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Centre National de la Recherche Scientifique, Unité 8162, Université de Paris XI, and Laboratoire de Recherches Médicales, Hôpital Marie Lannelongue, Le Plessis-Robinson, France (C.P.); Institut National de la Santé et de la Recherche Médicale, Unité 621, and Université Pierre et Marie Curie-Paris 6, Unité Mixte de Recherche S621, Paris, France (V.A., S.N.H., A.C.); and Division of Cardiovascular Diseases II, Centre de Recherche Pierre Fabre, Castres, France (S.S., B.LG, C.P., D.C., M.P.)
Received November 5, 2007; accepted March 6, 2008
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Abstract |
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). Indeed, thrombin can permeate vessels or endocardial barrier by extravasation and therefore can affect the first layers of cardiomyocytes. Thus, thrombin levels may be locally elevated at site of vascular injury and thrombus formation and reach an activity as high as 10 to 30 U/ml (Park et al., 1994). Then, thrombin has multiple cellular effects mediated by a family of G-protein-coupled protease-activated receptors (PARs), of which PAR1 is the prototype (Coughlin, 2000). PAR1 are expressed in human myocardium (Jiang et al., 1996) and activates a spectrum of biochemical signals leading to changes in contractile performance and alteration in gene expression (Glembotski et al., 1993), sarcomeric organization, and cardiomyocyte morphology (Sabri et al., 2002). As shown recently, theses phenomenons are clearly involved in human coronary atherosclerosis and can lead to endothelial dysfunction and vascular inflammation (Lavi et al., 2007). Thus, thrombin stimulates phosphoinositide hydrolysis, for instance by converting phosphatidylcholine into lysophosphatidylcholine (LPC), via PLA2 (Park et al., 1994; Sabri et al., 2000). Thrombin also activates the extracellular signal-regulated protein kinase (Sabri et al., 2002), facilitates rapid sodium current (Pinet et al., 2002), modulates calcium homeostasis (Steinberg et al., 1991, 2005), increases arrhythmias (Goldstein et al., 1994), and hastens recovery from an imposed acid load by activating Na+-H+ exchange (Avkiran and Haworth, 2003). Taken together, these signal events profoundly alter electrophysiological properties and contractile behavior and could induce cardiomyocyte toxicity during the myocardial ischemia-reperfusion injuries.
Increase in intracellular sodium during ischemia plays a key role in intracellular calcium overload, resulting in myocyte injuries. The involvement of thrombin-dependent activation of PAR1 in Na+ entry during ischemia has never been assessed. In a previous study, we reported that thrombin can reversibly act as direct agonist on INa. The main consequences of this action were the shift toward hyperpolarizing potentials of the activation-voltage relationship, the large increase in peak INa amplitude, and the consequent increase in the window of sodium current; however, this effect was independent of PAR1 activation (Pinet et al., 2002). The present study examines whether PAR1 signaling in human cardiomyocytes is involved in the effect of thrombin on sodium current specially the persistent sodium current (INaP) (Haigney et al., 1994; Maltsev et al., 1998; Fedida et al., 2006; Noble and Noble, 2006; Saint, 2006). The main result was that thrombin, by binding to PAR1 receptor, activates INaP.
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Materials and Methods |
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Cell Isolation. Human atrial myocytes were isolated enzymatically as described previously (Antoine et al., 1998). The same chunk procedure was used to isolate myocytes from the right ventricle of guinea pig hearts, except that elastase was omitted in the chunks predigestion bath. Only quiescent rod-shaped myocytes with clear cross-striations, sharp edges, and a well delineated cell membrane were chosen for experiments. Small myocytes were preferred to optimize spatial voltage-clamp.
Solutions and Drugs. For whole-cell current recordings, the intracellular pipette solution contained 5 mM NaCl, 130 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, 15 mM EGTA, 10 mM HEPES, and 4 mM MgATP, pH adjusted to 7.2 with CsOH; the basal external solution contained 25 mM NaCl, 108.5 mM CsCl, 2.5 mM CoCl2, 0.5 mM CaCl2, 2.5 mM MgCl2, 5 mM 4-aminopyridine, 10 mM HEPES, and 10 mM glucose, pH adjusted to 7.4 with CsOH. Hirudin (from leeches, 
2000 U/mg) was from Roche (Mannheim, Germany). Haloenol lactone suicide substrate (HELSS) from TEBU France, was dissolved in dimethyl sulfoxide that did not exceed 0.05%. The PAR1 agonist (SFLLR-NH2) and antagonist SCH 203099 were from the Division of Medicinal Chemistry IV (Centre de Recherche Pierre Fabre, Castres, France), 
-thrombin (human plasma 1000 U/mg), and other chemicals were purchased from Sigma (St. Quentin Fallavier, France). Commercially available lyophilized thrombin is provided as a sodium salt. The addition of 32 U/ml thrombin to the basal perfusion medium containing 25 mM NaCl increases the Na+ activity to 32.1 ± 0.3 mM (n = 10) (measured with a sodium ion-selective electrode). The control perfusion medium was therefore supplied with NaCl to keep the Na+ activity equivalent.
Current Recordings. Ionic currents were recorded by the whole-cell patch-clamp technique with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA). Patch pipettes (Corning Kovar Sealing code 7052; World Precision Instruments, Sarasota, FL) had resistances of 0.5 to 2.0 M
. Currents were filtered at 20 kHz (-3 dB, eight-pole, low-pass Bessel filter) and digitized at 50 kHz (Digidata 1200; Molecular Devices, Sunnyvale, CA).
Cell membrane capacitance was 48.2 ± 2.6 pF (n = 67 cells, from 62 donors) for human atrial myocytes and 62.4 ± 7.5 pA/pF (n = 18, from 10 hearts) for guinea pig right ventricular myocytes. Series resistance was compensated at 80 to 95%, resulting in voltage errors of <3 mV. Leakage current was compensated for, whereas cell membrane capacitive current was not. Peak INa and INaP amplitudes were monitored according to a steady-state pulse protocol: a 1000-ms depolarizing test pulse to -30 mV from a HP of -100 mV at 0.2 Hz. It is noteworthy that the test pulse chosen was beyond the upper limit of the potential range inside which the sodium window current has been observed to increase under the direct effect of thrombin on sodium channel (Pinet et al., 2002). To avoid a putative overlap between PAR1-induced INaP and thrombin-increased sodium window current, experiments were also performed with test pulse to -10 mV. An equilibration period was allowed until peak INa reached steady state and remained stable without evidence of a leftward shift of the availability-voltage relationship (h
-Vm). This protocol was designed to detect any such shifts. After each sequence of five depolarizing pulses, HP was set to -140 mV. Consequently, during the stabilization period, when peak current amplitude was higher after a HP at -140 mV compared with -100 mV, the recording was discarded. Likewise, because thrombin has no effect on the h-Vm relationship (Pinet et al., 2002), recordings showing an irreversible hyperpolarizing shift of h-Vm after thrombin application were also discarded. The steady-state pulse protocol was applied in control, thrombin, or when other substances were tested and washed out. Experiments were carried out at room temperature (22-25°C).
Action Potential Recording. Male guinea pigs were anesthetized by intraperitoneal pentobarbital sodium injection, and their hearts were quickly removed. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication no. 85-23, revised 1996). A total of 21 hearts were used for the AP study. Papillary muscles were dissected from the right ventricle and superfused (4-6 ml/min) with the Krebs' solution maintained at 36.0 ± 0.5°C in a 5-ml tissue bath. The Krebs' solution was oxygenated with 95% O2/5% CO2 and had the following composition: 113.1 mM NaCl, 4.6 mM KCl, 2.45 mM MgCl2, 3.5 mM NaH2PO4, 21.9 mM NaHCO3, and 5 mM glucose, pH 7.4. Action potentials (AP) were recorded by conventional "floating" glass microelectrodes (5-20 M) filled with 3 M KCl and were coupled to a high-input impedance preamplifier (VF 102; Biologic, Echirolles, France). The preparations were electrically stimulated (Pulsar BP; FHC, Bowdoin, ME) with 1-ms pulses at 1.5 times the threshold voltage through a bipolar Ag-electrode. APs were displayed on a dual-beam oscilloscope (TDS 2012; Tektronics, Heerenveen, The Netherlands) and simultaneously digitized and analyzed with interactive software (NOTOCORD-hem 3.4; Notocord Systems, Croissy Sur Seine, France). The preparations were allowed to equilibrate for at least 1 h at a stimulation rate of 1 Hz. A single impalement was maintained throughout control and compound superfusion periods. AP parameters measured were maximum upstroke velocity [(dV/dt)max], amplitude, resting membrane potential, and action potential duration (APD) at 50 and 90% repolarization levels (APD50 and APD90, respectively).
Data Analysis and Statistics. As much as possible, depending on the experimental protocol performed, only the TTX-inhibited PAR1-induced INaP was taken into account and determined by subtracting the current obtained under concomitant application of TTX and thrombin or SFLLR-NH2 from that previously recorded under thrombin alone. The amplitude of TTX-blocked thrombin-induced INaP was the mean current calculated from 80 to 180 ms after the beginning of the depolarization (Valdivia et al., 2002). Peak INa amplitude was measured with respect to current amplitude at the end of the test pulse. Data are expressed as means ± S.E.M. of n determinations or myocytes. Statistical analysis were performed by using paired or unpaired Student's t test or analysis of variance, as appropriate, and the null hypothesis was rejected at the 0.05 level; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
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Results |
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). Local perfusion of 32 U/ml thrombin on freshly isolated human atrial myocytes caused the already described marked increase in peak of INa (Fig. 1 A), which occurred rapidly (Fig. 1D) and was shown to be completely independent of the activation of PAR1 receptors (Pinet et al., 2002). In addition to this effect on peak INa, after a longer time of application, over 6 min, thrombin induced very slowly inactivating or persistent TTX-blocked sodium current (Fig. 1, B and D), termed INaP. Although the increase in peak INa was observed in all tested cells, the induction of INaP was observed in 51 of 67 cells, most probably as a consequence of the lost and/or damage caused to PAR1 receptors by the enzymatic procedure used for cell dissociation. Thrombin (32 U/ml) increased peak INa from -118.9 ± 12.6 to -191.7 ± 20.8 pA/pF (n = 23; p < 0.001, paired t test) and induced INaP in a concentration-dependent manner (Fig. 1C). At this concentration of thrombin, maximum increase in INaP was 1.8% of control peak INa. The density of INaP was -0.02 ± 0.01 pA/pF, n = 3, with 1 U/ml thrombin, -0.50 ± 0.02 pA/pF, n = 3, with 10 U/ml thrombin, -2.23 ± 0.54 pA/pF, n = 6, with 32 U/ml thrombin, and -3.69 ± 0.82 pA/pF, n = 4, with 100 U/ml thrombin. Estimated-maximal density of INaP was -3.98 pA/pF, and the apparent EC50 and the Hill coefficient were 28.4 U/ml and 1.98, respectively. This concentration-response relationship exhibits clear differences compared with the reported concentration-response of the effect on peak INa (Pinet et al., 2002) where EC50 and Hill coefficient were 91 U/ml and 0.75, respectively. Thrombin-induced INaP was not a residual current resulting from the thrombin-enhanced window sodium current (Pinet et al., 2002), because the increase in INaP, with 32 U/ml thrombin, was observed with test pulses to -10 mV, a membrane potential at which the window current could not be activated (INaP density was -1.71 ± 0.83 pA/pF; n = 5). These two distinct effects of thrombin on sodium current (increase in peak INa and in INaP) were suppressed by 50 µM TTX, a specific sodium channel blocker (Fig. 1, A and B). It is noteworthy that the difference in kinetics of thrombin effect on peak INa compared with the onset of INaP (Fig. 1D) and that the time to reach maximum thrombin effects was much longer for INaP (416.0 ± 40.4 s, n = 15) than for peak INa (61.7 ± 10.1 s, n = 12; Fig. 1E), suggesting distinct underlying mechanisms. This is also supported by the observation that after washout of thrombin, the amplitude of peak INa returned to the basal (Pinet et al., 2002) but not that of INaP (Fig. 1D), in accordance with the irreversibility of the cleavage of PAR1 by thrombin. The below of Fig. 1D exemplifies the prominence of thrombin effect on INaP over the effect on peak INa, when the latter effect wore out.
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) has been used. After human atrial cell preincubation with 10 µM SCH 203099 for at least 15 min, thrombin failed to induce INaP [Fig. 3, B-D; -0.30 ± 0.07 pA/pF (n = 14) versus -2.27 ± 0.51 pA/pF, n = 8 in absence of SCH 203099 (p < 0.001)], whereas the thrombin effect on peak INa was unaffected [Fig. 3, A and C; from -103.7 ± 14.1 pA/pF under SCH 203099 to -158.4 ± 21.6 pA/pF (n = 14) after addition of 32 U/ml thrombin (p < 0.001)]. These results suggest that PAR1 activation constitutes a key mechanism for the thrombin-induced INaP. Then, the ability of the PAR1 agonist peptide SFLLR-NH2 to putatively mimic the role of thrombin in induction of INaP via PAR1 was tested (Fig. 4). SFLLR-NH2 stimulated INaP [mean increased: -1.46 ± 0.63 pA/pF, n = 5, p < 0.05 versus control (Fig. 4, B and C)], exhibiting a fair reversibility upon washout (Fig. 4, B and D) but failed to increase peak INa (Fig. 4, A and D). Taken together, the present results demonstrate that the thrombin-induced INaP is mediated by PAR1 activation.
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). Several studies have reported that in cardiomyocytes, thrombin stimulates the Ca2+-independent phospholipase A2 (PLA2) via PAR1, which produced an intracellular lysophosphatidyl choline (LPC) accumulation (Park et al., 1994; Yan et al., 1995). Therefore, two inhibitors of PLA2 [bromoenol lactone (BEL) and haloenol lactone suicide substrate (HELSS)] were used to investigate whether PLA2 activity is involved in the enhancement of INaP, induced by PAR1 stimulation. After cell incubation with BEL for at least 30 min, thrombin had its usual effect on peak INa (Fig. 5A) but failed to stimulate INaP (Fig. 5B, -0.22 ± 0.11 pA/pF, n = 5 versus -2.27 ± 0.51 pA/pF, n = 8, in the absence of BEL; p < 0.01). A similar result was obtained with HELSS (Fig. 5C). The density of INaP activated in presence of thrombin and HELSS was -0.39 ± 0.14 pA/pF, n = 5 (p < 0.01). These results indicate that the PAR1-induced INaP required the activation of Ca2+-independent PLA2.
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Thrombin and PAR1 Agonist Peptide SFLLR-NH2 Increased Action Potential Duration. To examine the consequence of activation of INaP by thrombin and PAR1 agonist peptide SFLLR-NH2 on cardiac tissue electrical activity, we recorded action potentials in right ventricle papillary muscle from guinea pigs. We used the classic microelectrode technique, which allows a stable and reliable AP recording during prolonged period as required for the study of the delayed effect of thrombin on INaP. We first checked that, in this species and in ventricular cells, thrombin also activates INaP by performing patch-clamp experiments in isolated guinea pig ventricular myocytes. Test pulses to -10 mV from a holding potential of -80 mV were used to elicit current. Immediate application of thrombin (32 U/ml) increased peak INa (Fig. 6A, a and b) from -112.6 ± 11.5 to -166.9 ± 16.4 pA/pF, n = 11 (p < 0.001; paired t test), blocked by application of 50 µM TTX. Moreover, after 7 min of application, thrombin induced a TTX-sensitive INaP of -2.91 ± 1.1 pA/pF, n = 9 (Fig. 6A, b and d). The increase in INaP was 0.026% of control peak INa. This effect was blocked by the PAR1 antagonist ER 112787 (Barry et al., 2006; Chackalamannil and Xia, 2006) (Fig. 6B). After myocytepreincubation with 1 µM ER 112787 for at least 5 min, thrombin did not induced INaP [Fig. 6B, b, c, and d; -0.076 ± 0.05 pA/pF (n = 5) versus -2.91 ± 0.68 pA/pF, in absence of drug, n = 9; p < 0.001], whereas the effect of thrombin on peak INa was unaffected (Fig. 6A, a and c; -87.3 ± 6.1 pA/pF, n = 5, under ER 112787 to -123.4 ± 10.3 pA/pF, n = 9, after addition of thrombin; p < 0.05).
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Figure 7A, a, shows the effects of thrombin (32 U/ml) and SFLLR-NH2 on action potentials recorded from right papillary ventricle. After a 10-min application, thrombin and SFLLR-NH2 significantly increased APD measured at 50 and 90% of repolarization (Fig. 7A and Table 1). At these two levels of repolarization, the increases in APD were 15.3 and 12.9%, respectively, for thrombin and 12.4 and 12.1% for SFLLR-NH2 (Table 1). The increases in APD were barely reversible (Table 1). It is noteworthy that in presence of TTX (1 µM), applications of thrombin (32 U/ml) or SFLLR-NH2 (100 µM) failed to increase the action potential duration (Fig. 7A, b, and Table 2). We used a low concentration of TTX to minimize the consequences of the inhibition of peak INa on AP upstroke.
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To investigate the involvement of PAR1 on thrombin- and SFLLR-NH2-induced APD lengthening, two selective PAR1 antagonists, SCH 203099 and ER 112787, were then evaluated. Figure 7B, a and b, show that both SCH 203099 and ER 112787, in a concentration-dependent manner, reduced the thrombin- and SFLLR-NH2-induced APD prolongations. Thus, SCH 203099 (10 µM) and ER 112787 (1 µM) both fully blocked the APD prolongation induced by 100 µM SFLLR-NH2 (Fig. 7B), a result that was in complete agreement with the potency of this antagonist against PAR1 (Chackalamannil and Xia, 2006). Taken together, these results indicated that stimulation of the inward persistent Na+ current by protease-activated receptors after PAR1 activation can modify the AP duration.
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Discussion |
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Involvement of PAR1 Signaling Pathway in the Thrombin Effect on INaP. The conclusion that thrombin enhances INaP through the proteolytic cleavage of PAR1 is supported by the findings that this thrombin effect on current is mimicked by the PAR1 agonist peptide SFLLR-NH2 and is fully blocked by the PAR1 antagonists, SCH 203099 and ER 112787 (Ahn and Chackalamannil, 2001). Cleavage of the amino-terminal PAR1 exodomain unmasks a tethered ligand that binds the receptor body to trigger intracellular signaling. Consequently, PAR1 is irreversibly activated by this proteolytic cleavage, contributing to keep on its signal-transducing capability. This could explain the low reversibility of the thrombin effect on INaP upon washout (see Fig. 1). This was not the case for SFLLR-NH2 (see Fig. 4), an exogenous peptide that does not cleave the PAR1 and that exhibited an effect more sensitive to washout. Another evidence that thrombin effect on INaP is mediated by PAR1 signaling pathways is provided by the observation that this effect is suppressed by the inhibition of PLA2 by BEL and HELSS. There are previous studies showing that the increase in intracellular LPC levels plays an important role in the thrombin effect (Undrovinas et al., 1992; Park et al., 1994; Yan et al., 1995). For instance, the cleavage of PAR1 by thrombin stimulates the amphipathic lipids catabolism leading to an accumulation of LPC in the intracellular medium (Undrovinas et al., 1992; Park et al., 1994). However, the precise level of the signaling pathway at which PLA2 inhibitors were able to inhibit thrombin-induced INaP remains to be clarified.
Nature of the INaP Current. The intracellular accumulation of LPC could mediate the effect of thrombin on INaP. The LPC is well known to maintain sodium channel in bursting activity, thus giving rise to noninactivating sodium current (Undrovinas et al., 1992). The exact nature of this noninactivating sodium current is not yet fully elucidated. There is evidence that this persistent component of sodium current, INaP, could be generated by a small fraction of the "normal" transient-mode sodium channel population, that undergoes burst or/and late scattered mode(s). Such modes have been recently described by Maltsev and Undrovinas (2006) for the persistent Na+ current in human ventricular myocytes. Thus, thrombin could favor this bursting activity of voltagegated sodium channels, by cleaving PAR1 and stimulating the conversion of phosphatidylcholine into LPC, which, via PLA2 (Park et al., 1994; Sabri et al., 2000), increases intracellular LPC (Undrovinas et al., 1992; Park et al., 1994). In a previous study, we reported that thrombin stimulates the peak INa and the sodium-window current. However, this effect, which is fast and fully reversible, is not mediated by PAR1 (Pinet et al., 2002) as further demonstrated in the present study. Moreover, thrombin was able to induce INaP at membrane potentials as high as -10 mV, which is clearly not in membrane potential range of window current (from -85 to -40 mV). Finally, there are clear differences between the concentration-response curves of the effect of thrombin on peak INa (Pinet et al., 2002) and on INaP. One hypothesis currently tested concerning the direct effect of thrombin on peak INa is the involvement of a β-subunit (Herfst et al., 2003). The majority of Na+ channels in the heart correspond to the expression of the TTX-resistant NaV1.5 isoform, and this is the case for human atrial myocytes (Makielski et al., 2003). However, it cannot be excluded that thrombin-PAR1 activation targets another population of sodium channels (Brette and Orchard, 2006). The effect of thrombin on INaP that develops slowly is likely to have more significant consequences on cardiac myocyte.
Pathophysiological Consequences of the Activation by Thrombin of INaP. Both persistent and window sodium current are known to participate to action potential duration. Although the window sodium current, which activates in a restricted membrane potential range from -85 to -40 mV, is involved in the late phase of repolarization and in the control of resting membrane potential (Pinet et al., 2002), INaP, activated from -10 mV, is found to contribute to the regulation of the early phase of APD (Kiyosue and Arita, 1989; Maltsev et al., 1998; Sakmann et al., 2000; Fedida et al., 2006; Noble and Noble, 2006; Wu et al., 2006). It has been previously reported that blocking the INaP with TTX caused a 10 to 20% decrease of APD, although this current is of very small density in control condition (Kiyosue and Arita, 1989; Maltsev et al., 1998; Sakmann et al., 2000). Therefore, the progressive several-fold increase in INaP by thrombin is likely to have a significant effect on APD, whereas, despite a fast increase in peak INa (Fig. 1), thrombin is devoid of significant effect on (dV/dt)max. Indeed, thrombin and SFLLR markedly increased the duration of action potentials of guinea pig papillary muscle. This effect of thrombin or SFLLR-NH2 on AP duration can be largely explained by an increase of INaP, because it was abolished by TTX. However, it cannot be excluded that the thrombin/PAR1 pathway can affect other ionic currents indirectly via changes in [Na]i and [Ca]i. Previous studies have shown that thrombin lengthens APD and increases cesium-induced early afterdepolarizations and pro-arrhythmic events in canine Purkinje fibers (Steinberg et al., 1991) and in intact adult rat hearts during early reperfusion (Jacobsen et al., 1996; Woodcock et al., 1998). It has been shown also that suppression of the late sodium current can suppress EADs of myocytes isolated from failing hearts (Maltsev et al., 1998; Undrovinas et al., 2002; Valdivia et al., 2005; Fedida et al., 2006). Thus, blocking INaP by inhibiting PAR1 - PLA2 pathway may be a new pharmacological target to reduce thrombin-induced arrhythmic activity.
Yan et al. (1995) have demonstrated that activation of PAR1 by SFLLR-NH2 peptide induced a rapid and dramatic elevation in [Na+]i, which was associated with a concomitant increase in LPC content in isolated, blood-perfused rabbit hearts in response to ischemia (Lavi et al., 2007). The increase in [Na+]i could contribute to myocyte injury during ischemia as the result of intracellular calcium overload and the activation of Ca2+-dependent signaling cascades. This hypothesis is supported by the recent finding of the cardioprotective effects of the late sodium current inhibitor ranolazine (Belardinelli et al., 2006). Moreover, in in vivo and in vitro studies, Strande et al. (2007) have shown that a preventive and a curative treatments with a selective PAR1 antagonist reduced the infarct size and increased ventricular function recovery after ischemia reperfusion in an isolated heart model. It remains to determine whether the increase in [Na+]i induced by SFLLR-NH2 or by thrombin during ischemia is mediated by the activation of INaP.
In conclusion, this study describes a new regulatory mechanism of sodium current involving PAR1-PLA2 signaling pathway, which could be evoked by thrombin during cardiac ischemia and thrombus formation (Haigney et al., 1994; Maltsev et al., 1998). Selective antagonists of PAR1 receptor, which are very efficient to suppress this effect of thrombin, might represent a novel cardioprotective strategy in the clinical setting of myocardial ischemia and reperfusion (Strande et al., 2007).
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Acknowledgements |
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Footnotes |
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C.Pinet and V.A. contributed equally to this study.
ABBREVIATIONS: PAR, protease-activated receptor; HELSS, haloenol lactone suicide substrate; SCH 203099, N3-cyclopropyl-7-((4-(1-methylethyl)phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine; HP, holding potential; AP, action potential; APD, action potential duration; TTX, tetrodotoxin; PLA2, phospholipase A2; LPC, lysophosphatidyl choline; BEL, bromoenol lactone; ER 112787, 1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-[3-(3-ethyl-3-hydroxy-pentyl)-2-imino-2,3-dihydro-imidazol-1-yl]-ethanone.
1 Current affiliation: Centre National de la Recherche Scientifique UMR 6187, Institut de Physiologie et de Biologie Cellulaire, Université de Poitiers, Poitiers, France. 
Address correspondence to: Alain Coulombe, INSERM U-621, 91 Blvd de l'Hôpital, 75634 Paris cedex 13, France. E-mail: alain.coulombe{at}chups.jussieu.fr
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