Cell Calcium 55 (2014) 200–207
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Transient receptor potential ankyrin 1 (TRPA1) channel activation by the thienopyridine-type drugs ticlopidine, clopidogrel, and prasugrel夽 Anja Schulze 1 , Philipp Hartung 1 , Michael Schaefer, Kerstin Hill ∗ Rudolf-Boehm-Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Leipzig, Germany
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Article history: Received 1 November 2013 Received in revised form 24 January 2014 Accepted 14 February 2014 Available online 25 February 2014 Keywords: TRPA1 Serotonin Ticlopidine Clopidogrel Prasugrel Thienopyridine QGP-1
a b s t r a c t Transient receptor potential A1 (TRPA1) is widely expressed throughout the human and animal organism, including the dorsal root ganglia as well as the bladder, stomach and small intestine. Here, we examined the effect of three platelet aggregation inhibitors on TRPA1: ticlopidine, clopidogrel and prasugrel. Utilising fluorometric Ca2+ influx analysis and electrophysiological whole cell measurements in TRPA1-expressing HEK293 and in human enterochromaffin-like QGP-1 cells, we found that ticlopidine, clopidogrel and prasugrel are direct activators of TRPA1. Although this polymodal channel commonly contributes to the perception of pain, temperature and chemical irritants, recent studies provide evidence for its involvement in the release of serotonin (5-HT) from enterochromaffin cells. Therefore, we further investigated the ability of ticlopidine, clopidogrel and prasugrel to stimulate 5-HT release from QGP-1 cells. We could determine 5-HT in supernatants from cultured QGP-1 cells upon treatment with ticlopidine and clopidogrel but not with prasugrel. These findings indicate that a robust TRPA1 activation by ticlopidine and clopidogrel correlates with the stimulatory effect on the secretion of 5-HT. As recipients of ticlopidine and clopidogrel frequently complain about gastrointestinal adverse events such as nausea, vomiting and diarrhoea, an activation of TRPA1 may contribute to adverse effects of such drugs in the digestive system. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Thienopyridines such as ticlopidine, clopidogrel and prasugrel are important drugs to prevent platelet aggregation in coronary artery, peripheral vascular and cerebrovascular disease. The compounds do not exhibit direct antiaggregant activity in vitro, but have to be converted to their active form by hepatic metabolisation in vivo [1]. The active metabolites then form disulfide bonds with cysteine residues in extracellular loops of the P2Y12 receptor of platelets, and irreversibly inhibit aggregation by blocking the ADP-binding site [2,3]. Therapies of many diseases are commonly accompanied by adverse effects including gastrointestinal (GI) disorders. Nausea, vomiting and diarrhoea are the most prevalent adverse effects
夽 This work was supported by the Deutsche Forschungsgemeinschaft [HI 829/2-1 to Kerstin Hill and GRK1097 to Michael Schaefer]. ∗ Corresponding author at: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany. Tel.: +49 0341 9724 616; fax: +49 0341 9724 609. E-mail address: [email protected] (K. Hill). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.ceca.2014.02.014 0143-4160/© 2014 Elsevier Ltd. All rights reserved.
observed in clinical trials evaluating the platelet aggregation inhibitors ticlopidine and clopidogrel [4,5], while recipients of prasugrel, a third generation antiplatelet drug, show fewer incidences of GI disturbances. With clopidogrel being one of the world’s blockbuster drugs, this limitation may be important for its clinical use. In general, the mechanisms causing nausea and vomiting are diverse: afferent vagal neurones of the intestinal mucosa can be stimulated by serotonin (5-hydroxytryptamine, 5-HT), which is released from endocrine cells in response to various stimuli [6]. Excitement of vagal nerve ends leads to the stimulation of the chemoreceptive trigger zone (CTZ) in the area postrema, resulting in the activation of the vomiting centre, located in the dorsal portion of the lateral reticular formation [7,8]. Alternatively, nausea may result from a direct binding of drugs, toxins or neurotransmitters, such as 5-HT to receptors within the CTZ [9]. Other pathways include the involvement of the vestibular system or the higher cortical centre [10]. The Ca2+ -permeable ion channel transient receptor potential ankyrin1 (TRPA1) is activated by several drugs (clotrimazole, apomorphine), toxicants and pollutants (acrolein, formaldehyde) and food ingredients, which produce a pungent and sharp sensation (AITC, allyl isothiocyanate from mustard, allicin from garlic) [11–16]. TRPA1 is localised in sensory neurones of the dorsal root
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and trigeminal ganglia, where it contributes to the perception of chemical pain and possibly to cold and mechanical stimuli [17,18] but is also expressed in peripheral nerve fibres in the stomach, small intestine and colon of rat and mouse [19–24]. In addition, recent studies on the expression and physiological function of TRPA1 also described the presence of the channel in non-neuronal cells, e.g. in 5-HT-containing enterochromaffin (EC) cells, and in cholecystokinin-releasing endocrine cells of the GI tract [25–27]. In this context it has been shown, that the administration of AITC induced TRPA1-dependent vomiting in dogs and contractions in the colon of mice and dogs [28,29]. Furthermore, we recently demonstrated the involvement of TRPA1 in the secretion of 5-HT from the human enterochromaffin model cell line QGP-1 induced by apomorphine, a drug with strong emetic properties [12]. Here, we tested three generations of P2Y12 receptor antagonists, ticlopidine, clopidogrel and prasugrel with regard to their activation of TRPA1 in HEK293TRPA1 and QGP-1 cells. Each substance provoked an increase in Ca2+ influx through stimulation of TRPA1. The first generation thienopyridine ticlopidine caused the most pronounced TRPA1-evoked Ca2+ influx, the second and third generation drugs clopidogrel and prasugrel provoked weaker [Ca2+ ]i rises at similar concentrations. Application of ticlopidine and clopidogrel but not of prasugrel induced a TRPA1-dependent secretion of 5-HT from QGP-1 cells. Taken together, we suggest that a stable TRPA1 activation by ticlopidine and clopidogrel entails a release of 5-HT, and might therefore influence GI homeostasis in vivo.
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were carried out in pigmented clear-bottom 384-well plates (10,000 cells/well; Greiner, Frickenhausen, Germany). Activation of the respective channel was followed by measuring increases in the fluorescence intensity of fluo-4. Measurements were carried out using a custom-made fluorescence plate imaging device as described before [32]. 2.3. RT-PCR The expression of human TRPA1 in HEK293TRPA1 and QGP1 cells was analysed by reverse transcription (RT) of total RNA to cDNA followed by PCR. RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First strand cDNA synthesis was carried out by using M-MuLV reverse transcriptase (NEB, Ipswich, MA, USA). RNA was reverse transcribed using random hexamer primer. The amplification of cDNA (30 cycles) was accomplished with Taq polymerase (GoTaq, Promega, Fitchburg, WI, USA). Annealing conditions for TRPA1 and -actin primers were 58 ◦ C and 62 ◦ C for 30 s, respectively. Specific intron-spanning primer sets for PCR were designed as follows: 5 -CGG-AGG-ATT-TCA-AGG-AAT-CG-3 (TRPA1 forward primer) and 5 -CTC-CTC-TGC-TGA-GAA-GAA-AC-3 (TRPA1 reverse primer), and 5 -GGC-GGC-ACC-ACC-ATG-TAC-CCT-3 (-actin forward primer) and 5 -AGG-GGC-CGG-ACT-CGT-CAT-ACT-3 (-actin reverse primer). PCR products were analysed by agarose gel electrophoresis, stained with ethidium bromide, and imaged with a digital gel documentation system.
2. Materials and methods 2.1. Cell culture HEK293 cells were grown in Earle’s Minimum Essential Medium (MEM), supplemented with 10% foetal calf serum, 2 mM lglutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. For cultivation of HEK293 cells stably transfected with human TRPA1 (HEK293TRPA1 ), 1 mg/ml geneticin (G418) was added to the medium [30]. QGP-1 cells were a kind gift from B. Wiedenmann (Charite, Berlin, Germany) and were cultured in RPMI 1640 medium, supplemented with 10% foetal calf serum, 2 mM l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. 2.2. Fluorometric Ca2+ assay QGP-1 and HEK293 cells stably expressing TRPA1 channels were grown on 25-mm glass coverslips and allowed to attach for 24 h prior to analysis of the increase in [Ca2+ ]i . For cysteine mutation studies, the cDNA of human TRPA1 wild-type (WT) and a cysteine triple mutant C619S, C639S, C663S (TRPA1-3C-S) were transiently transfected into HEK293 cells utilising Fugene HD according to the manufacturer’s protocol (Roche, Mannheim, Germany). The construct plasmids were a kind gift from David Julius (Department of Physiology, San Francisco, CA, USA). After incubation with 2 M fura-2/AM in a HEPES-buffered solution (HBS; 10 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgCl2 , 5.5 mM glucose, 1 mM CaCl2 , pH 7.4) at 37 ◦ C for 30 min, cells were rinsed, and mounted in a bath chamber for monochromator-assisted (TILL-Photonics, Graefelfing, Germany) digital epifluorescence videomicroscopy, built around an inverted microscope (Zeiss Axiovert 100, Jena, Germany). The fluorescence of fura-2 was sequentially excited at 340, 358, and 380 nm through the imaging objective (Fluar 10×/0.5; Carl Zeiss, Jena, Germany). Emitted light was filtered through a 512-nm longpass filter, and recorded with a 12-bit cooled CCD camera (IMAGO, TILL-Photonics, Graefelfing, Germany). The [Ca2+ ]i was calibrated as described before [31]. For generation of concentration–response curves, cell suspensions were loaded with 5 M fluo-4/AM (Invitrogen). Experiments
2.4. Electrophysiology Whole cell recordings were performed using Multiclamp 200B or 700B amplifiers combined with a 1440A digitiser (Molecular Devices, Sunnyvale, CA, USA) under the control of the pCLAMP 10 software. Coverslips with HEK293TRPA1 or QGP-1 cells were transferred to a continuously perfused recording chamber (500 l volume) and mounted on the stage of an inverted microscope. Patch pipettes were fabricated from borosilicate glass with a typical resistance of 3–5 M, when filled with pipette solution. Whole cell series resistances were compensated by 70%. The bath solution consisted of 140 mM NaCl, 5 mM CsCl, 2 mM MgCl2 , 5 mM glucose and 10 mM HEPES, pH 7.4 adjusted with NaOH. For whole cell recordings of QGP-1 cells, the bath solution was supplemented with 1 mM CaCl2 . The pipette solution contained 140 mM CsCl, 4 mM MgCl2 , 10 mM HEPES, and 10 mM EGTA, pH 7.4 adjusted with CsOH. All experiments were performed at room temperature. Currents were filtered with a four-pole Bessel filter at 3 kHz and sampled continuously at 5 kHz. Voltage ramps were applied every second from −80 mV to +80 mV (500 ms duration). Results are shown as current densities. 2.5. 5-HT release experiments QGP-1 cells were seeded in 24-well plates at a density of 2 × 105 cells/well in 1 ml RPMI, supplemented with 10% FCS. After cultivating the cells for 96 h [27], the medium was removed and cells were washed with glucose-free HBS solution, containing 2 M fluoxetine and 0.1% BSA. The HBS solution was replaced with 0.25 ml glucose-free HBS, containing AITC, ticlopidine, clopidogrel or prasugrel at a final concentration of 100 M. In some experiments, the TRPA1 blocker HC-030031 [33,34] (100 M) was applied 10 min prior to the activators. Cells were incubated for 1 h at 37 ◦ C. The supernatants were collected, centrifuged and stored at −80 ◦ C until 5-HT measurements were performed using an enzyme immunoassay (EIA) kit according to the manufacturer’s protocol (Beckman Coulter, Krefeld, Germany). DMSO (1%) served as control.
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2.6. Chemicals HC-030031 and clopidogrel hydrochloride were obtained from Tocris (Wiesbaden-Nordenstadt, Germany). Ticlopidine hydrochloride, prasugrel and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA), if not stated otherwise.
between groups was assessed by Student’s t-test. Differences were considered significant at *p < 0.05 or **p < 0.01. 3. Results 3.1. Application of ticlopidine, clopidogrel and prasugrel causes Ca2+ influx in HEK293TRPA1 cells
2.7. Data analysis Data are presented as means ± SEM. Each experimental approach consisted of ≥3 independent experiments. For curve fitting and statistical analysis, OriginPro 8 software (OriginLab Corporation, Northampton, MA, USA) was used. Statistical significance
To examine the effective concentration of ticlopidine, clopidogrel and prasugrel for activating hTRPA1 channels, we performed fluo-4-based Ca2+ measurements (Fig. 1A–C) on HEK293TRPA1 cells. A half-maximal activation of TRPA1 (EC50 ) was observed at the following concentrations: ticlopidine 7.2 ± 0.6 M (n = 4);
Fig. 1. Activation of TRPA1 by ticlopidine, clopidogrel and prasugrel in HEK293TRPA1 cells. (A–C) Concentration–response curves of ticlopidine-, clopidogrel- or prasugrelinduced Ca2+ elevation in fluo-4-loaded HEK293TRPA1 (black symbols) and control HEK293 cells (grey symbols). Data represent means and SEM of four independent experiments together with the best fit to a three-parameter Hill equation. Inset: chemical structures of the respective compounds. (D–F) Representative single cell time-lapse analysis of [Ca2+ ]i in fura-2-loaded HEK293TRPA1 cells stimulated with ticlopidine (10 M), clopidogrel (50 M) or prasugrel (100 M), followed by the application of HC-030031 (10 M). Responses of single cells are shown in grey lines and their mean response as a black line. (G) Statistical analysis of peak [Ca2+ ]i of four independent experiments without (black bars) and with 10 M HC-030031 (grey bars). For comparison, activation of HEK293TRPA1 cells by 10 M AITC was added to the bar graph. Data represent means and SEM. Basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line in the figure.
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Fig. 2. Ticlopidine, clopidogrel and prasugrel induce TRPA1 currents in HEK293TRPA1 cells. (A, C, E) Whole cell current densities recorded in HEK293TRPA1 cells clamped at a holding potential of +80 mV (upper trace) and −80 mV (lower trace). Measurements were performed in nominally Ca2+ -free bath solutions during stimulation with ticlopidine (50 M), clopidogrel (50 M) or prasugrel (50 M), followed by HC-030031 (10 M). Data were extracted from voltage ramps such as in (B, D, F). (B, D, F) I/V curves of the thienopyridine-induced currents at the time points indicated in (A, C, E). Inset: statistical analysis of 5–7 independent experiments such as in (A, C and E). Data represent means and SEM.
clopidogrel 5.4 ± 1.0 M (n = 4) and prasugrel 16.6 ± 1.2 M (n = 4). Ticlopidine caused the highest maximal response, but the apparent EC50 might not be accurate, as saturating concentrations of the drug could not be achieved due to poor solubility in buffer. A parental HEK293 control cell line was insensitive to all substances tested. Fura-2-assisted [Ca2+ ]i imaging analysis further confirmed the activation of TRPA1 in single HEK293TRPA1 cells by ticlopidine, clopidogrel and prasugrel. Single HEK293TRPA1 cells showed a strong and persistent increase in [Ca2+ ]i after addition of 10 M ticlopidine (2223 ± 88 nM; n = 4) and 50 M clopidogrel (1603 ± 56 nM; n = 4; Fig. 1D–G). Notably, ticlopidine induced a stronger Ca2+ influx than AITC in HEK293TRPA1 (1657 ± 148 nM; n = 3; Fig. 1G). The Ca2+ increase, caused by prasugrel (100 M) was weaker (1373 ± 106 nM; n = 4; Fig. 1G). All compound-induced responses could be blocked by the TRPA1-specific blocker HC030031.
3.2. Ticlopidine, clopidogrel and prasugrel induce robust TRPA1 currents To prevent desensitisation of TRPA1 by extracellular Ca2+ , whole cell patch clamp recordings were carried out in nominally Ca2+ -free bath solutions [18,35,36]. Stimulation of TRPA1 by 50 M ticlopidine, clopidogrel or prasugrel revealed stable, outwardly rectifying currents in HEK293TRPA1 cells, typical to TRPA1 (Fig. 2B, D, and F). In line with measurements of [Ca2+ ]i , ticlopidine provoked a stronger TRPA1 activation compared to clopidogrel and prasugrel. Inward and outward currents could be completely abrogated by addition of 10 M HC-030031 to the bath solution (Fig. 2A–F). Many TRPA1 activators act on TRPA1 via a covalent modification of free sulfhydryl groups on cysteines. Three cysteines (C619, C639 and C663, equal C621, C641, C665 from GenBank Accession NP 015628.2) have been identified as being crucial
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Fig. 3. Elevation of [Ca2+ ]i by thienopyridines in QGP-1 cells. (A–C) Representative time-lapse analysis of [Ca2+ ]i in fura-2-loaded QGP-1 cells, stimulated with 100 M ticlopidine, clopidogrel or prasugrel. Shown are single cells (grey lines) and their mean response (black line). The number of cells responding to the substances and of all cells analysed is indicated. Cells were considered as responders when [Ca2+ ]i rose by more than threefold of the SEM of the mean basal [Ca2+ ]i . (D) Concentration–response curves of ticlopidine-, clopidogrel- or prasugrel-induced [Ca2+ ]i elevation in fura-2-loaded QGP-1 cells. Data were obtained from three independent experiments as shown in (A–C), and modelled with the best fit to a three-parameter Hill equation. The basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line. (E) Statistical analysis of several experiments as shown in (A–C). Data represent means and SEM of four independent experiments. Peak [Ca2+ ]i of all QGP-1 cells was determined after addition of 100 M ticlopidine, clopidogrel or prasugrel (black bar). Activation of QGP-1 cells by the thienopyridines was additionally examined after preincubation with 100 M HC-030031 or 0.2 M A-967079 (grey bars). The value for basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line.
for such an electrophilic activation. A mutant TRPA1, in which these cysteines were replaced by serines (TRPA1-3C-S) [37,38] could still be activated by the thienopyridines at a concentration of 50 M (Supplementary Fig. 1), indicating that a modification of at least these three cysteine residues is not crucial for TRPA1 activation by ticlopdine, clopidodgrel, and prasugrel. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2014.02.014.
3.3. Ticlopidine, clopidogrel and prasugrel induce an activation of TRPA1 in QGP-1 cells Applying RT-PCR, we confirmed that QGP-1 cells express the human TRPA1 gene, as described before [39]. An amplicon with the specific size was detected by reverse transcription following PCR in QGP-1 cells (Supplementary Fig. 2A). To confirm the presence of functional TRPA1 channels in QGP-1 cells, we tested known activators of TRPA1. 300 M cinnamaldehyde, 600 M 4-hydroxynonenal, and 100 M AITC evoked rises in [Ca2+ ]i of 578.1 ± 57.2 nM (n = 3), 444.6 ± 49.3 nM (n = 3), and 335.5 ± 20.2 nM (n = 3), respectively (Supplementary Fig. 2B–D). We next investigated whether also ticlopidine, clopidogrel or prasugrel can induce Ca2+ signals in QGP-1 cells. Approximately 86% and 80% of all cells analysed responded with a [Ca2+ ]i increase after addition of 100 M ticlopidine or clopidogrel, respectively (Fig. 3A and B). In contrast, prasugrel only caused a weak and transient influx of [Ca2+ ]i in 19% of all cells measured (Fig. 3C). Ticlopidine and clopidogrel showed a concentration-dependent activation of TRPA1 in QGP1 cells in QGP-1 cells, but as saturation could not be achieved, no EC50 values were determined (Fig. 3D). Similar to heterologously expressed TRPA1, ticlopidine caused the highest [Ca2+ ]i increase (493 ± 37 nM; n = 4) in QGP-1 cells, followed by clopidogrel
(413 ± 4 nM; n = 4). The increase in [Ca2+ ]i was indeed mediated by TRPA1, as preincubation of QGP-1 cells with 100 M HC-030031 or with another, more potent, TRPA1-selective blocker (200 nM A967079) [40] completely abrogated the responses to ticlopidine and clopidogrel (Fig. 3E). Prasugrel-induced [Ca2+ ]i did not significantly exceed basal [Ca2+ ]i before stimulation (118 ± 9.5 nM; n = 4; Fig. 3E). We further characterised the activation of TRPA1 in QGP1 cells by ticlopidine and clopidogrel in whole cell patch clamp recordings. Application of 100 M ticlopidine to the bath solution (supplemented with 1 mM CaCl2 ) evoked currents in 10 out of 18 cells recorded (Fig. 4A and B). Addition of 100 M clopidogrel also induced currents but only in 4 out of 17 QGP-1 cells (Fig. 4C and D). All currents could be completely blocked with 100 M HC-030031 (Fig. 4B and D, inset). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2014.02.014.
3.4. Effects of ticlopidine, clopidogrel and prasugrel on 5-HT release from QGP-1 cells To investigate whether also a ticlopidine-, clopidogrel- or prasugrel-mediated activation of TRPA1 leads to 5-HT secretion, we treated QGP-1 cells with 100 M of each substance and determined 5-HT in supernatants of cultured cells (Fig. 5). Ticlopidine and clopidogrel induced an increase in the 5-HT concentration which was inhibited by preincubation of QGP-1 cells with 100 M HC-030031. Moreover, the ticlopidine-induced 5-HT secretion (25.2 ± 0.9 ng/ml; n = 3) was about 1.5-fold higher compared to clopidogrel and AITC (15.7 ± 1.9 ng/ml; 16.6 ± 2.8 ng/ml; n = 3). In contrast, the release of 5-HT evoked by prasugrel did not exceed the basal release observed in cells treated with the vehicle control DMSO.
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Fig. 4. Ticlopidine and clopidogrel stimulate TRPA1-like currents in QGP-1 cells. Whole cell current densities recorded in a QGP-1 cell clamped at a holding potential of +80 mV and −80 mV during the addition of 100 M ticlopidine (A) or 100 M clopidogrel (C) followed by 100 M HC-030031. Data were extracted from voltage ramps such as in (B and D). (B and D) I/V curves for ticlopidine- and clopidogrel-induced current densities in QGP-1 cells before (black line) and after block with HC-030031 (grey line) were taken at the time points indicated in (A and C). Inset: statistical analysis of 10 (ticlopidine) and 4 (clopidogrel) independent recordings such as in (A) and (C), depicted as means and SEM. For statistical analysis only cells responding to the drug were included.
Fig. 5. Ticlopidine and clopidogrel but not prasugrel provoke 5-HT release in QGP-1 cells. 5-HT release from QGP-1 cells was determined in culture supernatants using an enzyme-linked immunosorbent assay. Cells were incubated for 1 h at 37 ◦ C in the presence of 100 M ticlopidine (T), clopidogrel (C), prasugrel (P) and AITC without addition of a TRPA1 blocker (black bars) or in the continuous presence of 100 M HC030031, which was applied 10 min before the activators (grey bars). Open bar: DMSO control (1%). Data represent means and SEM of three independent experiments performed in duplicates, each, *p < 0.05 and **p < 0.01.
4. Discussion Here, we present an activation of TRPA1 by the antiplatelet drugs ticlopidine, clopidogrel and prasugrel. In HEK293TRPA1 cells, the compounds evoke TRPA1-mediated intracellular Ca2+ signals and ionic currents. In whole cell recordings, all three compounds elicit currents with outwardly rectifying I/V curves, typical to TRPA1,
with ticlopidine being the strongest and prasugrel the weakest activator. Several TRPA1 activators such as menthol, nicotine and apomorphine exert a bimodal action on TRPA1, activating the channel in the low micromolar range but with antagonistic properties at higher concentrations [12,13,41]. We did not see inhibitory properties of ticlopidine, prasugrel, and clopidogrel but it has to be taken into account that concentrations above 100 M were not tested. The mechanisms of TRPA1 activation are diverse. Electrophilic substances have been shown to form disulfide bonds with cysteines in the N-terminus of the channel [37,38,42]. Other compounds such as menthol, nicotine and clotrimazole presumably activate TRPA1 independently of any covalent modification via a currently unknown mechanism [13,16,41]. As the thienopyridines do not possess electrophilic properties, we find it unlikely that the activation of TRPA1, we here describe, involves a covalent modification of TRPA1, Moreover, a TRPA1 mutant, which is largely insensitive to electrophiles (TRPA1-3C-S) [37] still showed TRPA1-mediated Ca2+ entry in response to ticlopidine, clopidogrel and prasugrel, albeit in a slightly reduced manner, which might reflect an altered expression of the TRPA1-3C-S construct in comparison with wild-type TRPA1. Besides its expression in neuronal cells, TRPA1 is also present in non-neuronal cells, like enterochromaffin cells [27]. Little is known about the physiological function of TRPA1 throughout the digestive system, but recent studies hint to a putative role in triggering the secretion of serotonin. For example, Doihara et al. demonstrated that enterochromaffin-like QGP-1 cells express TRPA1 and release 5-HT upon TRPA1 activation with AITC, cinnamaldehyde and acrolein [39]. The dispense of 5-HT into the GI lumen follows intraluminal distension, vagal nerve stimulation and ingestion of nutrition such as olfactants and tastants [43,44]. The most important mechanism behind the release of 5-HT from EC cells is an
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increase in [Ca2+ ]i [45]. Here, we report that upon stimulation with ticlopidine and clopidogrel, QGP-1 cells exhibit an increase in [Ca2+ ]i , which is prevented by two different TRPA1-specific blockers. In contrast, prasugrel-treated QGP-1 cells exhibited only very weak calcium signals, which were lower and more transient with fewer responding cells. No EC50 values for the activation of TRPA1 by the thienopyridines in QGP-1 cells could be determined but it is evident that in QGP-1 cells the channel is less sensitive to the drugs compared to the heterologous expression system. Furthermore, QGP-1 cells apparently display a higher degree of heterogeneity within the population, which was also evident from the patch clamp recordings. This inhomogeneity might be caused by a variable expression of TRPA1 and also of plasma membrane Ca2+ pumps and cytoplasmic Ca2+ buffering, which counteracts the TRPA1-mediated Ca2+ influx [46]. TRPA1 activation can be strongly potentiated by elevations of [Ca2+ ]i (EC50 900 nM) [36,47]. Therefore, depending on the channel density and strength of the respective activator, the resulting internal Ca2+ concentration may or may not trigger a secondary TRPA1 activation, causing an amplified response. Interestingly, when QGP-1 cells were challenged with ticlopidine or clopidogrel in patch clamp recordings, the resulting I/V curve was almost linear, possibly reflecting the dilated state of the TRPA1 channel [48]. Ticlopidine and clopidogrel only activated currents in a subset of QGP-1 cells, which might be due to the strong buffering of intracellular calcium in whole cell recordings. A rise in [Ca2+ ]i , as detected in our experiments after stimulation with the thienopyridine drugs, correlates with an excretion of 5-HT. Indeed, we could determine a release of 5-HT upon ticlopidine and clopidogrel treatment, which could be suppressed by HC-030031. No significant increase in the extracellular 5-HT concentration was evident when cells were treated with prasugrel. A possible reason might be that a strong and stable [Ca2+ ]i signal, that could not be obtained with prasugrel, is necessary to provoke 5-HT secretion. Obviously, relatively high concentrations of the platelet aggregation inhibitors were required to stimulate TRPA1 in QGP-1 cells. Reported peak plasma concentrations of the active metabolites are 3 M, 179 nM, and 233 nM after a dose of 250 mg ticlopidine twice daily, 300 mg load/75 mg daily clopidogrel and 60 mg load/10 mg daily prasugrel, respectively [3]. However, the effective pre-systemic concentrations of the prodrugs, acting on endocrine cells in the stomach and small intestine after oral administration are certainly higher than the peak plasma concentrations of the active metabolites, and possibly suffice to cause a TRPA1mediated 5-HT release in vivo. Released 5-HT exerts emesis by stimulation of the CTZ via vagal afferents [7,8]. Considering that ticlopidine and clopidogrel administration is frequently accompanied by adverse events such as nausea, vomiting and diarrhoea [4,5], it may be possible that these two platelet inhibitors trigger the release of 5-HT through a TRPA1 activation. This would be consistent with our finding that there is no association between taking prasugrel and the incidence of adverse effects in the GI tract. TRPA1 is not only involved in the secretion of 5-HT, but also in the release of cholecystokinin (CCK) from neuroendocrine STC-1 cells [26,49]. Although mainly responsible for the regulation of food digestion and satiety, high doses of intravenously infused CCK to healthy subjects produce nausea and abdominal discomfort [50]. Thus, GI-related adverse effects of TRPA1 activators might be mediated by more than one paracrine hormone. To summarise our results, we could show that the tested platelet inhibitors are direct activators of TRPA1 in HEK293TRPA1 and QGP-1 cells with varying efficacies and potencies: ticlopidine > clopidogrel > prasugrel. Furthermore, ticlopidine and clopidogrel stimulate QGP-1 cells to release 5-HT, reminiscent of the 5-HT release by apomorphine. Thus, TRPA1 may not only act as a versatile
irritant sensor in the skin and lung, but also sense irritants in the GI tract. Authors’ contribution Schulze, Hartung, Schaefer, Hill: participated in research design and wrote or contributed to the writing of the manuscript. Schulze, Hartung, Hill conducted experiments and performed data analysis. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements We thank Nicole Urban, Helga Sobottka and Marion Leonhardt for excellent technical assistance and Francesco Epifano (Chieti, Italy) for helpful discussion. References [1] P. Savi, J.M. Herbert, A.M. Pflieger, et al., Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel, Biochem. Pharmacol. 44 (1992) 527–532. [2] P. Savi, J.-L. Zachayus, N. Delesque-Touchard, et al., The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 11069–11074. [3] L. Wallentin, P2Y(12) inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use, Eur. Heart J. 30 (2009) 1964–1977. [4] M.J. Quinn, D.J. Fitzgerald, Ticlopidine and clopidogrel, Circulation 100 (1999) 1667–1672. [5] M.K. Ito, A.R. Smith, M.L. Lee, Ticlopidine: a new platelet aggregation inhibitor, Clin. Pharm. 11 (1992) 603–617. [6] M.D. Gershon, J. Tack, The serotonin signaling system: from basic understanding to drug development for functional GI disorders, Gastroenterology 132 (2007) 397–414. [7] M.D. Gershon, Plasticity in serotonin control mechanisms in the gut, Curr. Opin. Pharmacol. 3 (2003) 600–607. [8] R. Spiller, Recent advances in understanding the role of serotonin in gastrointestinal motility in functional bowel disorders: alterations in 5-HT signalling and metabolism in human disease, Neurogastroenterol. Motil. 19 (Suppl 2) (2007) 25–31. [9] P.L. Andrews, C.J. Davis, S. Bingham, H.I. Davidson, J. Hawthorn, L. Maskell, The abdominal visceral innervation and the emetic reflex: pathways, pharmacology, and plasticity, Can. J. Physiol. Pharmacol. 68 (1990) 325–345. [10] G.J. Wood, J.W. Shega, B. Lynch, J.H. von Roenn, Management of intractable nausea and vomiting in patients at the end of life: I was feeling nauseous all of the time..nothing was working, JAMA 298 (2007) 1196–1207. [11] D.M. Bautista, P. Movahed, A. Hinman, et al., Pungent products from garlic activate the sensory ion channel TRPA1, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 12248–12252. [12] A. Schulze, B. Oehler, N. Urban, M. Schaefer, K. Hill, Apomorphine is a bimodal modulator of TRPA1 channels, Mol. Pharmacol. 83 (2013) 542–551. [13] Y. Karashima, N. Damann, J. Prenen, et al., Bimodal action of menthol on the transient receptor potential channel TRPA1, J. Neurosci. 27 (2007) 9874–9884. [14] D. Vilceanu, C.L. Stucky, TRPA1 mediates mechanical currents in the plasma membrane of mouse sensory neurons, PLoS ONE 5 (2010) e12177. [15] P.G. Baraldi, D. Preti, S. Materazzi, P. Geppetti, Transient receptor potential ankyrin 1 (TRPA1) channel as emerging target for novel analgesics and antiinflammatory agents, J. Med. Chem. 53 (2010) 5085–5107. [16] V. Meseguer, Y. Karashima, K. Talavera, et al., Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole, J. Neurosci. 28 (2008) 576–586. [17] M. Bandell, G.M. Story, S.W. Hwang, et al., Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin, Neuron 41 (2004) 849–857. ˜ [18] K. Nagata, A. Duggan, G. Kumar, J. García-Anoveros, Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing, J. Neurosci. 25 (2005) 4052–4061. [19] S.M. Brierley, P.A. Hughes, A.J. Page, et al., The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli, Gastroenterology 137 (2009) 2084–2095, e3. [20] F. Cattaruzza, C. Johnson, A. Leggit, et al., Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice, Am. J. Physiol. Gastrointest. Liver Physiol. 304 (2013) G1002–G1012. [21] T. Kondo, T. Oshima, K. Obata, et al., Role of transient receptor potential A1 in gastric nociception, Digestion 82 (2010) 150–155. [22] A. Penuelas, K. Tashima, S. Tsuchiya, et al., Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine, Eur. J. Pharmacol. 576 (2007) 143–150.
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Transient receptor potential ankyrin 1 (TRPA1) channel activation by the thienopyridine-type drugs ticlopidine, clopidogrel, and prasugrel夽 Anja Schulze 1 , Philipp Hartung 1 , Michael Schaefer, Kerstin Hill ∗ Rudolf-Boehm-Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Leipzig, Germany
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Article history: Received 1 November 2013 Received in revised form 24 January 2014 Accepted 14 February 2014 Available online 25 February 2014 Keywords: TRPA1 Serotonin Ticlopidine Clopidogrel Prasugrel Thienopyridine QGP-1
a b s t r a c t Transient receptor potential A1 (TRPA1) is widely expressed throughout the human and animal organism, including the dorsal root ganglia as well as the bladder, stomach and small intestine. Here, we examined the effect of three platelet aggregation inhibitors on TRPA1: ticlopidine, clopidogrel and prasugrel. Utilising fluorometric Ca2+ influx analysis and electrophysiological whole cell measurements in TRPA1-expressing HEK293 and in human enterochromaffin-like QGP-1 cells, we found that ticlopidine, clopidogrel and prasugrel are direct activators of TRPA1. Although this polymodal channel commonly contributes to the perception of pain, temperature and chemical irritants, recent studies provide evidence for its involvement in the release of serotonin (5-HT) from enterochromaffin cells. Therefore, we further investigated the ability of ticlopidine, clopidogrel and prasugrel to stimulate 5-HT release from QGP-1 cells. We could determine 5-HT in supernatants from cultured QGP-1 cells upon treatment with ticlopidine and clopidogrel but not with prasugrel. These findings indicate that a robust TRPA1 activation by ticlopidine and clopidogrel correlates with the stimulatory effect on the secretion of 5-HT. As recipients of ticlopidine and clopidogrel frequently complain about gastrointestinal adverse events such as nausea, vomiting and diarrhoea, an activation of TRPA1 may contribute to adverse effects of such drugs in the digestive system. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Thienopyridines such as ticlopidine, clopidogrel and prasugrel are important drugs to prevent platelet aggregation in coronary artery, peripheral vascular and cerebrovascular disease. The compounds do not exhibit direct antiaggregant activity in vitro, but have to be converted to their active form by hepatic metabolisation in vivo [1]. The active metabolites then form disulfide bonds with cysteine residues in extracellular loops of the P2Y12 receptor of platelets, and irreversibly inhibit aggregation by blocking the ADP-binding site [2,3]. Therapies of many diseases are commonly accompanied by adverse effects including gastrointestinal (GI) disorders. Nausea, vomiting and diarrhoea are the most prevalent adverse effects
夽 This work was supported by the Deutsche Forschungsgemeinschaft [HI 829/2-1 to Kerstin Hill and GRK1097 to Michael Schaefer]. ∗ Corresponding author at: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany. Tel.: +49 0341 9724 616; fax: +49 0341 9724 609. E-mail address: [email protected] (K. Hill). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.ceca.2014.02.014 0143-4160/© 2014 Elsevier Ltd. All rights reserved.
observed in clinical trials evaluating the platelet aggregation inhibitors ticlopidine and clopidogrel [4,5], while recipients of prasugrel, a third generation antiplatelet drug, show fewer incidences of GI disturbances. With clopidogrel being one of the world’s blockbuster drugs, this limitation may be important for its clinical use. In general, the mechanisms causing nausea and vomiting are diverse: afferent vagal neurones of the intestinal mucosa can be stimulated by serotonin (5-hydroxytryptamine, 5-HT), which is released from endocrine cells in response to various stimuli [6]. Excitement of vagal nerve ends leads to the stimulation of the chemoreceptive trigger zone (CTZ) in the area postrema, resulting in the activation of the vomiting centre, located in the dorsal portion of the lateral reticular formation [7,8]. Alternatively, nausea may result from a direct binding of drugs, toxins or neurotransmitters, such as 5-HT to receptors within the CTZ [9]. Other pathways include the involvement of the vestibular system or the higher cortical centre [10]. The Ca2+ -permeable ion channel transient receptor potential ankyrin1 (TRPA1) is activated by several drugs (clotrimazole, apomorphine), toxicants and pollutants (acrolein, formaldehyde) and food ingredients, which produce a pungent and sharp sensation (AITC, allyl isothiocyanate from mustard, allicin from garlic) [11–16]. TRPA1 is localised in sensory neurones of the dorsal root
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and trigeminal ganglia, where it contributes to the perception of chemical pain and possibly to cold and mechanical stimuli [17,18] but is also expressed in peripheral nerve fibres in the stomach, small intestine and colon of rat and mouse [19–24]. In addition, recent studies on the expression and physiological function of TRPA1 also described the presence of the channel in non-neuronal cells, e.g. in 5-HT-containing enterochromaffin (EC) cells, and in cholecystokinin-releasing endocrine cells of the GI tract [25–27]. In this context it has been shown, that the administration of AITC induced TRPA1-dependent vomiting in dogs and contractions in the colon of mice and dogs [28,29]. Furthermore, we recently demonstrated the involvement of TRPA1 in the secretion of 5-HT from the human enterochromaffin model cell line QGP-1 induced by apomorphine, a drug with strong emetic properties [12]. Here, we tested three generations of P2Y12 receptor antagonists, ticlopidine, clopidogrel and prasugrel with regard to their activation of TRPA1 in HEK293TRPA1 and QGP-1 cells. Each substance provoked an increase in Ca2+ influx through stimulation of TRPA1. The first generation thienopyridine ticlopidine caused the most pronounced TRPA1-evoked Ca2+ influx, the second and third generation drugs clopidogrel and prasugrel provoked weaker [Ca2+ ]i rises at similar concentrations. Application of ticlopidine and clopidogrel but not of prasugrel induced a TRPA1-dependent secretion of 5-HT from QGP-1 cells. Taken together, we suggest that a stable TRPA1 activation by ticlopidine and clopidogrel entails a release of 5-HT, and might therefore influence GI homeostasis in vivo.
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were carried out in pigmented clear-bottom 384-well plates (10,000 cells/well; Greiner, Frickenhausen, Germany). Activation of the respective channel was followed by measuring increases in the fluorescence intensity of fluo-4. Measurements were carried out using a custom-made fluorescence plate imaging device as described before [32]. 2.3. RT-PCR The expression of human TRPA1 in HEK293TRPA1 and QGP1 cells was analysed by reverse transcription (RT) of total RNA to cDNA followed by PCR. RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First strand cDNA synthesis was carried out by using M-MuLV reverse transcriptase (NEB, Ipswich, MA, USA). RNA was reverse transcribed using random hexamer primer. The amplification of cDNA (30 cycles) was accomplished with Taq polymerase (GoTaq, Promega, Fitchburg, WI, USA). Annealing conditions for TRPA1 and -actin primers were 58 ◦ C and 62 ◦ C for 30 s, respectively. Specific intron-spanning primer sets for PCR were designed as follows: 5 -CGG-AGG-ATT-TCA-AGG-AAT-CG-3 (TRPA1 forward primer) and 5 -CTC-CTC-TGC-TGA-GAA-GAA-AC-3 (TRPA1 reverse primer), and 5 -GGC-GGC-ACC-ACC-ATG-TAC-CCT-3 (-actin forward primer) and 5 -AGG-GGC-CGG-ACT-CGT-CAT-ACT-3 (-actin reverse primer). PCR products were analysed by agarose gel electrophoresis, stained with ethidium bromide, and imaged with a digital gel documentation system.
2. Materials and methods 2.1. Cell culture HEK293 cells were grown in Earle’s Minimum Essential Medium (MEM), supplemented with 10% foetal calf serum, 2 mM lglutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. For cultivation of HEK293 cells stably transfected with human TRPA1 (HEK293TRPA1 ), 1 mg/ml geneticin (G418) was added to the medium [30]. QGP-1 cells were a kind gift from B. Wiedenmann (Charite, Berlin, Germany) and were cultured in RPMI 1640 medium, supplemented with 10% foetal calf serum, 2 mM l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. 2.2. Fluorometric Ca2+ assay QGP-1 and HEK293 cells stably expressing TRPA1 channels were grown on 25-mm glass coverslips and allowed to attach for 24 h prior to analysis of the increase in [Ca2+ ]i . For cysteine mutation studies, the cDNA of human TRPA1 wild-type (WT) and a cysteine triple mutant C619S, C639S, C663S (TRPA1-3C-S) were transiently transfected into HEK293 cells utilising Fugene HD according to the manufacturer’s protocol (Roche, Mannheim, Germany). The construct plasmids were a kind gift from David Julius (Department of Physiology, San Francisco, CA, USA). After incubation with 2 M fura-2/AM in a HEPES-buffered solution (HBS; 10 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgCl2 , 5.5 mM glucose, 1 mM CaCl2 , pH 7.4) at 37 ◦ C for 30 min, cells were rinsed, and mounted in a bath chamber for monochromator-assisted (TILL-Photonics, Graefelfing, Germany) digital epifluorescence videomicroscopy, built around an inverted microscope (Zeiss Axiovert 100, Jena, Germany). The fluorescence of fura-2 was sequentially excited at 340, 358, and 380 nm through the imaging objective (Fluar 10×/0.5; Carl Zeiss, Jena, Germany). Emitted light was filtered through a 512-nm longpass filter, and recorded with a 12-bit cooled CCD camera (IMAGO, TILL-Photonics, Graefelfing, Germany). The [Ca2+ ]i was calibrated as described before [31]. For generation of concentration–response curves, cell suspensions were loaded with 5 M fluo-4/AM (Invitrogen). Experiments
2.4. Electrophysiology Whole cell recordings were performed using Multiclamp 200B or 700B amplifiers combined with a 1440A digitiser (Molecular Devices, Sunnyvale, CA, USA) under the control of the pCLAMP 10 software. Coverslips with HEK293TRPA1 or QGP-1 cells were transferred to a continuously perfused recording chamber (500 l volume) and mounted on the stage of an inverted microscope. Patch pipettes were fabricated from borosilicate glass with a typical resistance of 3–5 M, when filled with pipette solution. Whole cell series resistances were compensated by 70%. The bath solution consisted of 140 mM NaCl, 5 mM CsCl, 2 mM MgCl2 , 5 mM glucose and 10 mM HEPES, pH 7.4 adjusted with NaOH. For whole cell recordings of QGP-1 cells, the bath solution was supplemented with 1 mM CaCl2 . The pipette solution contained 140 mM CsCl, 4 mM MgCl2 , 10 mM HEPES, and 10 mM EGTA, pH 7.4 adjusted with CsOH. All experiments were performed at room temperature. Currents were filtered with a four-pole Bessel filter at 3 kHz and sampled continuously at 5 kHz. Voltage ramps were applied every second from −80 mV to +80 mV (500 ms duration). Results are shown as current densities. 2.5. 5-HT release experiments QGP-1 cells were seeded in 24-well plates at a density of 2 × 105 cells/well in 1 ml RPMI, supplemented with 10% FCS. After cultivating the cells for 96 h [27], the medium was removed and cells were washed with glucose-free HBS solution, containing 2 M fluoxetine and 0.1% BSA. The HBS solution was replaced with 0.25 ml glucose-free HBS, containing AITC, ticlopidine, clopidogrel or prasugrel at a final concentration of 100 M. In some experiments, the TRPA1 blocker HC-030031 [33,34] (100 M) was applied 10 min prior to the activators. Cells were incubated for 1 h at 37 ◦ C. The supernatants were collected, centrifuged and stored at −80 ◦ C until 5-HT measurements were performed using an enzyme immunoassay (EIA) kit according to the manufacturer’s protocol (Beckman Coulter, Krefeld, Germany). DMSO (1%) served as control.
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2.6. Chemicals HC-030031 and clopidogrel hydrochloride were obtained from Tocris (Wiesbaden-Nordenstadt, Germany). Ticlopidine hydrochloride, prasugrel and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA), if not stated otherwise.
between groups was assessed by Student’s t-test. Differences were considered significant at *p < 0.05 or **p < 0.01. 3. Results 3.1. Application of ticlopidine, clopidogrel and prasugrel causes Ca2+ influx in HEK293TRPA1 cells
2.7. Data analysis Data are presented as means ± SEM. Each experimental approach consisted of ≥3 independent experiments. For curve fitting and statistical analysis, OriginPro 8 software (OriginLab Corporation, Northampton, MA, USA) was used. Statistical significance
To examine the effective concentration of ticlopidine, clopidogrel and prasugrel for activating hTRPA1 channels, we performed fluo-4-based Ca2+ measurements (Fig. 1A–C) on HEK293TRPA1 cells. A half-maximal activation of TRPA1 (EC50 ) was observed at the following concentrations: ticlopidine 7.2 ± 0.6 M (n = 4);
Fig. 1. Activation of TRPA1 by ticlopidine, clopidogrel and prasugrel in HEK293TRPA1 cells. (A–C) Concentration–response curves of ticlopidine-, clopidogrel- or prasugrelinduced Ca2+ elevation in fluo-4-loaded HEK293TRPA1 (black symbols) and control HEK293 cells (grey symbols). Data represent means and SEM of four independent experiments together with the best fit to a three-parameter Hill equation. Inset: chemical structures of the respective compounds. (D–F) Representative single cell time-lapse analysis of [Ca2+ ]i in fura-2-loaded HEK293TRPA1 cells stimulated with ticlopidine (10 M), clopidogrel (50 M) or prasugrel (100 M), followed by the application of HC-030031 (10 M). Responses of single cells are shown in grey lines and their mean response as a black line. (G) Statistical analysis of peak [Ca2+ ]i of four independent experiments without (black bars) and with 10 M HC-030031 (grey bars). For comparison, activation of HEK293TRPA1 cells by 10 M AITC was added to the bar graph. Data represent means and SEM. Basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line in the figure.
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Fig. 2. Ticlopidine, clopidogrel and prasugrel induce TRPA1 currents in HEK293TRPA1 cells. (A, C, E) Whole cell current densities recorded in HEK293TRPA1 cells clamped at a holding potential of +80 mV (upper trace) and −80 mV (lower trace). Measurements were performed in nominally Ca2+ -free bath solutions during stimulation with ticlopidine (50 M), clopidogrel (50 M) or prasugrel (50 M), followed by HC-030031 (10 M). Data were extracted from voltage ramps such as in (B, D, F). (B, D, F) I/V curves of the thienopyridine-induced currents at the time points indicated in (A, C, E). Inset: statistical analysis of 5–7 independent experiments such as in (A, C and E). Data represent means and SEM.
clopidogrel 5.4 ± 1.0 M (n = 4) and prasugrel 16.6 ± 1.2 M (n = 4). Ticlopidine caused the highest maximal response, but the apparent EC50 might not be accurate, as saturating concentrations of the drug could not be achieved due to poor solubility in buffer. A parental HEK293 control cell line was insensitive to all substances tested. Fura-2-assisted [Ca2+ ]i imaging analysis further confirmed the activation of TRPA1 in single HEK293TRPA1 cells by ticlopidine, clopidogrel and prasugrel. Single HEK293TRPA1 cells showed a strong and persistent increase in [Ca2+ ]i after addition of 10 M ticlopidine (2223 ± 88 nM; n = 4) and 50 M clopidogrel (1603 ± 56 nM; n = 4; Fig. 1D–G). Notably, ticlopidine induced a stronger Ca2+ influx than AITC in HEK293TRPA1 (1657 ± 148 nM; n = 3; Fig. 1G). The Ca2+ increase, caused by prasugrel (100 M) was weaker (1373 ± 106 nM; n = 4; Fig. 1G). All compound-induced responses could be blocked by the TRPA1-specific blocker HC030031.
3.2. Ticlopidine, clopidogrel and prasugrel induce robust TRPA1 currents To prevent desensitisation of TRPA1 by extracellular Ca2+ , whole cell patch clamp recordings were carried out in nominally Ca2+ -free bath solutions [18,35,36]. Stimulation of TRPA1 by 50 M ticlopidine, clopidogrel or prasugrel revealed stable, outwardly rectifying currents in HEK293TRPA1 cells, typical to TRPA1 (Fig. 2B, D, and F). In line with measurements of [Ca2+ ]i , ticlopidine provoked a stronger TRPA1 activation compared to clopidogrel and prasugrel. Inward and outward currents could be completely abrogated by addition of 10 M HC-030031 to the bath solution (Fig. 2A–F). Many TRPA1 activators act on TRPA1 via a covalent modification of free sulfhydryl groups on cysteines. Three cysteines (C619, C639 and C663, equal C621, C641, C665 from GenBank Accession NP 015628.2) have been identified as being crucial
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Fig. 3. Elevation of [Ca2+ ]i by thienopyridines in QGP-1 cells. (A–C) Representative time-lapse analysis of [Ca2+ ]i in fura-2-loaded QGP-1 cells, stimulated with 100 M ticlopidine, clopidogrel or prasugrel. Shown are single cells (grey lines) and their mean response (black line). The number of cells responding to the substances and of all cells analysed is indicated. Cells were considered as responders when [Ca2+ ]i rose by more than threefold of the SEM of the mean basal [Ca2+ ]i . (D) Concentration–response curves of ticlopidine-, clopidogrel- or prasugrel-induced [Ca2+ ]i elevation in fura-2-loaded QGP-1 cells. Data were obtained from three independent experiments as shown in (A–C), and modelled with the best fit to a three-parameter Hill equation. The basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line. (E) Statistical analysis of several experiments as shown in (A–C). Data represent means and SEM of four independent experiments. Peak [Ca2+ ]i of all QGP-1 cells was determined after addition of 100 M ticlopidine, clopidogrel or prasugrel (black bar). Activation of QGP-1 cells by the thienopyridines was additionally examined after preincubation with 100 M HC-030031 or 0.2 M A-967079 (grey bars). The value for basal [Ca2+ ]i before stimulation of the cells is shown as a dashed line.
for such an electrophilic activation. A mutant TRPA1, in which these cysteines were replaced by serines (TRPA1-3C-S) [37,38] could still be activated by the thienopyridines at a concentration of 50 M (Supplementary Fig. 1), indicating that a modification of at least these three cysteine residues is not crucial for TRPA1 activation by ticlopdine, clopidodgrel, and prasugrel. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2014.02.014.
3.3. Ticlopidine, clopidogrel and prasugrel induce an activation of TRPA1 in QGP-1 cells Applying RT-PCR, we confirmed that QGP-1 cells express the human TRPA1 gene, as described before [39]. An amplicon with the specific size was detected by reverse transcription following PCR in QGP-1 cells (Supplementary Fig. 2A). To confirm the presence of functional TRPA1 channels in QGP-1 cells, we tested known activators of TRPA1. 300 M cinnamaldehyde, 600 M 4-hydroxynonenal, and 100 M AITC evoked rises in [Ca2+ ]i of 578.1 ± 57.2 nM (n = 3), 444.6 ± 49.3 nM (n = 3), and 335.5 ± 20.2 nM (n = 3), respectively (Supplementary Fig. 2B–D). We next investigated whether also ticlopidine, clopidogrel or prasugrel can induce Ca2+ signals in QGP-1 cells. Approximately 86% and 80% of all cells analysed responded with a [Ca2+ ]i increase after addition of 100 M ticlopidine or clopidogrel, respectively (Fig. 3A and B). In contrast, prasugrel only caused a weak and transient influx of [Ca2+ ]i in 19% of all cells measured (Fig. 3C). Ticlopidine and clopidogrel showed a concentration-dependent activation of TRPA1 in QGP1 cells in QGP-1 cells, but as saturation could not be achieved, no EC50 values were determined (Fig. 3D). Similar to heterologously expressed TRPA1, ticlopidine caused the highest [Ca2+ ]i increase (493 ± 37 nM; n = 4) in QGP-1 cells, followed by clopidogrel
(413 ± 4 nM; n = 4). The increase in [Ca2+ ]i was indeed mediated by TRPA1, as preincubation of QGP-1 cells with 100 M HC-030031 or with another, more potent, TRPA1-selective blocker (200 nM A967079) [40] completely abrogated the responses to ticlopidine and clopidogrel (Fig. 3E). Prasugrel-induced [Ca2+ ]i did not significantly exceed basal [Ca2+ ]i before stimulation (118 ± 9.5 nM; n = 4; Fig. 3E). We further characterised the activation of TRPA1 in QGP1 cells by ticlopidine and clopidogrel in whole cell patch clamp recordings. Application of 100 M ticlopidine to the bath solution (supplemented with 1 mM CaCl2 ) evoked currents in 10 out of 18 cells recorded (Fig. 4A and B). Addition of 100 M clopidogrel also induced currents but only in 4 out of 17 QGP-1 cells (Fig. 4C and D). All currents could be completely blocked with 100 M HC-030031 (Fig. 4B and D, inset). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2014.02.014.
3.4. Effects of ticlopidine, clopidogrel and prasugrel on 5-HT release from QGP-1 cells To investigate whether also a ticlopidine-, clopidogrel- or prasugrel-mediated activation of TRPA1 leads to 5-HT secretion, we treated QGP-1 cells with 100 M of each substance and determined 5-HT in supernatants of cultured cells (Fig. 5). Ticlopidine and clopidogrel induced an increase in the 5-HT concentration which was inhibited by preincubation of QGP-1 cells with 100 M HC-030031. Moreover, the ticlopidine-induced 5-HT secretion (25.2 ± 0.9 ng/ml; n = 3) was about 1.5-fold higher compared to clopidogrel and AITC (15.7 ± 1.9 ng/ml; 16.6 ± 2.8 ng/ml; n = 3). In contrast, the release of 5-HT evoked by prasugrel did not exceed the basal release observed in cells treated with the vehicle control DMSO.
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Fig. 4. Ticlopidine and clopidogrel stimulate TRPA1-like currents in QGP-1 cells. Whole cell current densities recorded in a QGP-1 cell clamped at a holding potential of +80 mV and −80 mV during the addition of 100 M ticlopidine (A) or 100 M clopidogrel (C) followed by 100 M HC-030031. Data were extracted from voltage ramps such as in (B and D). (B and D) I/V curves for ticlopidine- and clopidogrel-induced current densities in QGP-1 cells before (black line) and after block with HC-030031 (grey line) were taken at the time points indicated in (A and C). Inset: statistical analysis of 10 (ticlopidine) and 4 (clopidogrel) independent recordings such as in (A) and (C), depicted as means and SEM. For statistical analysis only cells responding to the drug were included.
Fig. 5. Ticlopidine and clopidogrel but not prasugrel provoke 5-HT release in QGP-1 cells. 5-HT release from QGP-1 cells was determined in culture supernatants using an enzyme-linked immunosorbent assay. Cells were incubated for 1 h at 37 ◦ C in the presence of 100 M ticlopidine (T), clopidogrel (C), prasugrel (P) and AITC without addition of a TRPA1 blocker (black bars) or in the continuous presence of 100 M HC030031, which was applied 10 min before the activators (grey bars). Open bar: DMSO control (1%). Data represent means and SEM of three independent experiments performed in duplicates, each, *p < 0.05 and **p < 0.01.
4. Discussion Here, we present an activation of TRPA1 by the antiplatelet drugs ticlopidine, clopidogrel and prasugrel. In HEK293TRPA1 cells, the compounds evoke TRPA1-mediated intracellular Ca2+ signals and ionic currents. In whole cell recordings, all three compounds elicit currents with outwardly rectifying I/V curves, typical to TRPA1,
with ticlopidine being the strongest and prasugrel the weakest activator. Several TRPA1 activators such as menthol, nicotine and apomorphine exert a bimodal action on TRPA1, activating the channel in the low micromolar range but with antagonistic properties at higher concentrations [12,13,41]. We did not see inhibitory properties of ticlopidine, prasugrel, and clopidogrel but it has to be taken into account that concentrations above 100 M were not tested. The mechanisms of TRPA1 activation are diverse. Electrophilic substances have been shown to form disulfide bonds with cysteines in the N-terminus of the channel [37,38,42]. Other compounds such as menthol, nicotine and clotrimazole presumably activate TRPA1 independently of any covalent modification via a currently unknown mechanism [13,16,41]. As the thienopyridines do not possess electrophilic properties, we find it unlikely that the activation of TRPA1, we here describe, involves a covalent modification of TRPA1, Moreover, a TRPA1 mutant, which is largely insensitive to electrophiles (TRPA1-3C-S) [37] still showed TRPA1-mediated Ca2+ entry in response to ticlopidine, clopidogrel and prasugrel, albeit in a slightly reduced manner, which might reflect an altered expression of the TRPA1-3C-S construct in comparison with wild-type TRPA1. Besides its expression in neuronal cells, TRPA1 is also present in non-neuronal cells, like enterochromaffin cells [27]. Little is known about the physiological function of TRPA1 throughout the digestive system, but recent studies hint to a putative role in triggering the secretion of serotonin. For example, Doihara et al. demonstrated that enterochromaffin-like QGP-1 cells express TRPA1 and release 5-HT upon TRPA1 activation with AITC, cinnamaldehyde and acrolein [39]. The dispense of 5-HT into the GI lumen follows intraluminal distension, vagal nerve stimulation and ingestion of nutrition such as olfactants and tastants [43,44]. The most important mechanism behind the release of 5-HT from EC cells is an
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increase in [Ca2+ ]i [45]. Here, we report that upon stimulation with ticlopidine and clopidogrel, QGP-1 cells exhibit an increase in [Ca2+ ]i , which is prevented by two different TRPA1-specific blockers. In contrast, prasugrel-treated QGP-1 cells exhibited only very weak calcium signals, which were lower and more transient with fewer responding cells. No EC50 values for the activation of TRPA1 by the thienopyridines in QGP-1 cells could be determined but it is evident that in QGP-1 cells the channel is less sensitive to the drugs compared to the heterologous expression system. Furthermore, QGP-1 cells apparently display a higher degree of heterogeneity within the population, which was also evident from the patch clamp recordings. This inhomogeneity might be caused by a variable expression of TRPA1 and also of plasma membrane Ca2+ pumps and cytoplasmic Ca2+ buffering, which counteracts the TRPA1-mediated Ca2+ influx [46]. TRPA1 activation can be strongly potentiated by elevations of [Ca2+ ]i (EC50 900 nM) [36,47]. Therefore, depending on the channel density and strength of the respective activator, the resulting internal Ca2+ concentration may or may not trigger a secondary TRPA1 activation, causing an amplified response. Interestingly, when QGP-1 cells were challenged with ticlopidine or clopidogrel in patch clamp recordings, the resulting I/V curve was almost linear, possibly reflecting the dilated state of the TRPA1 channel [48]. Ticlopidine and clopidogrel only activated currents in a subset of QGP-1 cells, which might be due to the strong buffering of intracellular calcium in whole cell recordings. A rise in [Ca2+ ]i , as detected in our experiments after stimulation with the thienopyridine drugs, correlates with an excretion of 5-HT. Indeed, we could determine a release of 5-HT upon ticlopidine and clopidogrel treatment, which could be suppressed by HC-030031. No significant increase in the extracellular 5-HT concentration was evident when cells were treated with prasugrel. A possible reason might be that a strong and stable [Ca2+ ]i signal, that could not be obtained with prasugrel, is necessary to provoke 5-HT secretion. Obviously, relatively high concentrations of the platelet aggregation inhibitors were required to stimulate TRPA1 in QGP-1 cells. Reported peak plasma concentrations of the active metabolites are 3 M, 179 nM, and 233 nM after a dose of 250 mg ticlopidine twice daily, 300 mg load/75 mg daily clopidogrel and 60 mg load/10 mg daily prasugrel, respectively [3]. However, the effective pre-systemic concentrations of the prodrugs, acting on endocrine cells in the stomach and small intestine after oral administration are certainly higher than the peak plasma concentrations of the active metabolites, and possibly suffice to cause a TRPA1mediated 5-HT release in vivo. Released 5-HT exerts emesis by stimulation of the CTZ via vagal afferents [7,8]. Considering that ticlopidine and clopidogrel administration is frequently accompanied by adverse events such as nausea, vomiting and diarrhoea [4,5], it may be possible that these two platelet inhibitors trigger the release of 5-HT through a TRPA1 activation. This would be consistent with our finding that there is no association between taking prasugrel and the incidence of adverse effects in the GI tract. TRPA1 is not only involved in the secretion of 5-HT, but also in the release of cholecystokinin (CCK) from neuroendocrine STC-1 cells [26,49]. Although mainly responsible for the regulation of food digestion and satiety, high doses of intravenously infused CCK to healthy subjects produce nausea and abdominal discomfort [50]. Thus, GI-related adverse effects of TRPA1 activators might be mediated by more than one paracrine hormone. To summarise our results, we could show that the tested platelet inhibitors are direct activators of TRPA1 in HEK293TRPA1 and QGP-1 cells with varying efficacies and potencies: ticlopidine > clopidogrel > prasugrel. Furthermore, ticlopidine and clopidogrel stimulate QGP-1 cells to release 5-HT, reminiscent of the 5-HT release by apomorphine. Thus, TRPA1 may not only act as a versatile
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