A role for 5,6-epoxyeicosatrienoic acid in calcium entry by de novo conformational coupling in human platelets

Abstract

A major pathway for Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this capacitative Ca(2+) entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6-EET, might be a component of the de novo conformational coupling in human platelets. In these cells, 5,6-EET induces divalent cation entry without having any detectable effect on Ca(2+) store depletion. 5,6-EET-induced Ca(2+) entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca(2+) entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. We have found that 5,6-EET-induced Ca(2+) entry requires basal levels of H(2)O(2), which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca(2+) store towards the PM to facilitate de novo coupling between IP(3)R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP(3)R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.

Figure 1. Effect of 5,6-EET on Ca²⁺ release and entry in human platelets

A, fura-2-loaded human platelets were treated in a Ca²⁺-free medium (100 μm EGTA was added) with various concentrations of 5,6-EET (0.01–3 μm) or left untreated (dotted trace) followed by addition of CaCl₂ (final concentration 300 μm) to initiate Ca²⁺ entry. Modifications in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. B, bars indicate the amount of Ca²⁺ entry and release in the presence of different concentrations of 5,6-EET. Ca²⁺ entry and release were estimated using the integral of the rise in [Ca²⁺]_i for 3 min after addition of CaCl₂ or 5,6-EET, respectively, and expressed as nanomolar seconds (nm s) as described in Methods. Values are expressed as the mean ± s.e.m. from six independent experiments. *P < 0.05 compared to the resting level.

Figure 2. Effect of 5,6-EET on extracellular Mn²⁺ entry

Human platelets were loaded with fura-2 and resuspended in a Ca²⁺-free medium as described in Methods. Fura-2-fluorescence was measured with an excitation wavelength of 360 nm, the isoemissive wavelength. Platelets were stimulated with different concentrations of 5,6-EET (0.01–3 μm, traces a–d) 5 min before addition of MnCl₂ (final concentration 300 μm). Mn²⁺ was added to untreated control cells (trace e) or cells treated with 5,6-EET. Traces are representative of seven separate experiments.

Figure 3. 5,6-EET-induced Ca²⁺ entry is inhibited by 2-APB, SKF 96365, LaCl₃, NiCl₂ and the anti-hTRPC1 antibody

Fura-2-loaded human platelets were preincubated at 37°C with 100 μm 2-APB (A) or 10 μm SKF 96365 for 10 min (B), or with 10 μm NiCl₂ (C) or 100 μm LaCl₃ for 1 min (D), or with 15 μg ml⁻¹ anti-hTRPC1 antibody (αTRPC1 ab) for 10 min (E) or the vehicles as Control (bold traces) and then treated in a Ca²⁺-free medium (100 μm EGTA was added) for 5 min with 1 μm 5,6-EET followed by addition of CaCl₂ (300 μm) to initiate Ca²⁺ entry. F, bars indicate the percentage of Ca²⁺ entry under the different experimental conditions relative to their respective control. Ca²⁺ entry was determined as described in Methods and values are expressed as the mean ± s.e.m. from five to nine independent experiments. ***P < 0.001 compared to control.

Figure 4. Role of cytochrome P450 enzymes on CCE induced by low concentrations of TG

Fura-2-loaded human platelets were preincubated for 10 min at 37°C with 10 μm 17-ODYA (B and D) or the vehicle as Control (A and C) and then treated in a Ca²⁺-free medium (100 μm EGTA was added) for 5 min with TG (10 nm) in the absence or presence of 1 μm 5,6-EET, as indicated, followed by addition of CaCl₂ (300 μm) to initiate Ca²⁺ entry. E and F, bars indicate the percentage of Ca²⁺ release (E) and entry (F) under the different experimental conditions relative to their control (vehicle was added). Ca²⁺ release and entry were determined as described in Methods and values are expressed as the mean ± s.e.m. from nine independent experiments. *P < 0.05 compared to control.

Figure 5. Role of cytochrome P450 enzymes on CCE induced by treatment with TBHQ

Fura-2-loaded human platelets were preincubated for 10 min at 37°C with 10 μm 17-ODYA (B) or the vehicle as Control (A) and then treated in a Ca²⁺-free medium (100 μm EGTA was added) for 5 min with TBHQ (20 μm) followed by addition of CaCl₂ (300 μm) to initiate Ca²⁺ entry. Changes in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. Traces are representative of 11 separate experiments.

Figure 6. Effect of Cytochalasin D on 5,6-EET-induced Ca²⁺ entry

Fura-2-loaded human platelets were preincubated for 40 min at 37°C with 10 μm Cyt D or the vehicle as Control and then treated in a Ca²⁺-free medium (100 μm EGTA was added) for 5 min with 1 μm 5,6-EET (A) or 3 μm BN (B) followed by addition of CaCl₂ (300 μm) to initiate Ca²⁺ entry. Changes in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. Traces are representative of six separate experiments.

Figure 7. Effect of 5,6-EET on actin polymerization in platelets

Human platelets were treated with 1 μm 5,6-EET in a Ca²⁺-free medium (100 μm EGTA was added). Samples were removed 5 s before and 5, 10, 30 and 60 s after the addition of 5,6-EET and the actin filament content was determined as described in Methods. Values given are 5,6-EET-evoked actin filament formation as a percentage of control and results are expressed as mean ± s.e.m. of nine separate determinations. *P < 0.05, **P < 0.01 compared to control (resting cells).

Figure 8. Effect of catalase on 5,6-EET-induced Ca²⁺ entry

A, human platelets loaded with CM-H₂DCFDA were stimulated with 1 μm 5,6-EET in a Ca²⁺-free medium (100 μm EGTA was added). Traces are representative of eight independent experiments. B–D, fura-2-loaded human platelets were preincubated for 10 min at 37°C with 300 U ml⁻¹ catalase or the vehicle as Control and then treated in a Ca²⁺-free medium (100 μm EGTA was added) for 5 min with 10 μm H₂O₂ (B), 1 μm 5,6-EET (C) or 1 μm BN (D) followed by addition of CaCl₂ (300 μm) to initiate Ca²⁺ entry. Changes in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. Traces are representative of 10 separate experiments.

Figure 9. Role for protein tyrosine phosphorylation on 5,6-EET-induced Ca²⁺ entry in platelets

A, human platelets were treated with different concentrations of 5,6-EET (0.01–3 μm) in a Ca²⁺-free medium (100 μm EGTA was added). Samples were taken from the platelet suspension 5 s before and 5 min after the addition of different concentrations of 5,6-EET. Platelet proteins were analysed by 10% SDS-PAGE and subsequent Western blotting with a specific anti-phosphotyrosine antibody, and the presence of phosphotyrosine residues quantified by densitometry in Western blots as described. Molecular masses (M) indicated on the right were determined using molecular-mass markers run in the same gel. B, bars represent the integrated optical density for entire lanes under each condition. Results are expressed as fold-increases (mean ± s.e.m. of four separate experiments) over the integrated optical density of resting platelet. *P < 0.05, **P < 0.01 compared to control (resting cells). C, fura-2-loaded human platelets were incubated at 37°C with 1 μg ml⁻¹ M-2,5-DHC or the vehicle for 30 min. At the time of the experiment 100 μm EGTA was added. Cells were then stimulated with 5,6-EET (1 μm) and, 5 min later, CaCl₂ (final concentration 300 μm) was added to the medium to initiate Ca²⁺ entry. Elevations in [Ca²⁺]_i were monitored using the 340/380 nm ratio as described in Methods. Traces shown are representative of four others.

Figure 10. Role of 5,6-EET on TG-induced coupling between IP₃R II and hTRPC1 in human platelets

Platelets were preincubated for 10 min at 37°C in the presence (lane 4) or absence (lanes 1–3) of 10 μm 17-ODYA. Cells were then stimulated with either 5,6-EET (1 μm) or TG (10 nm). Samples were taken 5 s before and 3 min after the addition of 5,6-EET or TG and lysed. Whole cell lysates were immunoprecipitated with anti-hTRPC1 antibody. Immunoprecipitates were analysed by Western blotting using either anti-IP₃R type II polyclonal antibody (upper panel) or anti-hTRPC1 antibody (lower panel) as described in Methods. These results are representative of four independent experiments.