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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2012 Aug;166(7):2161–2175. doi: 10.1111/j.1476-5381.2012.01937.x

Pharmacological profile of phosphatidylinositol 3-kinases and related phosphatidylinositols mediating endothelinA receptor-operated native TRPC channels in rabbit coronary artery myocytes

J Shi 1, M Ju 1, WA Large 1, AP Albert 1
PMCID: PMC3402779  PMID: 22404177

Abstract

BACKGROUND AND PURPOSE

EndothelinA (ETA) receptor-operated canonical transient receptor potential (TRPC) channels mediate Ca2+ influx pathways, which are important in coronary artery function. Biochemical pathways linking ETA receptor stimulation to TRPC channel opening are unknown. We investigated the involvement of phosphatidylinositol 3-kinases (PI3K) in ETA receptor activation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels in rabbit coronary artery vascular smooth muscle cells (VSMCs).

EXPERIMENTAL APPROACH

A pharmacological profile of PI3K was created by studying the effect of pan-PI3K, pan-Class I PI3K and Class I PI3K isoform-selective inhibitors on ETA receptor-evoked single TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches from rabbit freshly isolated coronary artery VSMCs. The action of phosphatidylinositol 3-phosphate- [PI(3)P], 4-phosphate- [PI(4)P] and 5-phosphate- [PI(5)P] containing molecules involved in PI3K-mediated reactions were studied in inside-out patches. Expression of PI3K family members in coronary artery tissue lysates were analysed using quantitative PCR.

KEY RESULTS

ETA receptor-operated TRPC1/C5/C6 and TRPC3/C7 channel activities were inhibited by wortmannin. However, ZSTK474 and AS252424 reduced ETA receptor-evoked TRPC1/C5/C6 channel activity but potentiated TRPC3/C7 channel activity. All the PI(3)P-, PI(4)P- and PI(5)P-containing molecules tested induced TRPC1/C5/C6 channel activation, whereas only PI(3)P stimulated TRPC3/C7 channels.

CONCLUSIONS AND IMPLICATIONS

ETA receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channel activities are likely to be mediated by Class I PI3Kγ and Class II/III PI3K isoforms, respectively. ETA receptor-evoked and constitutively active PI3Kγ-mediated pathways inhibit TRPC3/C7 channel activation. PI3K-mediated pathways are novel regulators of native TRPC channels in VSMCs, and these signalling cascades are potential pharmacological targets for coronary artery disease.

Keywords: canonical transient receptor potential, endothelin, phosphatidylinositol 3-kinase, phosphatidylinositol, vascular smooth muscle

Introduction

Endothelin-1 (ET-1) is one of the most potent endogenous vasoconstrictors identified so far, and also it is associated with vascular remodelling and angiogenesis (Nguyen et al., 2010; Thorin and Webb, 2010). ET-1 acts at two GPCR subtypes, ETA and ETB, which are expressed on the plasmalemma of vascular smooth muscle cells (VSMCs). ETA receptors are thought to be predominantly involved in ET-1-evoked contraction, proliferation and pro-inflammatory effects on VSMCs. Blockade of ETA receptors increases blood flow and perfusion and reduces fatty streaks and endothelial dysfunction in coronary arteries, and therefore pharmacological agents that reduce ETA receptor-mediated pathways are candidates for prevention and treatment of coronary artery disease (Nguyen et al., 2010; Thorin and Webb, 2010). The significance of the present work is in further understanding of these ETA receptor-mediated signalling pathways, which enhances our knowledge of potential therapeutic targets for coronary artery disease.

It is evident that ETA receptor-mediated vascular effects are correlated with a rise in [Ca2+]i of VSMCs, which involves a significant contribution by Ca2+ influx (Tykocki and Watts, 2010). There is no clear picture on the mechanisms governing ETA receptor-mediated Ca2+ influx in VSMCs, although activation of canonical transient receptor potential (TRPC) channel proteins has been proposed (Bergdahl et al., 2003; Ko et al., 2004; Tykocki and Watts, 2010).

The family of TRPC1-C7 channel subunits form Ca2+-permeable non-selective cation channels, which are important regulators of Ca2+ influx in VSMCs (Abramowitz and Birnbaumer, 2009; Dietrich et al., 2010). It is apparent that two TRPC channel subgroups are expressed in VSMCs; TRPC1 channels (channels containing TRPC1 subunits) and channels composed of TRPC3/C6/C7 subunits (Albert et al., 2009; Large et al., 2009; Albert, 2011), with these two TRPC channel subgroups having distinctive activation mechanisms. TRPC1 channels are activated by receptor- and intracellular Ca2+ store-dependent pathways, which involve PKC-dependent phosphorylation of TRPC1 proteins, and obligatory roles for the phosphatidylinositols (PIs), PI 4,5-bisphosphate [PI(4,5)P2] and PI 3,4,5-trisphosphate [PI(3,4,5)P3] (Albert and Large, 2002; Saleh et al., 2006; 2008; 2009a,b; Albert et al., 2009; Large et al., 2009; Albert, 2011). TRPC3/C6/C7 channels are activated by a GPCR or constitutively active pathways, which involve phospholipase-mediated generation of DAG that induces channel opening via PKC-independent mechanisms (Helliwell and Large, 1997; Inoue et al., 2001; Albert et al., 2003; 2005; 2006; Peppiatt-Wildman et al., 2007; Large et al., 2009; Albert, 2011). DAG is proposed to activate TRPC3/C6/C7 channels by removal of an inhibitory action of PI(4,5)P2, which is bound to channel proteins at rest (Albert et al., 2008; Large et al., 2009; Ju et al., 2010; Albert, 2011). Moreover, TRPC3/C6/C7 channels are inhibited by PKC stimulation, which is produced by receptor-mediated generation of DAG (Albert and Large, 2004; Saleh et al., 2006) and TRPC1 channel-mediated Ca2+ influx (Shi et al., 2010). As such, TRPC3/C6/C7 channels are only activated by lower concentrations of receptor agonists, whereas TRPC1 channels are induced by a wide range of receptor stimuli concentrations (Large et al., 2009; Shi et al., 2010; Albert, 2011).

In coronary artery VSMCs, differences between biophysical properties, blocking actions of anti-TRPC antibodies, pharmacology, and co-immunoprecipitation indicate that stimulation of ETA receptors by low concentrations of ET-1 (1–10 nM) activate two distinct native TRPC channels composed of TRPC1/C5/C6 with a unitary conductance of about 3 pS (Saleh et al., 2008; 2009b; Albert et al., 2009) and TRPC3/C7 channels with four sub-conductance states between 15 and 70 pS (Peppiatt-Wildman et al., 2007; Albert et al., 2009). In contrast, ETA receptor activation with higher concentrations of ET-1 (100 nM) only activates TRPC1/C5/C6 channels (Saleh et al., 2009b). ETA receptor-induced TRPC1/C5/C6 channel activation is governed by phosphatidylinositol 3-kinase (PI3K)-mediated generation of PI(3,4,5)P3 whereas stimulation of these channels by ETB receptor activation requires PI(4,5)P2 (Saleh et al., 2009b). It is unclear how ETA receptors activate TRPC3/C7 channels (Peppiatt-Wildman et al., 2007).

The PI3K family consists of three classes of isoforms (Classes I, II and III), which phosphorylate PI, PI 4-phosphate [PI(4)P], PI 5-phosphate [PI(5)P] and PI(4,5)P2 at their D3 hydroxyl groups to generate, respectively, PI(3)P, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3 (Hawkins et al., 2006; Vanaesebroeck et al., 2010). PI3K-dependent pathways are considered to be important in vascular physiology and pathology (Oudit et al., 2004; Morello et al., 2009) and are proposed to mediate ET-1-evoked Ca2+ influx pathways (Kawanabe et al., 2004; Miwa et al., 2005; Ivey et al., 2008). The present work investigates the role of PI3K isoforms using selective pharmacological inhibitors, and associated PI substrates and products, in ETA receptor stimulation of native TRPC1/C5/C6 and TRPC3/C7 channels in freshly isolated coronary artery VSMCs.

Evidence was obtained showing that PI3K-mediated signalling pathways are pivotal in ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activation. The Class I PI3Kγ isoform are likely to couple ETA receptors to TRPC1/C5/C6 channel opening, whereas Class II and/or Class III PI3K isoforms are likely to mediate ETA receptor stimulation of TRPC3/C7 channels. Moreover, we showed for the first time that PI(3)P-, PI(4)P- and PI(5)P-containing molecules are novel gating ligands of native TRPC1 and TRPC3/C6/C7 channel subtypes.

Methods

All terms used for receptors, ion channels and enzymes are in accordance with the nomenclature of Alexander et al. (2011).

Cell isolation

All animal care and procedures were approved by St. George's, University of London animal welfare committee. Animals were allowed free access to food and water and kept on a 12–12 h light/dark cycle. New Zealand White rabbits (2–3 kg) were killed using i.v. sodium pentobarbitone (120 mg·kg–1) in accordance with the UK Animals Scientific Procedures Act, 1986. Right and left anterior descending coronary arteries were dissected free from fat and connective tissue, and the endothelium was removed with a cotton bud. Physiological salt solution contained (mM): NaCl (126), KCl (6), glucose (10), HEPES (11), MgCl2 (1.2), CaCl2 (1.5) and pH to 7.2 with 10 M NaOH. Enzymatic isolation of VSMCs was carried out using methods previously described (Peppiatt-Wildman et al., 2007; Saleh et al., 2008; 2009b).

Electrophysiology

Single channel cation currents using cell-attached and inside-out patches were made with an AXOpatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) at room temperature (20–23°C) as previously described (Peppiatt-Wildman et al., 2007; Saleh et al., 2008; 2009b). Single channel I/V relationships were obtained by manually altering the holding potential of −70 mV between −120 mV and +120 mV. Single TRPC1 and TRPC3/C6/C7 channel subgroups were analysed according to Shi et al. (2010). Single channel currents records were filtered at 0.1–0.5 kHz (-3 dB, low pass 8-pole Bessel filter, Frequency Devices, model LP02, Scensys Ltd., Aylesbury, UK) and acquired at 1–5 kHz [Digidata 1322A (Axon Instruments Inc., Union City, CA, USA) and pCLAMP v9.0 software (Molecular Devices, LLC, Sunnyvale, CA, USA)]. Single channel current amplitudes were calculated from idealized traces of ≥30 s in duration using the 50% threshold method and analysed using pCLAMP v9.0 software. Events lasting between <1.32 and <6.64 ms (×2 rise time of filtering used, see above) were excluded during creation of idealized traces to maximize the number of channel openings reaching their full current amplitude. Open probability (NPo) was used as a measure of channel activity and was calculated automatically by pCLAMP v9.0 software. Single channel current amplitude histograms were plotted from the event data of the idealized traces, using bin widths appropriate for the unitary amplitudes. Amplitude histograms were fitted using Gaussian curves, with peak values corresponding to channel open levels. Mean channel amplitudes at different membrane potential were plotted, and current–voltage relationships curves were fitted by linear regression with the gradient-determining conductances values. Figure preparation was carried out using MicroCal Origin software 6.0 (Originlab Corporation, Northampton, MA, USA) where inward single cation channel openings are shown as downward deflections.

Dot-blots

Dot-blots were performed using techniques described previously (Saleh et al., 2009b). Rabbit coronary arteries were mixed with radioimmunoprecipitation assay buffer (Sigma-Aldrich Company Ltd., Gillingham, UK) containing protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK) and sonicated at 4°C for 3 h. Tissue lysates were centrifuged at 6200×g at 4°C for 10 min, and 10 µL of tissue lysate supernatant was added to a PVDF membrane. Membranes were dried and placed into 5% blocking buffer and left on a rocker at room temperature for 1 h. Membranes were incubated with appropriate primary antibodies overnight at 4°C. Following removal of primary antibodies, PVDF membranes were washed for 1 h with PBS and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody diluted 1:5000. After three washes in PBS containing 0.1% Tween, PVDT membranes were treated with ECL chemiluminescence reagents (Pierce Biotechnology Inc., Rockford, IL, USA) for 1 min and exposed to photographic films.

RNA extraction and cDNA synthesis

Total RNA was extracted from rabbit fresh enzymatically-dispersed coronary arteries using the RNAqueous Small Phenol-Free Total RNA Isolation Kit (Life Technologies, Paisley, UK) according to the manufacturer's instruction. RNA quality was measured using Nanodrop ND1000 spectrophotometer (Thermo Scientific, Loughborough, UK) and RNA reverse-transcribed to cDNA using High Capacity RNA-to-cDNA Kit (Life Technologies). Negative controls were performed in the absence of reverse transcriptase (-RT) to check for genomic contamination.

End-point PCR

End-point PCR was performed using GoTag® DNA Polymerase (Promega, Southampton, UK) and under the following conditions: initial denaturation at 94°C for 2 min; PCR cycles: 94°C for 30 s, 55°C for 30 s and 72°C for 30 s; repeated for 40 cycles; final extension for 10 min. PCR product was checked on 1% agarose gel electrophoresis. If there were no visible bands, 5 µL of the PCR product was used as a template to perform a second round PCR with 20 cycles of 94°C for 30 s and 55°C for 30 s and 72°C for 30 s using the same pair of primers and same initial denaturation and final extension times. Negative control with no template was performed to check for contamination. PCR product amplified was confirmed by sequence analysis (Beckman Coulter Genomics, High Wycombe, UK) and checked for human analogues using the National Center for Biotechnology Information Basic Local Alignment Search Tool programme.

qPCR

qPCR was performed with The QuantiFast SYBR Green PCR Kit (Qiagen, Crawley, UK) and using a CFX96™ Real-Time PCR Detection System (Bio-Rad, Hemal Hempstead, UK). Duplicate reactions were carried out in 20 µL volumes including 1 µL of cDNA, 10 µL of SYBR Green Master Mix (Qiagen), 2 µL of sense primer and 2 µL of anti-sense primer. The cycling conditions were as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 10 s, combined annealing and extension at 65°C for 30 s. Melt curve analysis was performed to ensure that each primer set amplified a single product that shows a single peak in the melt curve. No template controls were applied to check for contamination. Cycle threshold (Ct) values were calculated using CFX65™ Manager Software (Bio-Rad). Standard curves were plotted using fourfold serial dilution of cDNA to determine the efficiency of amplification and R2 values. The expression of RNA relative to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated using equation 2-ΔCt.

Primers

The housekeeping genes β-actin and GAPDH were used as references. Primers (see Tables S1 and S2 in supplementary data) were designed using Primer 3 software. All primers designed flank a region that contains at least one intron to avoid genomic DNA contamination. All primers were synthesized by Invitrogen (Life Technologies).

Solutions and drugs

In cell-attached patch experiments, the membrane potential was set to approximately 0 mV by perfusing cells in a KCl external solution containing (mM): KCl (126), CaCl2 (1.5), HEPES (10) and glucose (11), and pH to 7.2 with 10 M KOH. Nicardipine (5 µM) was also included to prevent smooth muscle cell contraction by blocking Ca2+ entry through voltage-dependent Ca2+ channels. The bathing solution used in inside-out experiments (intracellular solution) contained (mM): CsCl (18), caesium aspartate (108), MgCl2 (1.2), HEPES (10), glucose (11), BAPTA (1), CaCl2 (0.48, free internal Ca2+ concentration approximately 100 nM as calculated using EQCAL software; Biosoft, Cambridge, UK), Na2ATP (1), NaGTP (0.2); pH 7.2 with Tris. The patch pipette solution used for both cell-attached and inside-out patch recording (extracellular solution) was K+ free and contained (mM): NaCl (126), CaCl2 (1.5), HEPES (10), glucose (11), TEA (10), 4-AP (5), iberiotoxin (0.0002), 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (0.1), niflumic acid (0.1) and nicardipine (0.005); pH to 7.2 with NaOH. The use of blockers of voltage dependent calcium channels (nicardipine), K+ currents (TEA, 4-AP, iberiotoxin), swell-activated Cl- currents (DIDS) and Ca2+-activated Cl- conductances (niflumic acid) allowed non-selective cation currents to be recorded in isolation.

All PI molecules were ordered in a water soluble diC8 form from Echelon Biosciences (Salt Lake City, UT, USA). Unless otherwise stated, all other drugs were purchased from Calbiochem (Nottingham, UK), Sigma Aldrich or Tocris (Bristol, UK), and agents were dissolved in distilled H2O or dimethyl sulfoxide (DMSO) (0.1%). DMSO alone had no effect on channel activity.

Statistical analysis

The values are presented as the mean of n cells ± SEM. Statistical analysis was carried out using Student's paired (comparing effects of agents on the same cell) or unpaired (comparing effects of agents between cells) t-test with the level of significance set at P < 0.05.

Results

Effect of wortmannin on two distinct ETA receptor-operated native TRPC channels in coronary artery VSMCs

In 46/80 cell-attached patches, stimulation of ETA receptors by bath application of 10 nM ET-1 (in the presence of the ETB receptor antagonist BQ788, 100 nM) evoked two distinct native cation channel currents in freshly isolated coronary artery VSMCs (Figure 1A). One channel had a unitary conductance of about 3 pS, whereas the second had four sub-conductance levels between 15 and 70 pS (data not shown). Both 3 pS and 15–70 pS channels had reversal potentials (Er) of about 0 mV. We have previously described the properties of these two channels and shown that the 3 pS channel is composed of heteromeric TRPC1/C5/C6 subunits (Saleh et al., 2008; 2009b), whereas the 15–70 pS channel consists of a heteromeric TRPC3/C7 channel structure (Peppiatt-Wildman et al., 2007). In the remaining 34/80 cell-attached patches, stimulation of ETA receptors with 10 nM ET-1 (also in the presence of 100 nM of BQ788) only evoked 3 pS channel currents (see Figure 2B). Stimulation of ETA receptors either activated TRPC1/C5/C6 and TRPC3/C7 channels or only TRPC1/C5/C6 channels is likely to be due to differences in expression levels of these two channel subtypes; not all patches contain TRPC3/C7 channels. Also, ETA receptor-mediated pathways may inhibit TRPC3/C7 channel activity as previously described for angiotensin II in mesenteric artery VSMCs (Saleh et al., 2006; Shi et al., 2010).

Figure 1.

Figure 1

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Effect of wortmannin on ETA receptor-evoked native TRPC1/C5/C6 and TRPC3/C7 channel activities in rabbit coronary artery VSMCs. (A) Bath application of 10 nM ET-1, in the presence of the ETB receptor antagonist 100 nM BQ788, activated TRPC1/C5/C6 and TRPC3/C7 channels in cell-attached patches held at −70 mV, which were unaffected by U73122 but blocked by wortmannin (Wort). Bold and dashed lines in insets show, respectively, closed and open levels. (B) and (C) Mean data showing that wortmannin significantly inhibited both ETA receptor-mediated TRPC1/C5/C6 and TRPC3/C7 channel activities. n≥ 6, **P < 0.01.

Figure 2.

Figure 2

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Differential effects of the selective pan-Class I PI3K inhibitor ZSTK474 on ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities. (A) ZSTK474 inhibited TRPC1/C5/C6 and potentiated TRPC3/C7 channel activities evoked by ETA receptor stimulation in cell-attached patches held at −70 mV. Moreover, ETA receptor-mediated TRPC3/C7 channel activity in the presence of ZSTK474 was inhibited by wortmannin. Note that the middle inset shows that TRPC1/C5/C6 channel openings are absent in the presence of ZSTK474. (B) Illustration of a cell-attached patch in which ETA receptor stimulation only induced TRPC1/C5/C6 channel activity, which was inhibited by ZSTK474. (C) Mean data showing that ZSTK474 significantly inhibited TRPC1/C5/C6 but potentiated TRPC3/C7 channel activities evoked by ETA receptor stimulation. n≥ 5, *P < 0.05, **P < 0.01.

It is generally considered that stimulation of TRPC channels in VSMCs is mediated by activation of GPCRs linked to phospholipase-dependent signalling pathways and generation of DAG, which activates channels via PKC-independent and -dependent mechanisms (see Introduction). However, PI3K-mediated processes have been proposed to mediate ET-1-evoked Ca2+ influx (Kawanabe et al., 2004; Miwa et al., 2005; Ivey et al., 2008) and ETA receptor-evoked TRPC1/C5/C6 channels in VSMCs (Saleh et al., 2009b). We compared the effect of PLC and PI3K inhibitors on ETA receptor-evoked TRPC1/C5/C6 and TRPC3/7 channel activities in cell-attached patches. ETA receptor-evoked TRPC1/C5/C5 and TRPC3/C7 channel activities were unaffected by co-application of the PLC inhibitor U73122 (2 µM) (Yule and Williams, 1992), whereas the pan-PI3K inhibitor wortmannin (50 nM) (Powis et al., 1994) reduced both TRPC1/C5/C6 and TRPC3/C7 channel activities by over 95% (Figure 1B and C).

Differential actions of ZSTK474, a pan-Class I PI3K inhibitor, on ETA receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channels

We studied the role of Class I PI3K isoforms in ETA receptor stimulation of TRPC1/C5/C6 and TRPC3/C7 channels using the pan-Class I PI3K inhibitor ZSTK474 (Kong and Yamori, 2007). Co-application of 100 nM ZSTK474 had differential actions on ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities; TRPC1/C5/C6 channel activity was inhibited by over 95% (Figure 2), whereas TRPC3/C7 channel activation was potentiated by over twofold (Figure 2). ZSTK474-induced augmentation of ETA receptor-evoked TRPC3/C7 was blocked by 50 nM wortmannin by 96 ± 2% (n= 4, P < 0.01, Figure 2A and C). ZSTK474-mediated inhibition of ETA receptor-evoked TRPC1/C5/C6 was similar in patches containing both TRPC1/C5/C6 and TRPC3/C7 channel activities (Figure 2A) or in patches containing only TRPC1/C5/C6 channel activity (Figure 2B).

Pharmacological profile of isoform-selective PI3K inhibitors on ETA receptor-induced native TRPC1/C5/C6 and TRPC3/C7 channels

The family of Class I PI3K consists of four isoforms of catalytic subunits (PI3Kα, β, δ and γ) (Hawkins et al., 2006; Vanaesebroeck et al., 2010). We investigated the effect of Class I PI3K isoforms in mediating ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities using established isoform-selective inhibitors, which we used at ≤5 times the concentration of IC50 values for other PI3K isoforms (Knight et al., 2006; Chaussade et al., 2007; Hayakawa et al., 2007). To clearly distinguish the effect of PI3K inhibitors on TRPC1/C5/C7 and TRPC3/C7 channel opening, we studied the effects of these inhibitors on cell-attached patches in which ETA receptor stimulation evoked either TRPC1/C5/C6 and TRPC3/C7 channel activities (Figure 3) or only TRPC1/C5/C6 channel activity (Figure 4). Figures 3 and 4 show that patches containing ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities or only TRPC1/C5/C6 channel activity were unaffected by TGX211 (100 nM), PIK75 (100 nM) and IC87114 (600 nM), which are selective inhibitors of PI3Kα, β and δ isoforms respectively. In contrast, AS252424, a PI3Kγ isoform inhibitor, had differential actions on TRPC1/C5/C6 and TRPC3/C7 channels; Figure 3(iv) and B show that 300 nM AS252424 potentiated TRPC3/C7 channel activity by over twofold, whereas Figure 4(iv) and B illustrate that 300 nM AS252424 reduced ETA receptor-evoked TRPC1/C5/C6 channel activity by over 95%. It should be noted that ZSTK474- and AS252424-mediated inhibition of ETA receptor-evoked TRPC1/C5/C6 was similar in patches containing both TRPC1/C5/C6 and TRPC3/C7 channel activities [Figures 2A and 3A(iv)] to that in patches containing only TRPC1/C5/C6 channel activity [Figures 2B and 4A(iv)].

Figure 3.

Figure 3

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Class I PI3Kγ isoform inhibitor potentiates ETA receptor-evoked TRPC3/C7 channel activity. (A) Effect of selective Class I PI3K isoform inhibitors (i) TGX221, (ii) PIK75, (iii) IC87114, and (iv) AS252424 on cell-attached patches held at −70 mV, which contained both ETA receptor-induced TRPC1/C5/C6 and TRPC3/C7 channel activities. (B) Mean data showing the effect of Class I PI3K isoform inhibitors on TRPC3/7 channel activity. n≥ 5, *P < 0.05. Note that only AS252424 potentiated ETA receptor-evoked TRPC3/C7 channel activity.

Figure 4.

Figure 4

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Class I PI3Kγ isoform inhibitor reduces ETA receptor-evoked TRPC1/C5/C6 channel activity. (A) Effect of selective Class I PI3K isoform inhibitors (i) TGX221, (ii) PIK75, (iii) IC87114, and (iv) AS252424 on cell-attached patches held at −70 mV, which contained only ETA receptor-induced TRPC1/C5/C6 activity. (B) Mean data showing the effect of Class I PI3K isoform inhibitors on TRPC1/C5/C6 channel activity. n≥ 5, **P < 0.01. Note that only AS252424 inhibited ETA receptor-evoked TRPC1/C5/C6 channel activity.

Effect of ZSTK474 and AS252424 on quiescent cell-attached patches

It is possible that the potentiating actions of ZSTK474 and AS252424 on ETA receptor-evoked TRPC3/C7 channel activity reflect channel inhibition by constitutive Class I PI3K isoform activity. We studied this hypothesis by investigating the effect of ZSTK474 and AS252424 on unstimulated cell-attached patches. Bath application of 100 nM ZSTK474 (Figure 5A) and 300 nM AS252424 (Figure 5B) activated channel currents with a mean NPO of, respectively, 0.86 ± 0.11 (n= 9) and 0.89 ± 0.09 (n= 9) in quiescent cell-attached patches held at −70 mV; both had four amplitude levels between −1 pA and −4.5 pA [Figure 5A(ii) (iii) and B(ii) (iii)] that related to sub-conductance states between 15 and 70 pS, which all had Er of about 0 mV, similar to ETA receptor-evoked TRPC3/C7 channel currents (Peppiatt-Wildman et al., 2007). In addition, ZSTK474- and AS252424-evoked TRPC3/C7 channel activities were both inhibited by co-application of 50 nM wortmannin by 97 ± 2% [n= 9, P < 0.01, Figure 5A(i)] and 95 ± 3% [n= 5, P < 0.05, Figure 5B(i)], respectively.

Figure 5.

Figure 5

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ZSTK474 and AS252424 activate TRPC3/C7 channel activity in unstimulated VSMCs. (A) (i) Bath application of ZSTK474 activated cation channel activity in quiescent cell-attached patches held at −70 mV, which was inhibited by wortmannin. (ii) Inset showing ZSTK474-evoked channel activity on a faster time scale. (iii) Amplitude histogram showing that ZSTK474-evoked channel currents illustrated in (i) had four sub-conductance levels between −1 and −4.5 pA at −70 mV. (B) Similar results were obtained with AS252424.

Effect of PI3K-generated PIs on quiescent inside-out patches

The differential effects of PI3K-mediated pathways on TRPC1/C5/C6 and TRPC3/C7 channels shown above imply that downstream products (Hawkins et al., 2006; Vanaesebroeck et al., 2010) are likely to have important actions on activation mechanisms of these two native TRPC channels. Bath application of the water soluble diC8-PI(3)P (10 µM) to the cytosolic surface of unstimulated inside-out patches held at −70 mV activated two distinct channels; one channel had an unitary conductance of 2.8 pS, whereas the other channel had four sub-conductance states of 16, 32, 49 and 68 pS, and both channel types had Er of about 0 mV (data not shown, Figure 6A). In contrast, PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3 (all at 10 µM) only activated channel currents with unitary conductances of about 3 pS (Figure 6B). Interestingly, substrates of PI3K-mediated pathways, PI(4)P, PI(5)P and PI(4,5)P2 (all at 10 µM) (Hawkins et al., 2006; Vanaesebroeck et al., 2010), also activated channels with conductances of about 3 pS (Figure 6C). Non-phosphorylated PI had no effect on quiescent inside-out patches (Figure 6D and E). All PIs were applied in the presence of 50 nM wortmannin and 2 µM U73122 to ensure that these were effects of the PI itself and not a metabolite of PI3K and PLC pathways, respectively (Saleh et al., 2009a,b).

Figure 6.

Figure 6

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Selective excitatory actions of PI(3)P and other PIs on TRPC1/C5/C6 and TRPC3/C7 channel activities. (A) Bath application of PI(3)P activated both TRPC1/C5/C6 and TRPC3/C7 channel activities in inside-out patches held at −70 mV. (B) and (C) Bath application of other PI(3)-containing molecules only activated TRPC1/C5/C6 channel activity in inside-out patches. (D) and (E) Mean data showing the effect of PI molecules on TRPC1/C5/C6 and TRPC3/C7 channel activities, respectively. Note that PI failed to activate either TRPC1/C5/C6 or TRPC3/C7 channels. All PIs used were diC8 molecules. (F) Dot-blots showing (i) the absence of PI(3)P in tissue lysates at rest and an increase in PI(3)P levels following ETA receptor stimulation, and (ii) ETA receptor-mediated increase in PI(3)P levels was prevented by pre-incubation with wortmannin (Wort).

To test the hypothesis that ETA receptor stimulation evokes TRPC1/C5/C6 and TRPC3/C7 channel activities through generation of PI(3)P, we carried out dot-blots studies that showed that ETA receptor stimulation leads to production of PI(3)P levels [Figure 6F(i)] through a wortmannin-sensitive process [Figure 6F(ii)].

Effect of PIs on PI(3)P-induced TRPC3/C7 channel activity

Earlier studies proposed that PI(4,5)P2, a substrate of PLC- and also PI3K-mediated pathways, has a pronounced inhibitory action on the TRPC3/C6/C7 channel subgroup in VSMCs (Albert et al., 2008; Ju et al., 2010), and therefore we examined if the excitatory actions of PI(3)P on TRPC3/C7 channel activity is regulated by equimolar concentrations of PI(4,5)P2. TRPC3/C7 channel activity induced by 10 µM diC8-PI(3)P in inside-out patches was almost abolished by co-application of 10 µM diC8-PI(4,5)P2 (Figure 7A and D). Moreover, other substrates of PI3K-mediated pathways, PI(4)P and PI(5)P, and also PI3K-generated products, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3 (all at 10 µM), produced marked inhibition of PI(3)P-induced TRPC3/C7 channel activity (Figure 7B–D). Unphosphorylated PI (10 µM) had no effect on PI(3)P-evoked TRPC3/C7 channel activity (Figure 7B and D).

Figure 7.

Figure 7

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PI(3)P-evoked TRPC3/C7 channel activity is inhibited by other PIs. (A) PI(3)P-activated TRPC3/C7 channel activity is inhibited by co-application of equimolar PI(4,5)P2 concentrations in inside-out patches held at −70 mV. Note that the insets show that TRPC1/C5/C6 channels are present with PI(3)P and also with PI(3)P + PI(4,5)P2 conditions. (B) Substrates of PI3K-mediated reactions, PI(4)P and PI(5)P, and (C) products of PI3K-mediated reactions inhibited PI(3)P-evoked TRPC3/C7 channel activities. (C) Mean data showing that PI molecules significantly inhibited PI(3)P-evoked TRPC3/C7 channel activity. n≥ 5, **P < 0.01. Note that PI had no effect on PI(3)P-induced TRPC3/C7 channel activity.

Effects of the G-protein βγ subunit inhibitor, gallein, on ETA receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channels

The present work proposes that ETA GPCR stimulation induces native TRPC1/C5/C6 and TRPC3/C7 channels via, respectively, Class I PI3Kγ and Class II and/or III PI3K isoforms in coronary artery VSMCs. We therefore examined if the established pathway of G-protein βγ subunits coupled to PI3K is responsible for linking ETA receptor to channel activation (Stephens et al., 1994; Clapham and Neer, 1997; Leopolt et al., 1998). ETA receptor stimulation of TRPC1/C5/C6 and TRPC3/C7 channel activities was inhibited by co-application of 20 µM gallein, a G-protein βγ subunit inhibitor (Seneviatne et al., 2011), by over 90% in cell-attached patches (Figure 8). In control experiments, gallein did not inhibit TRPC1/C5/C6 and TRPC3/C7 channel activities induced by 10 µM diC8-PI(3)P in inside-out patches (Figure 8B and C).

Figure 8.

Figure 8

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Effect of the G-protein βγ subunit inhibitor gallein on ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities. (A) Co-application of gallein inhibited both ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches held at −70 mV. (B) and (C) Mean data showing that gallein significantly inhibited ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches, respectively. In addition, gallein had no effect on PI(3)-evoked TRPC1/C5/C6 or TRPC3/C7 channel activities in inside-out patches. n≥ 5, **P < 0.01.

Expression of Class I, II and III PI3K isoforms in coronary artery tissue lysates

We carried out PCR studies to identify the expression of Class I, II and III PI3K isoforms in freshly dispersed coronary arteries. Coronary artery tissue lysates expressed transcripts for all known Class I, II and III PI3K isoforms after two sequential end-point PCR cycle steps (Figure 9A). Quantitative PCR using SYBR Green technology was used to assess the relative abundance of Class I, II and III PI3K isoforms in coronary artery tissue lysates. Melt-curve analysis was used to confirm specificity of primers (Figure 9B), and the efficiency of primer sets was established by the generation of standard curves from serial dilutions of cDNA from coronary artery tissue lysates (Figure 9C). The relative abundance of mRNA encoding PI3K isoforms was quantified on the basis of the expression ratio of target genes against two reference genes, β-actin [Figure 9D(i) and E] and GAPDH [Figure 9D(ii) and F]. Amplification plots of target genes against β-actin and GAPDH show that all known Class I, II, III PI3K isoforms are expressed in coronary artery tissue lysates, with PI3Kα expression levels considerably higher than levels of other PI3K isoforms (determined using two different primer sets, Figure 9G and H). Importantly, PI3Kγ and Class II and III PI3K isoforms are expressed in coronary artery tissue lysates.

Figure 9.

Figure 9

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Expression of PI3K isoforms in freshly dispersed coronary arteries. (A) End-point RT-PCR of (i) Class I and (ii) Class II/III (C2/3) PI3K isoforms following one 40-cycle plus one 20-cycle protocols. (B) Representative dissociation profile for the Class I PI3Kα pr 1 in serially diluted concentrations of rabbit coronary artery cDNA. (C) Standard curves for all PI3K, β-actin and GAPDH primers used. (D) Amplification plots for (i) Class I and (ii) Class II/III PI3K primer sets. (E) Relative expression of primer sets for Class I (excluding Class I PI3Kα), II and III PI3K isoforms, which are normalized to β-actin and (F) to GAPDH. (G) Amplification plots and (H) relative expression of two different Class I PI3Kα primer sets, using β-actin and GAPDH as controls. All values shown were determined from at least three different animals.

Discussion

PI3K mediates ETA receptor stimulation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels

The present study shows that low concentrations of wortmannin, a pan-PI3K inhibitor (Powis et al., 1994), almost abolished ETA receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channel activities, which were unaffected by the PLC inhibitor U73122 (Yule and Williams, 1992). The G-protein βγ subunit inhibitor gallein (Seneviatne et al., 2011) also markedly reduced ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channel activities, but gallein did not inhibit activation of TRPC1/C5/C6 and TRPC3/C7 channels by PI(3)P, a downstream product of PI3K. Figure 10 highlights our proposal that stimulation of ETA receptors coupled to G-protein βγ subunits leading to activation of different PI3K isoforms produces opening of two distinct native TRPC channels in freshly isolated rabbit coronary artery VSMCs. G-protein βγ-mediated activation of PI3K isoforms is a well-characterized biochemical pathway in many cell types (Stephens et al., 1994; Clapham and Neer, 1997; Leopolt et al., 1998).

Figure 10.

Figure 10

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Schematic representation of proposed activation mechanisms of ETA receptor-evoked TRPC1/C5/C6 and TRPC3/C7 channels. Stimulation of ETA GPCRs leads to Gβγ subunits activation of different PI3K isoforms. It is proposed that the PI3Kγ-mediated generation of PI(3)-containing molecules [PI(3)P, PI(3,4)P2, PI(3,5)P2, PI(3,4,5)P3] produce gating of TRPC1/C5/C6 channels and TRPC3/C7 channel inhibition. In contrast, Class II and /or III PI3K isoform-mediated generation of PI(3)P induced TRPC3/C7 channel gating. It is important to note that PI(3)P is represented has a direct activating ligand at TRPC3/C7 channels.

In VSMCs, receptor/Gαq/11-evoked PLC and constitutively active Gαi/o-evoked phospholipase D activities are currently thought to be the major signalling pathways involved in activating native TRPC channels (Abramowitz and Birnbaumer, 2009; Albert et al., 2009; Large et al., 2009; Dietrich et al., 2010; Albert, 2011). The present study, in conjunction with our earlier work (Saleh et al., 2009b), indicates that Gβγ-PI3K-mediated pathways must also be considered important in native TRPC channel activation.

PI3Kγ mediates ETA receptor-induced TRPC1/C5/C6 channel activation

ETA receptor stimulation leads to PI3K-mediated generation of PI(3,4,5)P3 and opening of TRPC1/C5/C6 channels in coronary artery VSMCs (Saleh et al., 2008; 2009b). The present work extends these ideas to suggest that the Class I PI3Kγ isoform mediates ETA receptor stimulation of TRPC1/C5/C6 channels (Figure 10). These conclusions are based upon marked inhibition of TRPC1/C5/C6 channel activity by the pan-Class I PI3K inhibitor, ZSTK474 (Kong and Yamori, 2007) and the selective Class I PI3Kγ blocker, AS252424 (Knight et al., 2006; Chaussade et al., 2007; Hayakawa et al., 2007). Inhibitors of Class I PI3Kα, β and δ isoforms had no effect on channel activity.

PI3K has been suggested to mediate the ET-1-evoked cation conductances involved in Ca2+ influx and vasoconstriction of rabbit basilar artery (Kawanabe et al., 2004; Miwa et al., 2005). Angiotensin II-evoked stimulation of L-type Ca2+ channels in rat portal vein is proposed to involve G-protein βγ subunits coupled to PI3Kγ and production of PI(3,4,5)P3 (Viard et al., 1999; Quignard et al., 2001; Le Blanc et al., 2004), and PI3Kγ is reported to be important for regulating Ca2+ oscillations and contraction of murine airway smooth muscle (Jiang et al., 2010).

Class II and/or Class III PI3K isoforms mediate ETA receptor-evoked TRPC3/C7 channel activity

In contrast to the inhibitory actions of ZSTK474 and AS252424 on TRPC1/C5/C6 channel activity, both these agents had excitatory actions on ETA receptor-stimulated TRPC3/C7 channel activity indicating that PI3Kγ-mediated pathways are involved in inhibiting these channels. Moreover, ZSTK474 and AS252424 also activated channels with similar conductance values as TRPC3/C7 channels in unstimulated cells. These results indicate that ETA receptor-mediated and constitutively active PI3Kγ exerts a powerful inhibitory action on native TRPC3/C7 channels in coronary artery VSMCs, which maintain these channels in a closed state (Figure 10).

ZSTK474- and AS252424-induced potentiation of ETA receptor-evoked TRPC3/C7 channel activity and activation of TRPC3/C7 channels in quiescent patches were both inhibited by low concentrations of wortmannin. These results indicate that non-Class I PI3K isoforms, most likely Class II and/or III PI3K isoforms, mediate activation of TRPC3/C7 channels. Moreover, they suggest that these Class II and/or Class III PI3K isoforms can be constitutively active. The qPCR studies confirmed that all known catalytic subunits of Class I, II and III PI3K isoforms are expressed in coronary artery tissue lysates. There are few established inhibitors of Class II and III PI3K isoforms, and so future experiments using protein knock-down approaches will be required to identify PI3K isoforms mediating ETA receptor-evoked TRPC3/C7 channel activation.

Gating of native TRPC1/C5/C6 channels by PI3K-mediated PI molecules

PI(4,5)P2 is obligatory for activation of native TRPC1/C5/C7 channels in portal vein VSMCs, through a process requiring PKC-dependent phosphorylation of TRPC1 proteins (Saleh et al., 2009a). In coronary artery VSMCs, ETA receptor-evoked activation of TRPC1/C5/C6 channels is proposed to be mediated by PI3K through generation of PI(3,4,5)P3, which acts as a gating ligand via a PKC-dependent mechanism (Saleh et al., 2008; 2009b).

The present work builds upon these earlier findings with PI(3,4,5)P3 to reveal that other signalling molecules generated by PI3K, PI(3)P, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3 activate TRPC1/C5/C6 channels in inside-out patches of coronary artery VSMCs, which indicates that these ligands are also gating molecules. Future studies will need to determine if TRPC1/C5/C6 channel opening by these PI3K-generated molecules requires PKC-dependent phosphorylation of TRPC1, and/or TRPC5/C6 subunits, and also to examine which TRPC subunit(s) confers activation by PI. Recent work has indicated that TRPC1 confers activation by PI(4,5)P2 and PI(3,4,5)P3 to a native heteromeric TRPC1/C5 channel in murine mesenteric artery (Shi et al., 2011).

PI(3)P is novel activator of TRPC3/C7 channels

The established view is that TRPC channels mediated by TRPC3/C6/C7 subunits are activated by phospholipase-generated DAG through a PKC-independent mechanism (Abramowitz and Birnbaumer, 2009; Dietrich et al., 2010). In VSMCs, DAG is proposed to gate channels composed of TRPC3/C6/C7 subunits by competing with PI(4,5)P2, which is bound to channel proteins at rest and acts as a physiological antagonist (Albert et al., 2008; Large et al., 2009; Ju et al., 2010; Albert, 2011). In agreement with these studies, previous evidence indicates that TRPC3/C7 channels in coronary artery VSMCs are activated by DAG via a PKC-independent mechanism (Peppiatt-Wildman et al., 2007). However, the present results indicate that PI3K, and not PLC, signalling pathways govern ETA receptor stimulation of TRPC3/C7 channels. The important conclusion from these findings is that although DAG can activate TRPC3/C7 channels (Peppiatt-Wildman et al., 2007), PI(3)P and not this triglyceride is involved in ETA receptor-mediated activation of native TRPC3/C7 channels in coronary artery VSMCs. It will be interesting to investigate whether PI(3)P is an activating ligand at other TRPC3/C6/C7 channel subgroups in different cell types.

We obtained compelling evidence that PI(3)P is the only PI3K-generated molecule that activates TRPC3/C7 channels. Furthermore, PI(3,4)PI2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 induced a pronounced inhibition of PI(3)P-evoked TRPC3/C7 channel activity. As PI(3)P is the only PI3K-generated molecule that activates TRPC3/C7, it is most likely that this PI mediates ETA receptor stimulation of TRPC3/C7 channel activity. In agreement with this hypothesis, dot-blot analysis showed that ETA receptor stimulation generates PI(3)P through a wortmannin-sensitive process in coronary artery tissue lysates. Moreover, PI(3)P is considered to be produced by the action of Class II/III PI3K isoforms (Hawkins et al., 2006; Vanaesebroeck et al., 2010). These results represent a significant advance in our understanding of the native TRPC3/C6/C7 channel subgroup; PI(3)P is a novel gating ligand (Figure 10). It will be important to further investigate interactions between PI(3)P (and other PIs) and DAG in activating the TRPC3/C6/C7 channel subgroup.

Substrates of PI3K-mediated pathways modulate TRPC1/C5/C6 and TRPC3/C7 channel activity

Our data indicate that substrates of PI3K-mediated pathways [PI(4)P, PI(5)P and PI(4,5)P2] activate TRPC1/C5/C6 channels but inhibit PI(3)P-evoked TRPC3/C7 channels in coronary artery VSMCs. These results suggest that specific PI(3) molecule-acting phosphatases (e.g. phosphatase and tensin homologue deleted on chromosome 10) acting at PI3K-mediated products to generated these substrates (Hawkins et al., 2006; Vanaesebroeck et al., 2010) are also likely to have important roles in regulating native TRPC channel regulation in VSMCs. PI molecules shown in the present study to activate TRPC1/C5/C6 and TRPC3/C7 channels and also inhibit PI(3)P-evoked TRPC3/C7 channel activity bind to expressed TRPC1, C5 C6 and C7 proteins (Kwon et al., 2007). Interestingly, the non-phosphorylated PI molecule, PI, which we showed has no effect on TRPC1/C5/C6 and TRPC3/C7 channel activities (see Figures 5 and 6), does not bind to expressed TRPC proteins (Kwon et al., 2007).

Importance of results

TRPC channels and PI3K have been independently reported to contribute to vascular tone, cell growth, proliferation, migration and survival of VSMCs and have both been implicated in vascular diseases such as hypertension and neointima hyperplasia (Huang and Kontos, 2002; Oudit et al., 2004; Huang et al., 2005; Abramowitz and Birnbaumer, 2009; Morello et al., 2009; Dietrich et al., 2010). To our knowledge, our results demonstrate for the first time that PI3Kγ and other Class II/III PI3K isoforms are likely to be important in regulating distinct native TRPC channels in VSMCs. Our findings provide an important rational for investigating the role of ETA receptor-PI3K-TRPC channel pathways in coronary artery function, for example, contraction, membrane potential regulation, cell migration and proliferation. Moreover, our data indicate that pharmacological manipulation of specific PI3K isoforms and TRPC channels involved in these ETA receptor-PI3K-TRPC pathways may be beneficial for the treatment and prevention of cardiovascular disease.

Of further value, our results highlight the novel roles of PI molecules in the regulation of native TRPC channels. PI molecules containing a phosphorylated D3, D4 or D5 hydroxyl group activate a native TRPC1 channel, whereas only PI(3)P activates a TRPC3/C6/C7 channel subtype. These novel findings provide an intriguing proposition for gating of the TRPC3/C6/C7 channel subgroup; PI containing D3 phosphorylation produces channel activation, whereas PI containing D4/D5 phosphorylations produces potent, and dominant, channel inhibition.

Acknowledgments

This work was supported by the British Heart Foundation Project grant, PG/07/079/23568 and PG/08/042/25066, to Anthony P. Albert and William A. Large. We would like to thank Ken Laing and Alison Davis for technical advice in qPCR.

Glossary

AS252424

5-[[5-(4-fluro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4-thiazolidinedione

BQ788

N-[(cis-2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine

IC87114

2-[6-amino-9H-purin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone

PIK75

N-((1E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N′-methyl-N″-(2-methyl-5-nitrobenzene)sulfonohyrazide

PI3K

phosphatidylinositol 3-kinase

PI

phosphatidylinositol

PI(3)P

phosphatidylinositol 3-phosphate

PI(4)P

phosphatidylinositol 4-phosphate

PI(5)P

phosphatidylinositol 5-phosphate

PI(3,4)P2

phosphatidylinositol 3,4-bisphosphate

PI(3,5)P2

phosphatidylinositol 3,5-bisphosphate

PI(4,5)P2

phosphatidylinositol 4,5-bisphosphate

PI(3,4,5)P3

phosphatidylinositol 3,4,5-trisphosphate

TGX221

(+-)-7-methyl-2-(morpholin-4-yl)-9-(1-phenylaminoethyl)-pyrido[1,2-a]-pyrimidin-4-one

TRPC

canonical transient receptor potential

U73122

1-[6-[[17β0-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1-H-pyrrole-2,5-dione

VSMC

vascular smooth muscle cells

ZSTK474

2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine

Conflicts of interest

None.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Table S1 Conventional PCR primers used for end-point PCR

Table S2 qPCR primers

bph0166-2161-SD1.doc (58.5KB, doc)

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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