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¹ Unité de Recherche de Biochimie, Institute Superieur de Biotechnologie, Monastir, Tunisia
² Department of Physiology, University of Extremadura, 10071 Cáceres, Spain

	Abstract

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A major pathway for Ca²⁺ entry in non-excitable cells is activated following depletion of intracellular Ca²⁺ stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca²⁺ stores has been proposed as the most likely mechanism to activate this capacitative Ca²⁺ 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 Ca²⁺ store depletion. 5,6-EET-induced Ca²⁺ entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca²⁺ 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 Ca²⁺ entry requires basal levels of H₂O₂, 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 Ca²⁺ store towards the PM to facilitate de novo coupling between IP₃R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP₃R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.

(Received 26 October 2005; accepted after revision 17 November 2005; first published online 24 November 2005)
Corresponding author J. A. Rosado: Department of Physiology, University of Extremadura, Cáceres 10071, Spain. Email: jarosado{at}unex.es

	Introduction

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Capacitative Ca²⁺ entry (CCE) is regulated by the filling state of the intracellular Ca²⁺ stores (Putney, 1986), although the mechanism underlying this process is still not fully understood. A number of hypotheses have been proposed in different cell types to account for the communication between the intracellular Ca²⁺ stores and the plasma membrane (PM), which can be grouped into those that assume the generation of a diffusible molecule, a calcium influx factor (CIF), that gates capacitative Ca²⁺ channels in the PM and those that propose a constitutive physical interaction between Ca²⁺ channels in the PM and inositol 1,4,5-trisphosphate receptors (IP₃R) in the membrane of the intracellular Ca²⁺ stores, the conformational coupling hypothesis (Putney et al. 2001; Venkatachalam et al. 2002).

Recently, a modification of the classical conformational coupling hypothesis has been presented in several non-excitable cells. De novo conformational coupling is proposed to be based on a reversible trafficking of portions of the Ca²⁺ stores towards the PM to facilitate de novo coupling between the IP₃R in the endoplasmic reticulum (ER) and Ca²⁺ channels in the PM (Rosado et al. 2005). In human platelets, where it has been demonstrated, coupling occurs between the type II IP₃R and naturally expressed human canonical transient receptor potential 1 (hTRPC1) (Rosado et al. 2000a, 2004a; Rosado & Sage, 2000a, 2001a). In this process, formerly called ‘secretion-like coupling’, the actin cytoskeleton plays a dual role. Although actin polymerization is required for the activation of CCE, since cytoskeletal disruption impairs Ca²⁺ entry, the cortical actin network acts as a negative modulator of the interaction between the ER and PM (Rosado et al. 2000a).

The proposed alternative to the conformational coupling involves the action of a CIF. Suggested CIFs include cGMP (Pandol & Schoeffield-Payne, 1990), tyrosine kinases (Sargeant et al. 1993), small GTP-binding proteins (Bird & Putney, 1993), a still uncharacterized non-protein CIF (Randriamampita & Tsien, 1993), and a product of cytochrome P450. Cytochrome P450 metabolites have been proposed to act as CIFs based on the finding that cytochrome P450 inhibitors prevent CCE (Alonso-Torre et al. 1993). In particular, 5,6-epoxyeicosatrienoic acid (5,6-EET), a metabolite of cytochrome P450 epoxygenases, has been presented as a CIF (Graier et al. 1995; Xie et al. 2002), although other isomers, such as 11,12-EET (Mombouli et al. 1999) or 14,15-EET (Alvarez et al. 2004), have also been proposed as messengers involved in the activation of CCE. This hypothesis has recently received support from studies that suggest an important role for a Ca²⁺-independent phospholipase A2 in the activation of CCE (Smani et al. 2003, 2004).

The de novo conformational coupling is a unique model that integrates some of the signalling molecules proposed as CIFs, such as tyrosine kinases or small GTP-binding proteins of the Ras family, with actin filament remodelling and conformational coupling between the IP₃R and hTRPC1 channels (Rosado & Sage, 2000b). Hence, we have investigated whether the cytochrome P450 epoxygenase metabolite 5,6-EET is involved in the activation of Ca²⁺ entry in human platelets, and, if this is the case, whether 5,6-EET might be a signalling molecule that participates in the de novo conformational coupling process in these cells.

	Methods

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Materials

Fura-2 acetoxymethyl ester (fura-2/AM), 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA), 2-(2,3-naphthalimino) ethyl trifluoromethanesulphonate (NT) and calcein-AM were from Molecular Probes (Leiden, the Netherlands). Apyrase (grade VII), aspirin, thapsigargin (TG), paraformaldehyde, Nonidet P-40, FITC-labelled phalloidin, ß-naphthoflavone (BN), 17-octadecynoic acid (17-ODYA), methyl 2,5-dihydroxycinnamate (M-2,5-DHC), catalase, valinomycin and bovine serum albumin (BSA) were from Sigma (Madrid, Spain). Cytochalasin D (Cyt D), SKF 96365 and 2-aminoethoxydiphenyl borate (2-APB) were from Calbiochem (Nottingham, UK). 5,6-Epoxyeicosatrienoic acid (5,6-EET) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) were from Alexis (Nottingham, UK). Anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology (Lake Placid, NY, USA). Horseradish peroxidase-conjugated ovine anti-mouse IgG antibody (NA931) was from Amersham (Buckinghamshire, UK). Anti-hTRPC1 polyclonal antibody was from Alomone Laboratories (Jerusalem, Israel). Anti-IP₃R type II polyclonal antibody (C-20), horseradish peroxidase-conjugated donkey anti-goat IgG antibody and horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were of analytical grade.

Platelet preparation

Fura-2-loaded platelets were prepared as previously described (Rosado et al. 2000a) as approved by Local Ethical Committees and in accordance with the Declaration of Helsinki. Briefly, blood was obtained from healthy drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (mM): 85 sodium citrate, 78 citric acid and 111 D-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 g and aspirin (100 µM) and apyrase (40 µg ml^–1) were added. Platelet-rich plasma was incubated at 37°C with 2 µM fura-2 acetoxymethyl ester for 45 min. Cells were then collected by centrifugation at 350 g for 20 min and resuspended in Hepes-buffered saline (HBS), pH 7.45, containing (mM): 145 NaCl, 10 Hepes, 10 D-glucose, 5 KCl, 1 MgSO₄, supplemented with 0.1% BSA and 40 µg ml^–1 apyrase.

Cell viability

Cell viability was assessed using calcein and trypan blue. For calcein loading, cells were incubated for 30 min with 5 µM calcein-AM at 37°C and centrifuged, and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 ml aliquots using a Cary Eclipse Spectrophotometer (Varian Ltd, Madrid, Spain). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. The results obtained with calcein were confirmed using the trypan blue exclusion technique. Ninety-five per cent of cells were viable in our platelet preparations, at least during the performance of the experiments.

Measurement of intracellular free calcium concentration ([Ca²⁺]_i)

Fluorescence was recorded from 2 ml aliquots of magnetically stirred platelet suspension (2x 10⁸ cells ml^–1) at 37°C using a fluorescence spectrophotometer with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca²⁺]_i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. (1985). Mn²⁺ influx was monitored as a quenching of fura-2 fluorescence at the isoemissive wavelength of 360 nm, which is presented on an arbitrary linear scale (Sage et al. 1989).

Ca²⁺ entry was estimated using the integral of the rise in [Ca²⁺]_i for 45 s after addition of CaCl₂ (Rosado et al. 2000a). Control experiments were performed for all experimental procedures in order to correct Ca²⁺ entry by subtraction of the [Ca²⁺]_i elevation due to leakage of the indicator. To calculate the initial rate of Ca²⁺ elevation after the addition of Ca²⁺ to the medium, the traces were fitted to the equation y=Ax (1 – e^–Kx), and to estimate the initial rate of fura-2 fluorescence quenching after the addition of Mn²⁺ to the medium, the traces were fitted to the equation y=Sx e^–Kx+A, where K is the slope, S is the span and A is the plateau. Ca²⁺ release was estimated using the integral of the rise in [Ca²⁺]_i for 3 min after the addition of 5,6-EET, BN or TG. Both Ca²⁺ entry and release are expressed as nanomolar taking a sample every second (nM· s), as previously described (Heemskerk et al. 1997; Rosado & Sage, 2000c).

Immunoprecipitation and Western blotting

The immunoprecipitation and Western blotting were performed as previously described (Rosado & Sage, 2000a). Briefly, 500 µl aliquots of platelet suspension (2 x 10⁹ cell ml^–1) were lysed with an equal volume of lysis buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM Na₃VO₄, 2 mM phenylmethylsulphonyl fluoride, 100 µg ml^–1 leupeptin and 10 mM benzamidine. Aliquots of platelet lysates (1 ml) were immunoprecipitated by incubation with 2 µg of anti-hTRPC1 polyclonal antibody and 25 µl of protein A–agarose overnight at 4°C on a rocking platform. The immunoprecipitates were resolved by 8% SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of IP₃R type II and hTRPC1 was achieved using the anti-IP₃RII polyclonal antibody diluted 1: 500 in TBST for 3 h or the anti-hTRPC1 antibody diluted 1: 200 in TBST. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG antibody or horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody diluted 1: 10 000 in TBST and then exposed to enhanced chemiluminescence reagents for 1 min. Blots were then exposed to photographic films. The density of bands on the film was measured using a scanning densitometry.

Protein tyrosine phosphorylation

Protein tyrosine phosphorylation was detected by gel electrophoresis and Western blotting (Rosado & Sage, 2000a). Platelets stimulation was terminated by mixing with an equal volume of 2 x Laemmli's buffer (Laemmli, 1970) with 10% dithiothreitol followed by heating for 5 min at 95°C. One-dimensional SDS-electrophoresis was performed with 10% polyacrylamide minigels, and separate proteins were electrophoretically transferred, for 2 h at 0.8 mA cm^–2, in a semidry blotter (Hoefer Scientific, Newcastle-under-Lyne, Staffordshire, UK) onto nitrocellulose for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of tyrosine phosphorylation was achieved using the anti-phosphotyrosine antibody 4G10 diluted 1: 2500 in TBST for 1 h. The primary antibody was removed and blots washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated ovine anti-mouse IgG antibody diluted 1: 10000 in TBST, washed six times in TBST, and exposed to enhanced chemiluminescence reagents for 5 min. Blots were then exposed to photographic films and the optical density of the entire lane was estimated using scanning densitometry.

Measurement of F-actin content

The F-actin content of resting and activated platelets was determined according to a previously published procedure (Rosado & Sage, 2000c). Briefly, washed platelets (2 x 10⁸ cells ml^–1) were activated in HBS. Samples of platelet suspension (200 µl) were transferred to 200 µl ice-cold 3% (w/v) formaldehyde in phosphate-buffered saline (PBS) for 10 min. Fixed platelets were permeabilized by incubation for 10 min with 0.025% (v/v) Nonidet P-40 detergent dissolved in PBS. Platelets were then incubated for 30 min with FITC-labelled phalloidin (1 µM) in PBS supplemented with 0.5% (w/v) bovine serum albumin. After incubation the platelets were collected by centrifugation for 90 s at 3000 g and resuspended in PBS. Staining of 2 x 10⁷ cells ml^–1 was measured using a fluorescence spectrophotometer. Samples were excited at 496 nm and emission was at 516 nm.

Intracellular ROS production through the oxidation of CM-H₂DCFDA

CM-H₂DCFDA is a ROS-sensitive probe that can be used to detect ROS production in living cells. It passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. CM-H₂DCFDA oxidation yields a fluorescent adduct, dichlorofluorescein (DCF) that is trapped inside the cell (Zhang et al. 2003). Cells were incubated at 37°C with 10 µM CM-H₂DCFDA acetyl ester for 30 min, then centrifuged and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 ml aliquots using a fluorescence spectrophotometer. Samples were excited at 488 nm and the resulting fluorescence was measured at 530 nm. ROS levels were quantified as the integral of the rise in DCF fluorescence for 5 min after platelet treatment.

Determination of EET production

EET production was determined following a method based on a previously published procedure (Nithipatikom et al. 2000). Briefly, platelets (2 x 10⁸ cells ml^–1) were preincubated in the absence or presence of 10 µM 17-ODYA. Cells were stimulated in with 10 nM TG or 20 µM TBHQ for 5 min at 37°C or left untreated and then fixed with ice-cold 3% (w/v) formaldehyde in PBS for 10 min, as described above, and sonicated. Freshly prepared NT (1 mM) in anhydrous acetonitrile was added to the samples and vortexed lightly for 2 s as previously described (Yue et al. 2004). The reaction tubes were placed in the dark at room temperature for 15 min and fluorescence was determined using a fluorescence spectrophotometer. Samples were excited at 259 nm and emission was at 394 nm.

Statistical analysis

Analysis of statistical significance was performed using Student's t test. P < 0.05 was considered to be significant for a difference.

	Results

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Treatment of platelets with 5,6-EET induces divalent cation entry

In the absence of extracellular Ca²⁺, treatment of fura-2-loaded human platelets in stirred cuvettes at 37°C with different concentrations of 5,6-EET has a negligible effect on Ca²⁺ release from the intracellular stores (Fig. 1). Only when platelets were stimulated with 3 µM 5,6-EET was a significant Ca²⁺ release from the intracellular stores detected (Fig. 1B; P < 0.05). Interestingly, subsequent addition of Ca²⁺ (300 µM) to the suspension of 5,6-EET-treated platelets resulted a concentration-dependent increase in [Ca²⁺]_i indicative of Ca²⁺ entry (Fig. 1). A significant Ca²⁺ entry was detected at 0.01 µM 5,6-EET (the integral of the rise in [Ca²⁺]_i above basal for 45 s after addition of Ca²⁺ taking data every second was 350 ± 86 nM· s) and reached a maximum after treatment of platelets with 3 µM 5,6-EET (the integral of the rise in [Ca²⁺]_i above basal was 1008 ± 157 nM· s; n= 6). The initial rate of Ca²⁺ elevation after the addition of Ca²⁺ to the external medium was similar for all the concentrations of 5,6-EET tested (the initial slope was 0.0589 ± 0.0057, 0.0571 ± 0.0052, 0.0575 ± 0.0055 and 0.0561 ± 0.0048 for 3, 1, 0.1 and 0.01 µM 5,6-EET). As observed in Fig. 1A, dashed trace, in the absence of stimulus Ca²⁺ entry in platelets is negligible, which indicates that the elevation in [Ca²⁺]_i above basal after addition of CaCl₂ to EET-treated cells was not due to leakage of the indicator.

View larger version (31K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

In order to investigate the nature of the cation entry induced by platelet treatment with 5,6-EET we have examined the effect of 2-APB and SKF 96365, two CCE blockers in human platelets (Jenner & Sage, 2000; Diver et al. 2001) and other cell types (Enfissi et al. 2004; Gratschev et al. 2004; Ng et al. 2005), on Ca²⁺ entry induced by 1 µM 5,6-EET, the highest concentration used that induces Ca²⁺ entry without any detectable Ca²⁺ release (see Fig. 1). As shown in Fig. 3A, B and F, in the presence of 100 µM 2-APB or 10 µM SKF 96365, 1 µM 5,6-EET was unable to induce Ca²⁺ entry (P < 0.001; n= 5–9), suggesting that this process exhibits pharmacological properties characteristics of CCE. To confirm this possibility we explored the effect of 5,6-EET on Ca²⁺ entry in the presence of La³⁺ or Ni²⁺, two well described blockers of CCE (Wang et al. 2004). As depicted in Figs 3C, D and F, both cations abolished Ca²⁺ entry induced by 1 µM 5,6-EET (P < 0.001; n= 5). These findings suggest that 5,6-EET-induced Ca²⁺ entry, in the range of 0.01–1 µM, shows characteristics of CCE in human platelets without any detectable Ca²⁺ release from the internal stores.

View larger version (22K): [in this window] [in a new window]   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 (TRPC1 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.

Role of cytochrome P450 enzymes in CCE in human platelets

We have recently identified two mechanisms for CCE in human platelets activated by depletion of two independent Ca²⁺ pools (Rosado et al. 2004b). The major store, presumably the dense tubular system (DTS), is sensitive to low concentrations of TG, while the acidic store is sensitive to TBHQ and high concentrations of TG (Lopez et al. 2005a). We have now investigated whether depletion of these stores is able to induce EET production determined using the fluorescent indicator NT, which reacts with EETs and forms highly fluorescent derivatives (Maier et al. 2000; Nithipatikom et al. 2000). Treatment of platelets with 10 nM TG to deplete the DTS (Lopez et al. 2005a) for 5 min significantly increased NT fluorescence to 121.3 ± 6.8% of control (P < 0.05; n= 12). In contrast TBHQ (20 µM), used to deplete the acidic stores, did not significantly modify NT fluorescence (NT fluorescence after treatment with TBHQ was 93.2 ± 4.7% of control; n= 12). To examine whether the increase in NT fluorescence induced by TG was due to EET production we repeated the experiments in the presence of 17-ODYA, a substrate inhibitor that selectively and irreversibly inhibits cytochrome P450 epoxygenases and -hydrolases (Dong et al. 1997). Preincubation of platelets for 10 min at 37°C with 10 µM 17-ODYA prevented the TG-evoked increase in NT fluorescence (in the presence of 17-ODYA, NT fluorescence in TG-treated cells was 100.6 ± 5.9% of control; n= 12) while having no significant effects on NT fluorescence in resting or TBHQ-treated platelets (NT fluorescence was 100.7 ± 9.6 and 95.5 ± 3.3% of control, in non-stimulated and TBHQ-treated platelets pretreated with 17-ODYA, respectively; n= 12). These findings suggest that the increase in NT fluorescence observed in platelets stimulated with TG is due to EET production by cytochrome P450 epoxygenases.

Hence, we have further investigated the role of these enzymes on CCE induced by low concentrations of TG in platelets. In the absence of extracellular Ca²⁺, addition of TG (10 nM) to fura-2-loaded human platelets in stirred cuvettes at 37°C evoked a prolonged elevation in [Ca²⁺]_i due to release of Ca²⁺ from internal stores. Subsequent addition of Ca²⁺ (300 µM) to the external medium induced a sustained increase in [Ca²⁺]_i indicative of CCE (Fig. 4A). Pretreatment of human platelets with 17-ODYA (10 µM), significantly reduced CCE induced by 10 µM TG by 42% (Fig. 4B and F; P < 0.05; n= 9). The initial slope for the rise in [Ca²⁺]_i after the addition of Ca²⁺ was significantly reduced from 0.0682 ± 0.0060 to 0.0422 ± 0.0042 in control and 17-ODYA-treated cells, respectively (P < 0.05). Treatment with 17-ODYA had no significant effects on Ca²⁺ release from the intracellular store, indicating that accumulation of Ca²⁺ in the DTS was unaffected by inhibition of cytochrome P450 epoxygenases (Fig. 4B and E; n= 9).

View larger version (26K): [in this window] [in a new window]   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.

To confirm that the inhibitory effect of 17-ODYA on CCE was mediated by inhibition of the cytochrome P450 epoxygenases we performed a series of experiments adding 5,6-EET (1 µM) at the same time as TG. As shown in Fig. 4C, E and F, treatment of platelets with a combination of TG plus 5,6-EET induced a similar Ca²⁺ mobilization as TG alone (Fig. 4A). In addition, platelet stimulation with TG + 5,6-EET overcame the effect of 17-ODYA on TG-induced CCE (Fig. 4D and F; n= 9). These findings indicate that the effect of 17-ODYA is mediated by inhibition of cytochrome P450 epoxygenases. Cytochrome P450 arachidonic acid epoxygenases catalyse the metabolism of endogenous arachidonic acid to 5,6-EET, 8,9-EET, 10,11-EET and 14,15-EET (Roman, 2002). In addition, these observations indicate that the product of cytochrome P450 epoxygenases, specifically 5,6-EET, is required for the activation of CCE by low concentrations of TG in human platelets.

We have further investigated the role of cytochrome P450 epoxygenases and 5,6-EET in CCE evoked by TBHQ. As shown in Fig. 5A, treatment of platelets with 20 µM TBHQ in the absence of extracellular Ca²⁺ resulted in a small rise in [Ca²⁺]_i as the acidic Ca²⁺ stores depleted and the subsequent addition of CaCl₂ (300 µM) resulted in a rise in [Ca²⁺]_i indicative of CCE (n= 11). Preincubation of platelets for 10 min with 10 µM 17-ODYA did not significantly modify TBHQ-induced Ca²⁺ release or entry, which indicates that the products of cytochrome P450 epoxygenases are not required either for Ca²⁺ release or for the activation of CCE induced by depletion of the acidic stores in human platelets (Fig. 5B; n= 11). These findings are consistent with the lack of EET production by TBHQ reported above. In addition, these findings suggest that 17-ODYA is not a Ca²⁺ chelators or Ca²⁺ channel blocker.

View larger version (14K): [in this window] [in a new window]   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.

Both pathways for the activation of CCE in human platelets are differentially modulated by the actin cytoskeleton, so that actin disassembly by Cyt D impairs CCE mediated by depletion of the DTS by low concentrations of TG while facilitates Ca²⁺ entry stimulated by depletion of the acidic stores using TBHQ (Rosado et al. 2004b; Lopez et al. 2005b). To further investigate whether 5,6-EET is required solely for CCE induced by low TG concentrations we examine the effect of Cyt D on Ca²⁺ entry evoked by 5,6-EET.

As shown in Fig. 6A, pretreatment of platelets for 40 min with 10 µM Cyt D abolished Ca²⁺ entry induced by 5,6-EET (1 µM; P < 0.001; n= 6). A similar result was observed when 5,6-EET was endogenously generated by using the cytochrome P450 inducer BN (Graier et al. 1995; Xie et al. 2002). Induction of cytochrome P450 by BN (3 µM) led to a significant Ca²⁺ entry in human platelets, similar to that found with (1 µM) 5,6-EET, whereas intracellular Ca²⁺ release remained unchanged. In Cyt D-pretreated cells, BN was unable to induce Ca²⁺ entry (Fig. 6B; P < 0.001; n= 6). These findings indicate that inhibition of actin polymerization prevents Ca²⁺ entry mediated by 5,6-EET.

View larger version (13K): [in this window] [in a new window]   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.

In human platelets, depletion of the DTS mediates the activation of CCE by a de novo conformational coupling between the IP₃R type II and hTRPC1 (Rosado et al. 2005). A number of cellular events have been shown to be involved in the activation of this process, including H₂O₂ generation, activation of tyrosine kinases and actin filament reorganization (Rosado & Sage, 2000d; Xie et al. 2002; Rosado et al. 2004a, 2005). We have now investigated whether the involvement of 5,6-EET in Ca²⁺ entry in platelets might be consistent with the de novo conformational coupling.

We have described above that Cyt D impairs 5,6-EET-evoked Ca²⁺ entry, which indicates that actin filament reorganization is required for this mechanism. In addition, we have now further investigated the involvement of 5,6-EET in the de novo conformational coupling model by exploring the role of 5,6-EET on actin filament reorganization, a process required for the activation of CCE by de novo conformational coupling in platelets. Treatment of human platelets with 1 µM 5,6-EET induced a rapid increase in the actin filament content, which was maximal 10 s after 5,6-EET stimulation and then decreased (Fig. 7).

View larger version (12K): [in this window] [in a new window]   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).

View larger version (28K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Cytochrome P450 metabolites, such as EET isomers (EETs), have been presented as components of the CIF pathway (Alonso-Torre et al. 1993). Numerous physiological roles have been suggested for the EETs and, collectively, EETs appear to have clear effects on ion channels (Campbell et al. 1996). The 5,6-EET has been reported as a diffusible messenger involved in the activation of CCE in different cell types, including astrocytes (Rzigalinski et al. 1999) and endothelial cells (Graier et al. 1995; Xie et al. 2002). Consistent with this, we have now found that 5,6-EET is likely to be a CCE activator in human platelets. In these cells, 5,6-EET, as well as the cytochrome P450 inducer BN, induces Ca²⁺ entry through cation channels, also permeable to Mn²⁺, similar to those conducting CCE. Ca²⁺ entry by 5,6-EET was found to be inhibited by 2-APB, SKF 96365 and the divalent and trivalent cations Ni²⁺ and La³⁺, which indicates that this process shows properties characteristic of CCE. Furthermore, 5,6-EET-stimulated Ca²⁺ entry was blocked by incubation with the anti-hTRPC1 antibody, which we have previously reported to impair CCE in human platelets (Rosado et al. 2002), providing evidence for a role of hTRPC1 in the conduction of Ca²⁺ entry by 5,6-EET.

It has recently been reported that there are two functionally separated Ca²⁺ stores in human platelets, an IP₃-sensitive major store with high affinity to TG, corresponding to the DTS, and an acidic store with low affinity to TG but sensitive to TBHQ and NAADP (Cavallini et al. 1995; Kovacs et al. 1997; Lopez et al. 2005a, b). We now report that depletion of the DTS induces EET production, and using the substrate inhibitor of cytochrome P450 epoxygenases, 17-ODYA, we have found that 5,6-EET is involved in CCE induced by depletion of the DTS but not in CCE induced by discharge of the acidic stores. Cytochrome P450 enzymes produce a number of EET isomers; however, our results indicate that among them, 5,6-EET is involved in Ca²⁺ entry in human platelets since its addition overcame the effect of 17-ODYA on TG-induced CCE.

These results are confirmed by the effect of Cyt D on Ca²⁺ entry induced by 5,6-EET. We have previously shown that CCE induced by depletion of the DTS by TG and the acidic stores by TBHQ are differentially regulated by the actin cytoskeleton, which plays a dual role in the activation of CCE by TG: a positive role as a support for the transport of portions of the Ca²⁺ store to the proximity of the PM to allow coupling to occur, which is impaired by Cyt D, and a negative effect provided by the membrane-associated cytoskeleton to prevent constitutive activation of Ca²⁺ entry (Rosado et al. 2004b). In contrast, TBHQ-induced CCE is regulated by the membrane cytoskeleton, which acts only as a physical actin barrier to prevent coupling between the Ca²⁺ stores and PM under resting conditions and therefore is facilitated by Cyt D (Rosado et al. 2004b). If the 5,6-EET is involved in TG-induced CCE one would postulate that Cyt D would inhibit 5,6-EET-stimulated Ca²⁺ entry. In contrast, an increase in Ca²⁺ entry activated by 5,6-EET would be expected if 5,6-EET were a component of the TBHQ-activated pathway. The inhibitory effect of Cyt D on Ca²⁺ entry induced by exogenous or endogenously generated 5,6-EET clearly confirms that 5,6-EET is a component of the mechanism involved in the activation of CCE upon depletion of the DTS by low concentrations of TG, the so called de novo conformational coupling.

We have found that 5,6-EET-mediated Ca²⁺ entry, as well as the de novo conformational coupling (Rosado et al. 2004a), requires a favourable redox state, where H₂O₂, a powerful oxidizing compound (Törnquist et al. 2000), plays an important role. Although speculative, H₂O₂ might maintain an oxidative redox state that delays 5,6-EET degradation. These findings might provide an explanation for the requirement of H₂O₂ for the activation of Ca²⁺ entry in human platelets (Rosado et al. 2004a).

To further support the involvement of 5,6-EET in the de novo conformational coupling, we provide evidence supporting a role for 5,6-EET in the activation of protein tyrosine kinases and actin filament polymerization. A role for tyrosine kinases in CCE has been suggested in several cell types (Yule et al. 1994; Camello et al. 1999), including platelets (Vostal et al. 1991; Sargeant et al. 1993, 1994), where we have recently reported the involvement of tyrosine kinases in the de novo conformational coupling mediated by the reorganization of the actin cytoskeleton (Rosado et al. 2000b). We show that 5,6-EET induces a concentration-dependent increase in the phosphotyrosine content of platelet proteins, which together with the effect of the tyrosine kinase inhibitor, M-2,5-DHC, supports that tyrosine kinases are involved in 5,6-EET-evoked Ca²⁺ entry. We have previously reported that the de novo conformational coupling requires a mechanical support provided by the cytosolic actin filament network (Rosado et al. 2000a). Inhibition of actin reorganization by Cyt D prevented EET-induced Ca²⁺ entry, which supports that the actin polymerizing mechanism activated by 5,6-EET is likely to be involved in 5,6-EET-induced Ca²⁺ entry.

The most clear evidence of the involvement of 5,6-EET in the activation of Ca²⁺ entry by a de novo conformational coupling in platelets comes from the findings that demonstrate that 5,6-EET activates the coupling between the IP₃R type II and hTRPC1 at the concentration that induce Ca²⁺ entry, which is impaired by 17-ODYA. The effect of 5,6-EET on the coupling between the IP₃R type II and hTRPC1 was smaller than that induced by TG suggesting that an independent pathway must be involved in this process. We have recently demonstrated in platelets that TG-induced coupling between the IP₃R type II and hTRPC1, and subsequently CCE, is mediated by both cytoskeleton-dependent and -independent pathways (Rosado & Sage, 2001b). Since inhibition of actin polymerization by Cyt D resulted in complete inhibition of 5,6-EET-induced Ca²⁺ entry, this messenger might only be a component of the cytoskeleton-dependent branch of the cellular machinery for the activation of coupling between the IP₃R type II and hTRPC1 and CCE by TG.

In summary, we have shown that 5,6-EET induces Ca²⁺ entry without having any effect on Ca²⁺ store depletion in human platelets. 5,6-EET is likely to be a messenger molecule involved in Ca²⁺ entry mediated by depletion of the DTS, compatible with the de novo conformational coupling, where store depletion might stimulate the synthesis of 5,6-EET, which, in turns, induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton. Actin filament remodelling might provide a support for the transport of portions of the Ca²⁺ store towards the PM to allow the de novo coupling of IP₃R type II to hTRPC1, which we have suggested may underlie the activation of CCE in human platelets (Rosado et al. 2000a, 2002, 2004a, 2005; Rosado & Sage, 2000a, 2001a).

	References

Top Abstract Introduction Methods Results Discussion References

 
Alonso-Torre SR, Alvarez J, Montero M, Sanchez A & Garcia-Sancho J (1993). Control of Ca²⁺ entry into HL60 and U937 human leukaemia cells by the filling state of the intracellular Ca²⁺ stores. Biochem J 289, 761–766.[Medline]

Alvarez DF, Gjerde EA & Townsley MI (2004). Role of EETs in regulation of endothelial permeability in rat lung. Am J Physiol Lung Cell Mol Physiol 286, 445–451.[CrossRef]

Beech DJ, Muraki K & Flemming R (2004). Non-selective cationic channels of smooth muscle and the mammalian homologues of Drosophila TRP. J Physiol 559, 685–706.[Abstract/Free Full Text]

Bergdhal A, Gómez MF, Dreja K, Xu SZ, Adner M, Beech DJ, Broman J, Hellstrand P & Sward K (2003). Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca²⁺ entry dependent on TRPC1. Circ Res 93, 839–847.[Abstract/Free Full Text]

Bird GS & Putney JW (1993). Inhibition of thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement of a small G-protein in capacitative calcium entry. J Biol Chem 268, 21486–21488.[Abstract/Free Full Text]

Bode HP & Goke B (1994). Protein kinase C activates capacitative Ca²⁺ entry in the insulin secreting cell line RINm5F. FEBS Lett 339, 307–311.[CrossRef][Medline]

Camello C, Pariente JA, Salido GM & Camello PJ (1999). Sequential activation of different Ca²⁺ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells. J Physiol 516, 399–408.[Abstract/Free Full Text]

Campbell WB, Gebremedhin D, Pratt PF & Harder DR (1996). Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78, 415–423.[Abstract/Free Full Text]

Cavallini L, Coassin M & Alexandre A (1995). Two classes of agonist-sensitive Ca²⁺ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin. Biochem J 310, 449–452.[Medline]

Diver JM, Sage SO & Rosado JA (2001). The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca²⁺ entry channels in human platelets: cautions for its use in studying Ca²⁺ influx. Cell Calcium 30, 323–329.[CrossRef][Medline]

Dong H, Waldron GJ, Galipeau D, Cole WC & Triggle CR (1997). NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery. Br J Pharmacol 120, 695–701.[Medline]

Enfissi A, Prigent S, Colosetti P & Capiod T (2004). The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells. Cell Calcium 36, 459–467.[CrossRef][Medline]

Fasolato C, Hoth M & Penner R (1993). A GTP-dependent step in the activation mechanism of capacitative calcium influx. A GTP-dependent step in the activation mechanism of capacitative calcium. Influx J Biol Chem 268, 20737–20740.

Graier W, Simecek S & Sturek M (1995). Cytochrome P450 mono-oxygenase-regulated signalling of Ca²⁺ entry in human and bovine endothelial cells. J Physiol 482, 259–274.[Medline]

Gratschev D, Blom T, Bjorklund S & Tornquist K (2004). Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry. J Biol Chem 279, 49816–49824.[Abstract/Free Full Text]

Grynkiewicz G, Poenie M & Tsien RY (1985). A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260, 3440–3450.[Abstract/Free Full Text]

Heemskerk JW, Feijge MA, Henneman L, Rosing J & Hemker HC (1997). The Ca²⁺-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets – concentration and time effects of thrombin-induced Ca²⁺ signaling. Eur J Biochem 249, 547–555.[Medline]

Jenner S & Sage SO (2000). Two pathways for store-mediated calcium entry in human platelets. Platelets 11, 215–221.[CrossRef][Medline]

Kovacs T, Berger G, Corvazier E, Paszty K, Brown A, Bobe R et al. (1997). Immunolocalization of the multi-sarco/endoplasmic reticulum Ca²⁺ ATPase system in human platelets. Br J Haematol 97, 192–203.[CrossRef][Medline]

Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]

Liu XB, Wang WC, Singh BB, Lockwich T, Jadlowiec J, O'Connell B, Wellner R, Zhu MX & Ambudkar IS (2000). Trp1, a candidate protein for the store-operated Ca²⁺ influx mechanism in salivary gland cells. J Biol Chem 275, 3403–3411.[Abstract/Free Full Text]

Lopez JJ, Camello-Almaraz C, Pariente JA, Salido GM & Rosado JA (2005a). Ca²⁺ accumulation into acidic organelles mediated by Ca²⁺- and vacuolar H⁺-ATPases in human platelets. Biochem J 390, 243–252.[CrossRef][Medline]

Lopez JJ, Redondo PC, Pariente JA, Salido GM & Rosado JA (2005b). Two distinct Ca²⁺ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets. Cell Signal 18, 373–381.[CrossRef][Medline]

Luo Y, Umegaki H, Wang X, Abe R & Roth GS (1998). Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem 273, 3756–3764.[Abstract/Free Full Text]

Ma R, Pluznick J, Kudlacek P & Sansom SC (2001). Protein kinase C activates store-operated Ca²⁺ channels in human glomerular mesangial cells. J Biol Chem 276, 25759–25765.[Abstract/Free Full Text]

Mahaut-Smith MP, Rink TJ, Collins SC & Sage SO (1990). Voltage-gated potassium channels and the control of membrane potential in human platelets. J Physiol 428, 723–735.[Abstract/Free Full Text]

Maier KG, Henderson L, Narayanan J, Alonso-Galicia M, Falck JR & Roman RJ (2000). Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. Am J Physiol Heart Circ Physiol 279, H863–H871.[Abstract/Free Full Text]

Mombouli JV, Holzmann S, Kostner GM & Graier WF (1999). Potentiation of Ca²⁺ signaling in endothelial cells by 11,12-epoxyeicosatrienoic acid. J Cardiovasc Pharmacol 33, 779–784.[CrossRef][Medline]

Ng LC, Wilson SM & Hume JR (2005). Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca²⁺ entry in isolated canine pulmonary arterial smooth muscle cells. J Physiol 563, 409–419.[Abstract/Free Full Text]

Nithipatikom K, Pratt PF & Campbell WB (2000). Determination of EETs using microbore liquid chromatography with fluorescence detection. Am J Physiol Heart Circ Physiol 279, H857–H862.[Abstract/Free Full Text]

Pandol SJ & Schoeffield-Payne MS (1990). Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell. J Biol Chem 265, 12846–12853.[Abstract/Free Full Text]

Parekh AB & Putney JW Jr (2005). Store-operated calcium channels. Physiol Rev 85, 757–810.[Abstract/Free Full Text]

Putney JW Jr (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1–12.[Medline]

Putney JW Jr, Broad LM, Braun FJ, Lievremont JP & Bird GS (2001). Mechanisms of capacitative calcium entry. J Cell Sci 114, 2223–2229.[Medline]

Randriamampita C & Tsien RY (1993). Emptying of intracellular Ca²⁺ stores releases a novel small messenger that stimulates Ca²⁺ influx. Nature 364, 809–814.[CrossRef][Medline]

Redondo PC, Lajas AI, Salido GM, González A, Rosado JA & Pariente JA (2003). Evidence for secretion-like coupling involving pp60^src in the activation and maintenance of store-mediated Ca²⁺ entry in mouse pancreatic acinar cells. Biochem J 370, 255–263.[CrossRef][Medline]

Roman RJ (2002). P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82, 131–185.[Abstract/Free Full Text]

Rosado JA, Brownlow SL & Sage SO (2002). Endogenously expressed Trp1 is involved in store-mediated Ca²⁺ entry by conformational coupling in human platelets. J Biol Chem 277, 42157–42163.[Abstract/Free Full Text]

Rosado JA, Graves D & Sage SO (2000b). Tyrosine kinases activate store-mediated Ca²⁺ entry in human platelets through the reorganization of the actin cytoskeleton. Biochem J 351, 429–437.[CrossRef][Medline]

Rosado JA, Jenner S & Sage SO (2000a). A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling. J Biol Chem 275, 7527–7533.[Abstract/Free Full Text]

Rosado JA, Lopez JJ, Harper AG, Harper MT, Redondo PC, Pariente JA et al. (2004b). Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets. J Biol Chem 279, 29231–29235.[Abstract/Free Full Text]

Rosado JA, Redondo PC, Sage SO, Pariente JA & Salido GM (2005). Store-operated Ca²⁺ entry: Vesicle fusion or reversible trafficking and de novo conformational coupling?J Cell Physiol 205, 262–269.[CrossRef][Medline]

Rosado JA, Redondo PC, Salido GM, Gomez-Arteta E, Sage SO & Pariente JA (2004a). Hydrogen peroxide generation induces pp60^src activation in human platelets, evidence for the involvement of this pathway in store-mediated calcium entry. J Biol Chem 279, 1665–1675.[Abstract/Free Full Text]

Rosado JA & Sage SO (2000a). Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca²⁺ stores are depleted. Biochem J 350, 631–635.[CrossRef][Medline]

Rosado JA & Sage SO (2000b). A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Trends Cardiovasc Med 10, 327–332.[CrossRef][Medline]

Rosado JA & Sage SO (2000c). Farnesylcysteine analogues inhibit store-regulated Ca²⁺ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton. Biochem J 347, 183–192.[CrossRef][Medline]

Rosado JA & Sage SO (2000d). The actin cytoskeleton in store-mediated calcium entry. J Physiol 526, 221–229.[Abstract/Free Full Text]