover, PI3Kγ was able to mimic Ang II-induced stimulation of Ca2+ channel.7 Here, we checked whether these Ang II-activated responses were dependent on PI(3,4,5)P3 production. In Figure 3, we show that 10 μg/mL anti-PI(3,4,5)P3 antibody (anti-PIP3 Ab) completely inhibited Ang II-induced stimulation of L-type Ca2+ channel current. This inhibition was specific because intracellular infusion of the boiled anti-PIP3 Ab did not prevent the stimulation of Ba2+ current by Ang II. Moreover, we have previously shown that intracellular infusion of 10 μg/mL antibodies targeted against irrelevant proteins, ie, PI3Kα, or proteins that are not expressed in myocytes, ie, PI3Kβ, did not prevent Ang II-induced stimulation of Ca2+ channel currents.17

Figure 3. Inhibition of Ang II-induced stimulation of L-type Ca2+ channel by anti-PIP3 antibody (anti-PIP3 Ab). A, Typical Ba2+ currents elicited by depolarizations to +10 mV from a holding potential of −40 mV before (Ic) and during application of 100 nM Ang II (IAII) in a control cell (capacitance 32 pF) and in the intracellular presence of 10 μg/mL anti-PIP3 Ab (cell capacitance 30 pF). B, Compiled data showing the effects of anti-PIP3 Ab and the boiled antibody on the stimulation of Ca2+ channel by Ang II (IAII/Ic). Data are given as means±SEM, with the number of experiments in parentheses. Peak Ba2+ currents were measured 4 to 5 minutes after breakthrough into the whole-cell recording mode and expressed as a fraction of the control current measured before ejection of Ang II (IAII/Ic).

A peptide derived from neurogranin (W-NG28–43 peptide) previously described to bind to and scavenge PI(3,4,5)P319 has also been assessed for its ability to inhibit Ang II-induced stimulation of Ca2+ channel. W-NG28–43 peptide did inhibit the Ang II-induced increase in Ca2+ channel current; however, the peptide also inhibited the unstimulated Ba2+ currents, which invalidates its use in our experiments (not shown).

To further explore the role of PI(3,4,5)P3 in Ang II-mediated effects, we used the same anti-PI(3,4,5)P3 antibody in Ca2+ measurement experiments. As shown in Figure 4, increasing concentrations of anti-PIP3 Ab decreased the Ang II-induced Ca2+ responses without modifying the basal intracellular calcium concentration. Unspecific effect of the antibody is unlikely because the boiled antibody failed to decrease Ang II-induced Ca2+ signals. Another control is provided in Figure 4C, showing that the same antibody did not change the Ca2+ responses activated on the same cell batches of portal vein myocytes by norepinephrine (NE). The NE-induced Ca2+ responses have been largely studied and characterized on these cells as a classical Gq-PLC-IP3-mediated Ca2+ release mechanism from the endoplasmic reticulum. This additional control provides evidence that the anti-PIP3 Ab is specific, because it did not block PI(4,5)P2-hydrolysis by PLC on stimulation of the myocytes by NE.

Figure 4. Effect of anti-PIP3 Ab on Ang II-induced and norepinephrine (NE)-induced Ca2+ responses. A, Increases in [Ca2+]i (illustrated by increase in Fura-2 ratio) induced by Ang II (100 nM) in voltage-clamped (−50 mV) single myocytes, in control conditions, and in the presence of the intact or boiled anti-PIP3 Ab (10 μg/mL). B, Effect of anti-PIP3 Ab on both the basal Fura-2 ratio (left panel) and the Ang II-induced calcium responses (right panel, change in Fura-2 ratio) measured as the difference between the resting basal level and the peak of the Ca2+ transient. C (left panel), increases in Fura-2 ratio induced by NE (10 μmol/L) in voltage-clamped (−50 mV) single myocytes, in control conditions, and in the presence of anti-PIP3 Ab (10 μg/mL). C (right panel), Mean effect of anti-PIP3 Ab on the NE-induced change in Fura-2 ratio. Data are given as means±SEM, with the number of experiments in parentheses. ★, Values significantly different from those obtained in control conditions. Fura-2 ratios were measured 4 to 5 minutes after breakthrough into the whole-cell recording mode.

PI3Kγ Lipid-Kinase-Mediated Effects of Ang II

Experiments performed with anti-PIP3 Ab strongly suggest that PI(3,4,5)P3 is required for Ang II-induced Ca2+ responses; we have further checked this hypothesis by using PI3Kγ mutants that are lacking either the lipid-kinase activity (PI3KγFRAP) or both the lipid-kinase and serine-kinase activity (PI3KγKR). These mutants in their membrane targeted form (-CAAX) were expressed in native portal vein myocytes, which were then challenged for their ability to respond to Ang II.

As shown in Figure 5, the expression of PI3Kγ-CAAX did not modify the Ang II-induced Ca2+ responses in native myocytes. Basal Fura-2 ratios (0.82±0.03, n=5 in untransfected cells versus 0.85±0.06, n=5 in PI3Kγ-CAAX transfected cells) were not affected by PI3Kγ-CAAX expression. The bar graph (Figure 5C) shows that the Ang II-induced Ca2+ responses display the same amplitude and sensitivity to dihydropyridine (oxodipine) than in the untransfected cells, indicating that the trigger signal activated by Ang II corresponds to Ca2+ entry through L-type Ca2+ channels as previously described on this cell type. On the same cells, oxodipine did not inhibit the NE-induced Ca2+ release from IP3-sensitive Ca2+ stores (Figure 5A and 5B).

Figure 5. Oxodipine-sensitivity of Ang II-induced Ca2+ responses. Increases in [Ca2+]i (illustrated as change in Fura-2 ratio) induced by Ang II (100 nM) in single myocytes untransfected (A) or transfected with the membrane-targeted PI3Kγ (PI3Kγ-CAAX) (B), in control conditions (left panels), and in the presence of 10 μmol/L oxodipine to inhibit L-type channels. NE (10 μmol/L)-induced Ca2+ response was tested as a control after oxodipine treatment in transfected and in untransfected cells. C, Bar graph illustrating the effect of oxodipine on Ang II-induced Ca2+ responses in untransfected cells or cells transfected with PI3Kγ-CAAX. Cells were not patch-clamped. Data are given as means±SEM, with the number of experiments in parentheses. ★, Values significantly different from the control values (P<0.05).

Transfection of both FRAP and KR mutants of PI3Kγ clearly inhibited Ang II-induced Ca2+ responses obtained in untransfected cells from the same slides (Figure 6). In contrast, expression of the membrane-targeted PI3Kγ-CAAX or GFP alone did not affect the Ang II-induced change in Fura-2 ratio when compared with untransfected cells. These results suggest that the lipid-kinase activity of the PI3Kγ is required and that the serine-kinase activity alone (expression of PI3KγFRAP-CAAX) is not sufficient for transducing the Ang II-induced response. Further suppression of the serine-kinase activity in addition to suppression of the lipid-kinase activity (expression of PI3KγKR-CAAX) did not lead to a stronger inhibitory effect on Ang II-induced Ca2+ response (Figure 6).

Figure 6. Effects of PI3K mutants on Ang II-induced increase in [Ca2+]i. A, Typical traces of increases in [Ca2+]i (illustrated as change in Fura-2 ratio) induced by Ang II (100 nM) in single myocytes transfected with PI3Kγ-CAAX, a lipid-kinase-inactive mutant PI3KγFRAP-CAAX, or lipid-kinase and serine-kinase-deficient mutant PI3KγKR-CAAX. B, Bar graph compiling data illustrating the effects of PI3K mutants on Ang II-induced Ca2+ responses. Responses obtained in transfected cells were compared with responses obtained in untransfected cells from the same slides. Cells were not patch-clamped. Data are given as means±SEM, with the number of experiments in parentheses.★, Values significantly different from the control values (P<0.05).

Discussion

Our results show that among the 2 enzymatic activities of PI3K, the lipid-kinase activity is responsible for stimulation of Ca2+ channel and increase in [Ca2+]i induced by Ang II on smooth muscle myocytes. The main findings leading to this conclusion are: (1) the lipid product of PI3K is able to stimulate L-type Ca2+ channel current and to increase [Ca2+]i; (2) a specific anti-PIP3 Ab inhibits Ang II-induced Ca2+ channel stimulation and Ca2+ responses; and (3) mutant of PI3Kγ expressing only the serine-kinase activity is not able to transduce Ang II-induced Ca2+ responses.

The first finding is supported by experiments in which various PIs were assayed for their ability to stimulate L-type Ca2+ channel current densities. Intracellular infusion of either PI(3,4,5)P3 or PI(3,4)P2 resulted in a concentration-dependent increase of the maximal peak current density that was not observed with PI(3,5)P2 or PI(4,5)P2. Moreover, PI(3,4,5)P3 was able to increase basal [Ca2+]i, whereas PI(3,5)P2 did not change [Ca2+]i. These results are concordant with previous results showing that the PI3K-mediated stimulation of Ca2+ channels underlies the increase in [Ca2+]i observed on Ang II stimulation of the cells.7,17

In these experiments, PI(3,5)P2 and PI(4,5)P2 have been used as controls for PI(3,4,5)P3-mediated/PI(3,4)P2-mediated effects for different reasons. PI(3,5)P2 differs from the 4-OH-phosphorylated PIs by its synthesis pathway and cellular localization because it is formed and localized in endosomal intracellular membranes, whereas PI(4,5)P2, PI(3,4,5)P3, or PI(3,4)P2 are localized at the plasma membrane.20 PI(3,5)P2 is the result of PI(3)P phosphorylation by a PIP 5-kinase, whereas PI(3,4,5)P3 and PI(3,4)P2 result from PI3K-dependent phosphorylation of PI(4)P and PI(4,5)P2.3 A substantial fraction of cellular PI(3,4)P2 results from further dephosphorylation of PI(3,4,5)P3 by type II 5-phosphatases. Although type II 5-phosphatases are able to reduce some PI3K-mediated effects, they are not always terminating signals.3 Rather, they have the unique potential to shift PI3K-generated signals to other pathways or to maintain the intracellular effects of PI(3,4,5)P3 when the target of the second messenger is also activated by PI(3,4)P2. This is the case for some isoforms of PDK1 and PKC that might be activated by either PI(3,4,5)P3 or PI(3,4)P2.21,22 Our results suggest that the PI3K-mediated stimulation of Ca2+ channels requires an intermediate effector, which can be activated by lipid second messengers, ie, PI(3,4,5)P3 or PI(3,4)P2. This is in agreement with previous results showing that the stimulation of Ca2+ channel by Ang II, Gβγ, or recombinant PI3Kγ are blocked by PKC inhibitors but not by PKA inhibitors.7,18

PI(4,5)P2 has also been tested in its ability to stimulate L-type Ca2+ channels for several reasons. First, it has been shown to have complex and intriguing effects on a wide range of ion channels and transporters.23 Second, PI(4,5)P2 is the preferential substrate of PI3K and could thereby increase the basal activity of endogenous PI3K. However, it was not able to significantly increase L-type Ca2+ channel current density when infused into primary smooth muscle cells. The lack of effect of PI(4,5)P2 on L-type Ca2+ channel current density suggests that the basal activity of endogenous PI3Kγ is very low in portal vein myocytes and that it has to be stimulated by Gβγ to significantly increase intracellular PI(3,4,5)P3 concentration, as already suggested in a previous study.8 These results also contrasts with the ability of PI(4,5)P2 to modulate N-type and P-type/Q-type of Ca2+ channels, as described by Wu et al.24 The authors proposed a model of N-type or P-type/Q-type Ca2+ channels displaying 2 PI(4,5)P2 binding sites: 1 high-affinity binding site that confers stability and 1 low-affinity binding site that confers reluctant voltage-gated properties to the channels. Our results suggest that L-type Ca2+ channels may lack PI(4,5)P2-binding sites because acute intracellular PI(4,5)P2 application is not sensed as a stimulatory second messenger by the Ca2+ channel (or Ca2+ channel-regulating effector proteins), and Ang II-induced decrease in PI(4,5)P2 concentration by PI3K-dependent phosphorylation of PI(4,5)P2 into PI(3,4,5)P3 leads to a stimulation of L-type Ca2+ channel current, which contrasts with the inhibition of N-type current observed on luteinizing hormone releasing hormone-induced hydrolysis of PI(4,5)P2.24

Our second finding is based on the inhibition of the effect of Ang II by infusing a specific anti-PIP3 antibody able to scavenge PI(3,4,5)P3 into the cells and leading to the conclusion that PI(3,4,5)P3 was required for a full effect of Ang II. This antibody shows 30-fold selectivity for PI(3,4,5)P3 versus PI(4,5)P225 and immunofluorescent staining with this anti-PIP3 Ab showed a distribution identical to that of PH-Akt-GFP.26 This antibody clearly inhibited both Ang II-induced stimulation of Ca2+ channel and Ang II-induced Ca2+ responses. Interestingly, the same antibody was unable to inhibit NE-induced Ca2+ responses on the same batches of cells. These results are in agreement with the specificity of the antibody toward the various PIs, as previously shown by Chen et al,25 and with a series of previous results showing that Ang II and NE use 2 separate pathways to increase [Ca2+]i in venous myocytes. Ang II activates a G13/PI3K/L-type Ca2+ channel-dependent influx of Ca2+, whereas NE transduces through the classical Gq/PLC/IP3R-dependent Ca2+ release.16,18,27–29

The third argument in favor of the essential role of PI(3,4,5)P3 in generating Ca2+ signals is the use of lipid-kinase inactive mutants of PI3Kγ. We show here that expression of PI3Kγ-CAAX in venous myocytes did not significantly change both the basal [Ca2+]i and Ang II-induced Ca2+ responses. This contrasts with studies showing that expression of the membrane targeted form of PI3Ks increases the basal activity of PI3K14 and decreases hormone-induced PI3K-mediated cellular responses.30 However, these latter studies have been performed in cell cultures starved of serum before experiments to reveal the CAAX box-related activity of PI3K in resting cells. In contrast, in the present study, we used freshly isolated cells that require serum to fix to the glass slide. In addition, [Ca2+]i is highly controlled and regulated by a wide range of ion exchanges, channels, and pumps to maintain intracellular Ca2+ homeostasis, which is crucial for cell survival. Therefore, the downstream effect of the expected long-lasting increased basal activity of PI3Kγ-CAAX is not visible in terms of variation of basal [Ca2+]i, although it is visible during the acute application of PI(3,4,5)P3 through the patch pipette. The present experiments also suggest that the membrane-targeted PI3Kγ-CAAX is further stimulated by Gβγ on Ang II stimulation. This is in agreement with results showing that PI(3,4,5)P3 production measured by intracellular PIP3-sensor in PI3Kγ-CAAX transfected HEK cells is stimulated by GPCR stimulation.11

The main result from this set of experiments is that the lipid-kinase inactive mutant largely inhibited Ang II- induced Ca2+ responses, which were not further decreased by the serine-kinase inactive mutant. These results are concordant with previous results showing that stimulation of L-type Ca2+ channel by Ang II is independent of MAPK,17 with this latter enzyme being phosphorylated and activated by the serine-kinase activity of PI3Kγ.14 The effect of the lipid-kinase inactive mutant of PI3Kγ is in good agreement with the fact that PI(3,4,5)P3 is able, on its own, to reproduce the effect of Ang II on both Ca2+ channel and Ca2+ response. Therefore, in contrast to the antilipolytic effect of insulin in adipocytes that requires both lipid-kinase and serine-kinase activities of PI3K,31 Ang II seems to require only the lipid-kinase activity of PI3K to elicit a complete Ca2+ response in vascular myocytes.

Although an increasing number of studies describe PI3K-dependent regulation of Ca2+ entries, only 1 study so far has clearly identified that PI(3,4,5)P3 activates a Ca2+ entry through a receptor-activated Ca2+ channel in Jurkat T cells.32 In excitable cells, PI3K has been shown to stimulate voltage-gated Ca2+ channels, for example, in neurons,6 in vascular myocytes,7–8,17,33 and, recently, in neonatal cardiomyocytes.34 However, the present study is the first proposal of a dual approach to resolve the role of PI(3,4,5)P3 in these stimulatory mechanisms of voltage-gated Ca2+ channels. Together, reconstitution and inhibitory strategies lead to the conclusion that PI(3,4,5)P3 is necessary and sufficient to transduce hormone-activated and PI3K-dependent regulation of Ca2+ entry through voltage-gated Ca2+ channels.

Original received February 25, 2004; revision received June 18, 2004; accepted June 24, 2004.

The authors thank Dr. J. Mironneau for his support, critical reading of the manuscript, and helpful discussions. This work was supported by grants from the Conseil Régional d’Aquitaine and the Centre National de la Recherche Scientifique, France.

Footnotes

Correspondence to Nathalie Macrez, Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail

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