Short-Term Stimulation of Calcium-Permeable Transient Receptor Potential Canonical 5–Containing Channels by Oxidized Phospholipids
Objective— To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC).
Methods and Results— Oxidized phospholipids are key factors in inflammation and associated diseases, including atherosclerosis; however, the initial reception mechanisms for cellular responses to the factors are poorly understood. Low micromolar concentrations of PGPC and POVPC evoked increases in intracellular calcium in human embryonic kidney 293 cells that overexpressed human transient receptor potential canonical 5 (TRPC5) but not human TRP melastatin (TRPM) 2 or 3. The results of electrophysiological experiments confirmed stimulation of TRPC5. To investigate relevance to endogenous channels, we studied proliferating vascular smooth muscle cells from patients undergoing coronary artery bypass surgery. PGPC and POVPC elicited calcium entry that was inhibited by anti-TRPC5 or anti-TRPC1 antibodies or dominant-negative mutant TRPC5. Calcium release did not occur. The effect was functionally relevant because it enhanced cell migration. The actions of PGPC and POVPC depended on Gi/o proteins but not on previously identified G protein–coupled receptors for oxidized phospholipids.
Conclusion— Stimulation of calcium-permeable TRPC5-containing channels may be an early event in cellular responses to oxidized phospholipids that couples to cell migration and requires an unidentified G protein–coupled receptor.
One-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphor-ylcholine (PAPC) is a phospholipid that is a common component of mammalian cell membranes and lipoproteins.1,2 The susceptibility of PAPC and other constituent lipids to oxidation through myeloperoxidase, lipoxygenase, and other enzyme activities leads to bioactive oxidation products (oxidized phospholipids).3,4 Two important products of PAPC are 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC)1,2,5; they are 2 examples of oxidized phospholipids that are recognized to constitute a diverse family of signaling lipids that accumulate during oxidative stress, apoptosis, and necrosis. They are often associated with inflammatory conditions, such as rheumatoid arthritis and atherosclerosis. There are also suggestions of physiological roles for these lipids, including leukocyte-endothelial interactions and pattern recognition in innate immunity.6,7 Both pro- and anti-inflammatory effects of oxidized phospholipids have been detected,8 suggesting context-dependent actions and critical importance of the free-lipid concentration. Although the importance of oxidized phospholipids is increasingly established, understanding of the initial reception and signaling mechanisms is limited. Perhaps surprisingly, given the pivotal roles of Ca2+ signaling in mammalian cells, information is lacking on the effects of oxidized phospholipids on Ca2+ channels or other Ca2+ transport mechanisms.
Mammalian transient receptor potential (TRP) proteins are ion pore-forming subunits of cationic channels with wide-ranging roles in physiology and disease.9 The canonical subfamily has 7 members that are generally considered to form heteromultimeric assemblies in native cells.9–12 Although the channels may have constitutive activity, they are also stimulated by various chemical factors, potentially acting as polymodal integrators of Ca2+ movement across cell membranes.10,11 TRP canonical (TRPC) 5 is a commonly studied example of the TRPC subfamily.11–13 It is stimulated by factors that include lanthanide ions, protons, reduced thioredoxin, and sphingosine-1-phosphate (S1P).11–14 It is also stimulated by lysophosphatidylcholine,15 a major phospholipid on oxidized low-density lipoproteins. Because of the effect of lysophosphatidylcholine, we investigated chemically related phospholipids with relevance to cardiovascular disease. Herein, we report on the effects of POVPC and PGPC on TRPC5 overexpressed in the human embryonic kidney (HEK) 293 cell line and on endogenous TRPC5-containing channels in vascular smooth muscle cells (VSMCs), which are targets for the actions of oxidized phospholipids.16–18
Conditional Channel Expression in HEK 293 Cells
HEK 293 cells, stably expressing tetracycline-regulated human TRPC5, have been described.10 Cells were grown in DMEM-F12 medium containing 10% fetal calf serum (FCS), 100-U/mL penicillin, and 100-μg/mL streptomycin. Cells were maintained at 37°C in 95% air and 5% CO2 and selected with 250-μg/mL zeocin and 10 μg/mL blasticidin. TRP channel expression was induced by 1-μg/mL tetracycline (Tet+). Noninduced cells without the addition of tetracycline were controls (Tet−). Cells were replated on poly-d-lysine–coated black 96-well plates (Corning or BD Biosciences) or 13-mm glass coverslips 24 hours before the experiments.
Vascular Smooth Muscle Cells
Freshly discarded human saphenous vein segments were obtained anonymously and with informed consent from patients undergoing open heart surgery in the General Infirmary at Leeds. Approval was granted by the Leeds Teaching Hospitals Local Research Ethics Committee. The investigation conforms to the principles outlined in the Declaration of Helsinki. Proliferating VSMCs were prepared using an explant technique19 and grown in DME medium supplemented with 10% FCS, a combination of penicillin and streptomycin, and l-glutamine at 37°C in a 5% CO2 incubator. Experiments were performed on cells at passage 3 to 4.
Intracellular Ca2+ Measurement
Cells were incubated with 2-μmol/L of the Ca2+ fluo4-AM or fura2-AM for 1 hour at 37°C, followed by a 0.5-hour wash at room temperature. The loading buffer was standard bath (extracellular) solution (SBS), which contained the following: 130-mmol/L NaCl, 5-mmol/L potassium chloride, 8-mmol/L d-glucose, 10-mmol/L HEPES, 1.5-mmol/L calcium chloride, and 1.2-mmol/L magnesium chloride, titrated to a pH of 7.4 with sodium hydroxide. The loading buffer included 2-μmol/L probenicid (fluo4-AM only) and 8-μmol/L pluronic acid. Measurements were at room temperature (mean±SEM, 21±2°C) on an inverted fluorescence microscope (Supplemental Material; available online at http://atvb.ahajournals.org) or a 96-well fluorescence plate reader (FlexStation II384; Molecular Devices). All Ca2+ data derived from the 96-well system except those of Figure 3C. With either system, changes in intracellular Ca2+ concentration were indicated by the change in fluo4 fluorescence (F, in arbitrary units) or the ratio of fura2 emission intensities for 340- and 380-nm excitation (F ratio). Fluo4 fluorescence values were divided by 104 (F*). Wells within columns of 96-well plates were loaded alternately for test and control conditions. The extracellular solution was SBS unless otherwise indicated; Ca2+-free solution was SBS without Ca2+ but with 0.4-mmol/L EGTA.
Membrane Current Recording
Recordings were made under voltage clamp using the whole cell configuration of the patch-clamp technique at room temperature. Signals were amplified and sampled using an amplifier (Axopatch 200B) and software (pCLAMP) (Molecular Devices). The extracellular solution was SBS, and the patch pipette solution contained the following: 135-mmol/L cesium chloride, 2-mmol/L magnesium chloride, 1-mmol/L EGTA, 10-mmol/L HEPES, 5-mmol/L Na2ATP, and 0.1-mmol/L Na2GTP. This solution was titrated to pH 7.2 with sodium hydroxide and filtered using a 0.2-μm filter. The voltage paradigm was a 200-millisecond ramp protocol (−100 to 100 mV) applied every 10 seconds from a holding potential of 0 mV. Data were filtered at 2 kHz and digitally sampled at 4 kHz.
Linear Wound Assay
After transfection, cells were serum starved for 16 hours, wounded, washed, and photographed; then, they were treated with vehicle or PGPC before being photographed and analyzed. For more details, see the Supplemental Material.
Unless otherwise indicated, reagents were from Sigma UK. Stocks of chemicals were reconstituted in an appropriate vehicle: fura2-AM and fluo4-AM (Invitrogen) were dissolved at 1 mmol/L in dimethyl sulfoxide (DMSO); lysophosphatidylcholine was dissolved at 50 mmol/L in methanol; PGPC, POVPC (Cayman Chemicals, Ann Arbor, Mich), PAPC (Hycult Biotechnology), and oleic acid were dissolved at 16.40 to 16.84 mmol/L; and S1P was dissolved at 10 mmol/L in ethanol. Oxidized PAPC (Hycult Biotechnology) had an unknown molecular mass but was assumed to have the same mass as PAPC and was similarly dissolved in ethanol. 9-Nitro oleate (Cayman Chemicals) was dissolved at 1.53 mmol/L in ethanol. N-Oleoylethanolamide and oleamide were dissolved at 50 mmol/L in DMSO. Stocks of prostaglandin-E2 (PGE2) and (±)-15-deoxy-16R-hydroxy-17-cyclobutyl PGE1 ((R)-butaprost) at 10 mmol/L and 3-(4-[2-chlorophenyl]-9-methyl-6H-thieno[3,2-f](1,2,4)triazolo[4,3-α]diazepine-2-yl)-1-propanone (WEB2086; Tocris Bioscience) at 100 mmol/L were prepared in ethanol. Vascular endothelial growth factor (VEGF) was dissolved in water. The stock of pertussis toxin was 0.2 mg/mL in glycerol; denatured pertussis toxin was generated by boiling the pertussis toxin for 10 minutes. Two-Aminoethoxydiphenylborate was dissolved at 75 mmol/L in DMSO. Pluronic acid F-127 (Invitrogen) was stored at 10% wt/vol in DMSO at room temperature. Probenecid was freshly prepared in SBS. Final working concentrations of solvents were as follows: 0.01% methanol, 0.05% ethanol or less, and 0.06% DMSO or less. Functional anti-TRPC5 and anti-TRPC1 antibodies have been described.12,14,20 For control experiments, preadsorption with antigenic peptide, 10 μmol/L, occurred overnight at 4°C before use. Functional anti-TRPC1 was used at a 1:500 dilution, and functional anti-TRPC5 was used at a 1:100 dilution. Cells were exposed to antibody during loading with Ca2+ indicator and for the 0.5-hour wash period. Subsequently, cells were washed with fresh SBS to remove excess antibody. For the dominant-negative (DN) TRPC5 construct and vector, see the Supplemental Material.
Data Analysis and Presentation
Data are presented as mean±SEM, where n represents the number of independent experiments and N represents the number of wells of a 96-well plate used in a single experiment. For patch-clamp experiments, n was the number of recordings from individual cells. t Tests were used for comparisons between pairs of data. Data from linear wound assays were analyzed by ANOVA, followed by a Newman-Keuls multiple comparison test. P<0.05 indicated a significant difference. Data were analyzed and presented using computer software (Origin; Microcal Inc).
Ca2+ Signals in Cells Overexpressing TRPC5
Stimulation of Ca2+ entry in HEK 293 cells conditionally expressing human TRPC5 was investigated in 96-well Ca2+ indicator assays. In half of each plate, cells were induced to express TRPC5 by tetracycline (Tet+), whereas in the other half, cells were not exposed to tetracycline (Tet−) to provide controls for TRPC5-independent effects. Cells were exposed to the test substance while maintaining the vehicle constant. Small effects occurred on solution application (Figure 1A and B), but much larger increases in Ca2+ were observed on exposure of TRPC5 (Tet+) cells to 10-μmol/L oxidized PAPC, 3-μmol/L PGPC, or 3-μmol/L POVPC (Figure 1A–D). There were no stimulatory effects of PAPC or the vehicle (Figure 1A and B). There were also no effects of PGPC or POVPC in control (Tet−) cells (Figure 1A–E). Therefore, PGPC and POVPC did not have TRPC5-independent effects, such as evoking Ca2+ release. In TRPC5 Tet+ cells, the effects of PGPC occurred at greater than 0.1 μmol/L (Figure 1E). The effects of POVPC mostly occurred at similar concentrations to those of PGPC, except that some experiments showed additional effects at 10 nM (Figure 1F). The data suggest that PGPC and POVPC are relatively potent stimulators of TRPC5.
Ionic Current in Cells Overexpressing TRPC5
An alternative method for measuring TRPC channel activity is whole cell patch-clamp recording. Large ionic currents were evoked in TRPC5 (Tet+) cells by PGPC (Figure 2A and B). Currents were blocked by the nonspecific TRPC5 inhibitor 2-aminoethoxydiphenylborate (Figure 2A), and they reversed polarity near 0 mV, consistent with nonselective cationic permeability of the underlying channels (Figure 2C). More important, the evoked current-voltage relationships had double-rectifying characteristics expected of TRPC5 (Figure 2C).14,15,21 POVPC evoked similar responses (Figure 2D). The effects of PGPC and POVPC appeared to occur in 2 phases (early and late) (Figure 2A, C, and D), as previously observed for receptor activation of TRPC5.21 The data are consistent with PGPC and POVPC stimulating TRPC5 and suggest activation via a receptor-coupling mechanism.
Lack of Effect on Other TRP Channels in the Same Expression System
As further controls for the expression system and to investigate specificity, PGPC and POVPC were tested against other TRP channels expressed via the same conditional system as TRPC5. No stimulation of Ca2+ entry was observed in response to PGPC or POVPC in TRPM2- or TRPM3-expressing cells (supplemental Figure I).
Ca2+ Signals in VSMCs Endogenously Expressing TRPC5
Endogenous expression of TRPC5 usually occurs concomitant with expression of other TRP channels, leading to heteromultimeric channels. To investigate TRPC5 in such a context, we used human saphenous vein VSMCs that express TRPC5 along with other TRP proteins, including TRPC1.12,20 PGPC and POVPC elicited Ca2+ responses in cells from about half of the donors (Figure 3A and supplemental Figure II). Effects occurred at concentrations greater than 0.1 μmol/L (Figure 3B); however, as in HEK 293 cells overexpressing TRPC5, there were anomalous responses to 10-nM POVPC (Figure 3B).
TRPC-Dependent Ca2+ Entry
Responses to PGPC and POVPC in VSMCs were often transient, resembling the course of Ca2+ events caused by Ca2+ release (Figure 3A). Similar data were obtained using 2 different Ca2+ indicator dyes, fura2 (Figure 3A) and fluo4 (data not shown); or when recording signals from individual cells using microscope-based imaging (Figure 3C). However, unlike Ca2+ release events, responses to PGPC and POVPC were absent when Ca2+ was omitted from the extracellular medium (Figure 3D and E). ATP-evoked Ca2+ release was unaffected by the absence of extracelullar Ca2+ (Figure 3E). The data suggest that PGPC and POVPC evoke predominantly Ca2+ entry in VSMCs.
To investigate if TRPC5-containing channels were involved in PGPC- and POVPC-evoked Ca2+ entry in VSMCs, we first used anti-TRPC5 and anti-TRPC1 antibodies that target extracellular loops and inhibit channel function.12,14,20 Either antibody inhibited responses to PGPC (Figure 4A–C) and POVPC (Figure 4D and E) in paired experiments using antibodies preadsorbed to antigenic peptides as controls. As an independent test of the contribution of TRPC5-containing channels, we used an ion-pore mutant of TRPC5 (DN-TRPC5) that fails to pass current and acts as a dominant negative, presumably because it damages ion permeation by entering in the heteromultimeric complex.12 Transfection of this mutant into VSMCs inhibited PGPC-evoked Ca2+ entry (Figure 4F) (P<0.05, n=8). The data suggest that endogenous channels containing TRPC5 and TRPC1 contributed to Ca2+ entry evoked by PGPC and POVPC.
Positive Impact on Cell Migration
TRPC channel activity has been linked to cell migration.12 Therefore, the effect of PGPC on VSMC migration was investigated in linear wound assays with and without the presence of DN-TRPC5 (Figure 5A). PGPC enhanced cell movement into the wounded area (Figure 5B and supplemental Figure III). Transfection of VSMCs from 8 patients with DN-TRPC5 did not affect baseline cell movement; however, it prevented the stimulatory effect of PGPC (Figure 5B). The data suggest that PGPC promotes VSMC migration via a mechanism that depends on ion permeation through TRPC channels.
Dependence on Gi/o Signaling
Lipid factors may act directly on TRPC channels or through intermediate signaling pathways. For example, S1P acts via a G protein–coupled receptor whereas lysophosphatidylcholine acts relatively directly.12,15 In the case of S1P, receptor coupling occurs via a Gi or Go protein, as indicated by sensitivity to pertussis toxin,12 which ADP ribosylates Gi/o proteins and inactivates them.
Pertussis toxin strongly inhibited the effects of PGPC and POVPC on TRPC5 (Figure 6A and B). Inhibition of the PGPC effect tended to be less than inhibition of the POVPC effect, as shown by comparison of the summary data (Figure 6A and B). In confirmation of previous results,15 pertussis toxin lacked effect on TRPC5 stimulation by lysophosphatidylcholine (Figure 6C). The data suggest that PGPC and POVPC acted on TRPC5 through a Gi/o protein signaling pathway and that lysophosphatidylcholine acted via a different mechanism.
In VSMCs, pertussis toxin partially inhibited responses to PGPC (Figure 6D and E) and almost abolished responses to POVPC (Figure 6E). The data suggest that PGPC and POVPC couple to endogenous TRPC channels substantially through a Gi/o signaling pathway and presumably because they bind and activate a membrane receptor.
Investigation of the Receptor Type
Previous studies3,8,22,23 have suggested that oxidized phospholipids are ligands at prostaglandin E (PGE) and platelet-activating factor (PAF) G protein–coupled receptors. However, neither PGE2 (n=3, data not shown) nor the synthetic PGE2 receptor agonist (R)-butaprost stimulated TRPC5 (supplemental Figure IV), and a high concentration of the PAF receptor antagonist WEB2086 failed to inhibit PGPC-evoked responses in VSMCs (Figure 6F). VEGF receptors are suggested to be involved in oxidized phospholipid effects24; however, we confirmed that the VSMCs and Tet+ HEK 293 cells show no Ca2+ responses to 100-ng/mL VEGF, in contrast with endothelial cells from the saphenous vein (data not shown). The data indicate that previously suggested oxidized phosholipid receptors are not involved in mediating TRPC channel stimulation.
To obtain more information on the character of the receptor, we tested 3-μmol/L 9-nitro-oleate, 3-μmol/L oleic acid, 10-μmol/L oleoylethanolamide, and 10-μmol/L oleamide in Ca2+ measurement assays of TRPC5 Tet+ cells, using 3-μmol/L PGPC as a positive control. None of these additional lipid factors evoked TRPC5 activity (3 independent experiments for each lipid) (data not shown).
The present study reveals that the bioactive oxidation products of a common membrane phospholipid have short-term stimulatory effects on Ca2+ entry via TRPC1/5 channels and a G protein–signaling mechanism, without causing Ca2+ release. The mechanism is relevant to proliferating VSMCs.
The dominant-negative TRPC5 mutant had no effect on basal migration of VSMCs; however, it prevented migration evoked by PGPC. The result adds to an expanding picture of positive roles of TRPC1/5 in remodeling.12,20,25,26 The effect is consistent with a proatherogenic effect of the phospholipids and may be important in physiological events of formation, remodeling, and response to injury of blood vessels. PGPC and POVPC were potent stimulators of the channels, acting at concentrations considerably lower than those used in many studies2–5,27 or detected in animals fed atherogenic diets.
PGPC and POVPC generated relatively sustained Ca2+ signals in the TRPC5 Tet+ cells but mostly transient signals in VSMCs. The difference may have arisen because of the more complex channel composition in the VSMCs. TRPC1 was involved in the VSMC signal and is susceptible to Ca2+-induced inactivation28; therefore, it may confer the decay in the PGPC and POVPC responses. A previous study6 described opposite effects of PGPC and POVPC on endothelial cells. Our data show equivalent effects of the oxidized phospholipids on VSMCs, except for 2 features: greater inhibition of POVPC responses by pertussis toxin and anomalous responses to 10-nM POVPC. The 10-nM POVPC effect occurred in HEK 293 cells induced to express TRPC5 but not noninduced cells, suggesting dependence on TRPC5. A separate intriguing observation was made with lysophosphatidylcholine, which is chemically closely related to PGPC and POVPC and functionally active in similar contexts.29 Unlike PGPC and POVPC, lysophosphatidylcholine stimulated TRPC5 independently of G protein signaling (Figure 6).15 Chemical features in PGPC and POVPC presumably prevented these lipids from mimicking the effect of lysophosphatidylcholine.
The receptors mediating the TRPC effects of PGPC and POVPC were not identified. PGE2 and PAF receptors are unlikely to be involved. PGE2 and (R)-butaprost were ineffective, and POVPC was previously found to lack effect at PGE2 receptors.22 The PAF antagonist WEB2086 failed to block the action of PGPC; a previous study23 found that WEB2086 inhibited the effects of POVPC but not of PGPC. A candidate receptor for the PGPC and POVPC effects in our experiments was an S1P receptor because S1P also acts via a Gi/o pathway.12 However, PGPC and POVPC could not have been agonists at S1P receptors because, unlike S1P, they did not cause Ca2+ release. Although oxidized phospholipids bind to scavenger receptors like CD36, these receptors are not thought to couple to G protein signaling.
The data suggest that TRPC channels are components of an initial reception mechanism enabling cellular responses to biologically active oxidized phospholipids. The mechanism is indicated to involve binding to a previously unrecognized G protein–coupled receptor that couples to the channels but not to Ca2+ release. An identified functional consequence is the promotion of VSMC migration, consistent with the prior suggestion that oxidized phospholipids play key roles in the proliferating phenotype of VSMCs.16
Sources of Funding
This study was supported by the Wellcome Trust and the British Heart Foundation.
Received on: September 18, 2009; final version accepted on: March 31, 2010.
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