Inhibition of endothelial cell Ca²⁺ entry and transient receptor potential channels by Sigma-1 receptor ligands

Abstract

Background and purpose: The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.

Experimental approach: Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies.

Key results: In endothelial cells, 10-100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3.

Conclusions and implications: The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

Figure 1

Inhibition of histamine-induced Ca²⁺ entry in endothelial cells by Sig1R ligands. (A) Two-dimensional chemical structures of four Sig1R ligands. (B–F) Intracellular Ca²⁺ measurement data from SVECs. (B–C) Example of data showing effects of 10 μM histamine in the presence of 100 μM BD1047 (B) or 1–100 μM BD1063 (C) compared with vehicle controls (N = 4 for each). The ligands were applied 30 min before testing histamine and maintained throughout the experiments. (D–F) Mean data for the types of experiment illustrated in (B, C), showing analysis of the sustained (5 min) response to 10 μM histamine, 100 μg·mL⁻¹ VEGF, or 1 mM H₂O₂ in the presence of 100 μM of the Sig1R ligand indicated (n/N = 3/12 for each experiment). The 4-IBP experiments have a separate vehicle control because they used DMSO rather than water as the solvent.

Figure 2

Inhibition of TRPC5 by Sig1R ligands. (A) Example of Ca²⁺ measurement data for TRPC5-expressing cells, showing responses to 20 μM Gd³⁺ in the presence of 100 μM BD1047 or the vehicle control (N = 6 for each). Also shown is the lack of effect of Gd³⁺ in cells without TRPC5 expression (Tet–). (B) Mean data for the type of experiment illustrated in (A) but showing analysis of the maximum response to 10 μM LPC (in place of Gd³⁺) and effects of 100 μM of the Sig1R ligand indicated (n/N = 3/18 for each condition). (C–F) Whole-cell voltage clamp data from TRPC5-expressing cells. (C) Time series plot of outward and inward currents stimulated by 20 μM Gd³⁺, showing repeated effects of 100 μM BD1047. 2-aminoethoxydiphenyl borate (2-APB, 75 μM) was applied at the end of the experiment. (D) Mean data for Gd³⁺-stimulated TRPC5 currents in the presence of 0, 10, 30 and 100 μM BD1047 applied cumulatively (n = 13). (E) Current–voltage relationships for spontaneous currents show the effects of 6, 25 and 100 μM BD1047 applied cumulatively. (F) Mean data for spontaneous TRPC5 currents in the presence of 0, 6, 25 and 100 μM BD1047 applied cumulatively (n = 5).

Figure 3

Inhibition of TRPM3 but not TRPM2 by Sig1R ligands. (A) Mean Ca²⁺ measurement data for TRPM2 cells, showing responses to 1 mM H₂O₂ in the presence of 100 μM of the specified inhibitor or the vehicle control (n/N = 3/4 for each). (B–E) Data from TRPM3-expressing cells. (B) Example of Ca²⁺ measurement data showing responses to 20 μM nifedipine in the continuous presence of 100 μM BD1047 or the vehicle control (N = 6 for each). Also shown is the lack of effect of nifedipine in cells without TRPM3 expression (Tet–). (C) Mean data for the types of experiment illustrated in (B), showing analysis of the maximum responses to nifedipine in the presence of 100 μM BD1047, BD1063, SKF10047 or 4-IBP (n/N = 3/18 for each condition). The 4-IBP experiments used DMSO rather than water as the solvent. (D, E) Whole-cell voltage clamp data from TRPM3-expressing cells. (D) Time series plot of outward and inward currents, showing stimulation by 5 μM PregS and the effect of 100 μM BD1047. Horizontal bars indicate the periods of compound application. (E) As for (D) but mean data showing the percentage of PregS-induced current remaining after application of BD1047 (n = 5).

Figure 4

Lack of role of the Sig1R. (A) Example of Ca²⁺ measurement results for the effect of 50 μM BD1063 (30 min treatment) on 10 μM histamine-evoked Ca²⁺ signals in SVECs treated with Sig1R siRNA (Sig1R.si). (B) Mean Ca²⁺ measurement data for the maximum responses to 10 μM LPC in TRPC5-expressing HEK 293 cells treated with sc.si (control) or Sig1R.si and then exposed to 100 μM BD1047 or vehicle control (n/N = 3/16 for each condition). (C, D) Merged images showing typical non-induced HEK 293 cells transiently expressing TRPC5-GFP (C) or TRPM3-YFP (D). Fluorescence from the GFP or YFP is in green. Cells were stained with anti-Sig1R antibody (red) and the nuclear stain DAPI (blue). Cells were pretreated with 100 μM SKF10047 (C) or BD1047 (D). The scale bars are 10 μm.