Short-term stimulation of calcium-permeable transient receptor potential canonical 5-containing channels by oxidized phospholipids

Abstract

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 G(i/o) proteins but not on previously identified G protein-coupled receptors for oxidized phospholipids.

Conclusions: 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.

Figure 1. Ca²⁺-entry stimulated by PGPC and POVPC in TRPC5-expressing HEK 293 cells

Ca²⁺ measurement data, where black symbols indicating responses to OxPAPC, PGPC or POVPC in TRPC5-expressing (Tet+) cells and white symbols indicating PAPC, vehicle controls or responses in Tet− cells. The ordinate scale is the change (Δ) in fluo4 fluorescence divided by 10⁴ (F*) in arbitrary units. (a) Responses to bath-applied 10 μM OxPAPC or 10 μM PAPC (N=6 each). (b) Responses to bath-applied 3 μM PGPC or vehicle (N=6 each). (c, d) Mean data for PGPC, POVPC and vehicle responses in Tet+ and Tet− cells (n/N=3/18 for each point). (e, f) Concentration-response data for PGPC and POVPC in Tet+ and Tet− cells (n/N=3/9 for each point). Curves are Hill equations fitted to Tet+ cell data with mid-points at 2.24 μM (e) and 1.52 μM (f).

Figure 2. Ionic current stimulated by PGPC or POVPC in TRPC5-expressing HEK 293 cells

Whole-cell patch-clamp recordings from Tet+ cells under voltage-clamp. (a) Time-series plot showing the effect of bath-applied 3 μM PGPC in the continuous presence of the ethanol vehicle, and then inhibition by 75 μM 2-APB. (b) Mean time-matched data comparing current responses to the vehicle (ethanol) and 3 μM PGPC at −80 mV and +80 mV (n=6 each). (c) For the experiment of (a), current-voltage relationships (I-Vs) for currents induced by PGPC at the early and late stages of the response. (d) Response to bath-applied 3 μM POVPC shown as I-Vs for currents induced by POVPC.

Figure 3. Responses of VSMCs to PGPC and POVPC

Data are from intracellular Ca²⁺ measurements. (a) Typical response to bath-applied 3 μM PGPC compared with vehicle control. (b) Concentration-response data for PGPC and POVPC (n/N=4/12 for each point). Mid-points of the fitted Hill equations were at 1.5 μM (PGPC) and 1.09 μM (POVPC). (c) Example single VSMC responses to 3 μM PGPC observed using the microscope system (4 cells, labeled i-iv). (d) Responses to 3 μM PGPC in the presence and absence of extracellular Ca²⁺ (N=6 for each). (e) As for (d) but mean data from independent experiments for 3 μM PGPC, 3 μM POVPC and 100 μM ATP (n/N=3/18 for each).

Figure 4. Contribution of endogenous TRPC5 and TRPC1

Data are from intracellular Ca²⁺ measurements from VSMCs. (a, b) Responses to 3 μM PGPC after pretreatment with anti-TRPC antibody alone or anti-TRPC antibody preadsorbed to its antigenic peptide (+pep.) (N=5 each): T5E3 anti-TRPC5 antibody (a); T1E3 anti-TRPC1 antibody (b). (c) As for (a, b) but mean data for independent experiments (n/N=3/15). (d, e) As for (a, c) but using POVPC instead of PGPC (n/N=3/15). (f) Example from 8 independent paired experiments of the response to 3 μM PGPC after transfection with dominant negative mutant TRPC5 (DN-TRPC5) or vector control (N=16 for each).

Figure 5. VSMC migration evoked by PGPC and depending on TRPC channel function

(a) Representative images showing VSMCs at time zero (0 hr) and 48 hr after scratch wound in the presence of 3 μM PGPC. The images show a paired comparison of cells from the same patient transfected with DNA vector or vector expressing DN-TRPC5. In each image, VSMCs before migration are on the left of the thin line that delineated the origin (0 mm); the parallel thin line to the right defines the 0.2 mm distance required for cells to migrate before counting. The thick dark shadow is the 1 mm grid. (b) For experiments of the type shown in (a), mean data for VSMCs of 8 patients after transfection with DNA vector or vector containing the dominant negative (DN) TRPC5 insert. VSMCs were treated with vehicle (ethanol) or 3 μM PGPC and the numbers of migrated VSMCs were measured.

Figure 6. Role of G_i/o-signaling but not PAF receptors

Data are from intracellular Ca²⁺ measurements from TRPC5-expressing HEK 293 cells (a-c) and VSMCs (d-f). Cells were pretreated with 1 μg ml⁻¹ pertussis toxin (PTX) or denatured (denat.) PTX for 4 hr at 37 °C. (a-c) Responses were evoked by 3 μM PGPC (a, n/N=3/9), 5 μM POVPC (b, n/N=3/9) or 5 μM lysophosphatidylcholine (c, n/N=3/18). (d) Representative paired experiment showing responses to 3 μM PGPC after treatment with PTX or denatured PTX (N=5 each). (e) Responses were evoked by 3 μM PGPC or 5 μM POVPC (n/N=4/20 each). (f) Responses to 3 μM PGPC in cells pre-incubated and maintained in the presence of 100 μM WEB2086 or its vehicle (N=6 each and representative of 3 independent experiments).