Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine.

Scientific RepoRts | 5:17969 | DOI: 10.1038/srep17969 is unknown if Na v channel inhibition could contribute to the vasorelaxant effect of ranolazine. Na v channels are potential targets for ranolazine due to their role in regulating arterial contraction 18,19 . The present work aimed to explore the vascular effects of ranolazine and to elucidate the underlying molecular mechanisms.

Results
Effects of ranolazine on Na + current in rat aortic SMCs. I Na was evoked in rat aortic SMCs using either a voltage-ramp protocol or square depolarizations. In order to promote the current with sustained activation during depolarization, we used the Na v agonist veratridine. In presence of veratridine (100 μ M), I Na activated at voltages positive to − 30 mV and peaked around − 10 mV (Fig. 1). We used the specific Na v blocker tetrodotoxin (TTX) to validate that this current originated from Na v , and to quantify and specify the effect of ranolazine. In the presence of 1 μ M TTX, all currents were blocked (Fig. 1A). Ranolazine (20 μ M) blocked the TTX-inhibited I Na at its maximal amplitude (Fig. 1A,B), reducing the current by 40%. In sharp contrast with the blocking effect of TTX, ranolazine inhibition of I Na increased markedly with depolarization ( Fig. 1B, right panel).
Effects of ranolazine on intracellular Ca 2+ in rat aortic myocytes. In primary cultured rat aortic SMCs, veratridine (100 μ M) induced a transient and reproducible increase in [Ca 2+ ] i (Fig. 2). Ranolazine (20 μ M) and TTX (1 μ M) similarly inhibited the veratridine-induced [Ca 2+ ] i increase (Fig. 2). The veratridine response was completely blocked by TTX and was antagonized by 82.6 ± 6.2% by ranolazine. No antagonistic effect of either ranolazine or TTX was observed on the basal level of [Ca 2+ ] i suggesting that Na v channels were not activated at rest.

Figure 1. Ranolazine antagonizes veratridine-induced I Na in rat aortic myocytes. (A) (Left panel)
Representative I Na current traces obtained on cultured SMCs in control (Ctl) and in the presence of 20 μ M ranolazine (+ ranolazine) and 1 μ M tetrodotoxin (+ TTX). The current was revealed by a 40 ms ramp from − 100 to + 40 mV, following a 2 sec prepulse at − 100 mV, from a holding potential of − 80 mV in presence of veratridine (100 μ M). Arrows indicate the activation and the maximal amplitude of the current with the corresponding voltages. (Right panel) Bar graph showing the averaged data expressed as mean ± sem (n = 10). (B) (Left panel) I Na current-voltage relationships, obtained as described in A, for control (⚬, Ctl) and in the presence of ranolazine ( ) or TTX (• ) (n = 10). The inset shows representative traces of the I Na recorded for each condition at 0 mV from a HP of − 80 mV. (Right panel) Bar graph showing I Na block at various voltages, in the presence of ranolazine and TTX. Values expressed as percentage were calculated after subtraction of the TTX-insensitive current (n = 10). *p < 0.05, ***p < 0.001, one-way Anova followed by Bonferroni post-test.  M). Arrows indicate the time of application of veratridine. (C) Bar graph representing the (Ca +2 ) i increase induced by veratridine under various conditions. Changes in the fluorescence ratio induced by veratridine were determined under basal conditions and in the presence of ranolazine or TTX. Data are expressed as percent of the response induced by a first application of veratridine on the same cellular field and represent the mean ± sem of 6 different cell cultures (4 cover glasses/fields for each experimental condition per cell culture). **p < 0.01, Kruskal-Wallis one-way analysis of variance followed by Dunn's test. Ranolazine inhibited Na v channel-dependent aortic contraction. In aortic rings, veratridine (100 μ M) triggered an increase in tension corresponding to 44 ± 3% of the maximal contraction induced by phenylephrine (Phe, 10 μ M) in the presence of endothelium and to 56 ± 2% without endothelium (Fig. 3A). The subsequent addition of ranolazine induced a dose-dependent relaxation at concentrations ranging from 0.1 to 100 μ M, both in aortic rings with an intact endothelium (IC 50 2.5 ± 0.9 μ M, n = 6) and in endothelium-free preparations (IC 50 2.9 ± 1.3 μ M, n = 6) (Fig. 3A). Prior incubation with ranolazine (20 μ M) abolished the contractile response to veratridine (not shown). These results showed that ranolazine prevents and reverses veratridine effects in an endothelium-independent manner and initiates vasorelaxation of the artery.
We next investigated the effects of ranolazine on the vascular smooth muscle contractility according to experimental protocols that we have previously designed to unmask Na v channels contribution to contractile function 18 . Thereby, we compared responses to increasing concentrations of KCl by cumulative additions ranging between 2 and 40 mM in the absence or presence of ranolazine following or not α 1 -adrenergic receptor blockade with prazosin (10 μ M). We observed that ranolazine (20 μ M) prevented the contraction induced by low KCl concentrations (less than 10 mM and below EC 50 value) (Fig. 3B) both in the absence and in the presence of prasozin. The inhibitory effect of ranolazine induced a rightward shift in the dose response curves with slight increases in the EC 50 values: 7.5 ± 0.6 mM vs.6.1 ± 0.3 mM (p = 0.0316, t-test) in the absence of prazosin and 8.9 ± 0.7 mM vs. 7.1 ± 0.4 mM (p = 0.0349, t-test) in the presence of prazosin. Prazosin was also used in combination with a Na v channels antagonist (TTX) to unmask the contribution of SMCs Na v channels to the contraction induced by low KCl concentrations. In the presence of TTX (1 μ M), the KCl response was rightward shifted for concentrations below 10 mM, reflecting Na v channel inhibition. The same effect was obtained with ranolazine (20 μ M). There was no additional inhibition of ranolazine in the presence of TTX (Fig. 3C). The same inhibitory profiles were obtained with KB-R7943 (10 μ M), a blocker of the reverse mode of the NCX 26 . We observed no difference between contractile responses to low KCl concentrations either in presence of ranolazine, KB-R7943 or KB-R7943 plus ranolazine (Fig. 3C). Ranolazine had no additional effect after NCX blockade. In Fig. 3C, the bar graph demonstrates that the maximal contractile response to 80 mM KCl either in presence of TTX, ranolazine or KBR was unchanged while a robust inhibition was observed in presence of nifedipine (1μ M), a Ca 2+ channel blocker.
Ranolazine inhibited α 1 -adrenergic-dependent rat aortic contraction. Since antagonistic effects of ranolazine on the α 1 -adrenergic receptor have been reported, we investigated if this pathway is involved in the effects of ranolazine on arterial contraction in our model. We observed that ranolazine induced a dose-dependent relaxation (IC 50 8.4 ± 1.3 μ M; n = 6) of rat aorta previously contracted with a non-maximally active concentration of Phe (1 μ M) (Fig. 4A). In the presence of ranolazine (20 μ M), the dose-dependent response to Phe was shifted to the right (Fig. 4B), consistent with a competitive inhibition that was likewise correlated to ranolazine concentration (not shown). Furthermore, no effect of ranolazine was observed on the maximal response to Phe (Fig. 4B-inset).
The competitive antagonization of the α 1 -adrenergic receptor with ranolazine was confirmed on [Ca 2+ ] i levels in cultured SMCs (Fig. 4C) and on the binding of a α 1 -adrenergic agonist in situ on rat aortic SMCs (Fig. 4D). We observed that Phe induced a transient and reproducible increase in [Ca 2+ ] i (Fig. 4C). This response was antagonized by ranolazine (20 μ M), suppressed by the positive control prazosin (10 μ M) and insensitive to TTX (1 μ M) both in absence and presence of ranolazine (Fig. 4C). Prazosin binds the α 1 -adrenergic receptor, as illustrated by the fluorescent signal reflecting BODIPY FL-Prazosin binding at the SMCs level and widely distributed through the media (Fig. 4D, CTL). This fluorescence signal was strongly reduced in the presence of ranolazine (Fig. 4D, ranolazine) as well as in the presence of non-fluorescent control antagonists (Fig. 4D, prazosin and Phe).

Effect of ranolazine on human uterine arteries.
To investigate the potential therapeutic relevance of our results, we performed experiments in human arteries (Fig. 5). In human uterine artery, ranolazine (20 μ M) prevented the contractile response to low KCl concentrations (less than 30 mM and below EC 50 value) similarly to that seen on rat aorta (Fig. 5A). In the presence of ranolazine, the dose response curve of KCl was rightward shifted and the EC 50 value was increased (21.4 ± 0.8 m Mvs. 26 ± 1.7 mM, p = 0.0127, t-test). No inhibitory effect of ranolazine was observed on the maximal contractile response to KCl (Fig. 5A-inset). This effect reflected, at least partially, inhibition of Na v . Additionally, ranolazine induced a vasorelaxation of human uterine arteries contracted after application of a non-saturating concentration of Phe (10 μ M) ( Fig. 5B-a). The effect of ranolazine was dose-dependent with an IC 50 value of 2.5 ± 0.5 μ M consistent with therapeutic concentrations. In the presence of ranolazine (20 μ M), the dose-dependent response to Phe was significantly shifted to the right, reflecting competitive inhibition of the α 1 -adrenergic receptor ( Fig. 5B-b) whereas no effect was observed on the maximal contractile response to Phe ( Fig. 5B-

Discussion
The antianginal properties of ranolazine have been attributed primarily to the inhibition of the persistent I Na in cardiomyocytes [5][6][7][8]27 . In the present study, we show that the vasorelaxant effect of ranolazine in arteries involves antagonism of α 1 -adrenergic receptors and inhibition of Na v channels at the smooth muscle level.
One major finding of our study is that Na v channels, present in arteries, are possible targets of ranolazine and could participate in the vasorelaxant effects of the drug. Previously, we had evidenced a TTX-sensitive component of tension in the rat aorta which is comprised of two mechanisms 18 (Fig. 6). One mechanism involves Na v channels isoforms from the vascular myocytes. Na + entry through the SMCs Na v channels triggers Ca 2+ influx through the reverse mode of the NCX and, thereby, promotes contraction [17][18][19] . The other mechanism involves the activity of Na v channels at sympathetic perivascular nerve terminals and impacts catecholamine release with subsequent α 1 -adrenergic receptor activation. Both mechanisms were potentially inhibited by ranolazine.  We have shown an inhibitory effect of ranolazine on SMC Na v channels, both directly on a persistent I Na (Fig. 1) and indirectly by prevention or abolition of the intracellular Ca 2+ rise (Fig. 2) and contraction (Fig. 3) promoted by the alkaloid Na v agonist veratridine. Veratridine prevents the inactivation and deactivation of the Na v channel, thereby promoting persistent Na + influx and consequently a rise in [Ca 2+ ] i via a cascade of pathways which elicits contraction 18,19 and involves the NCX reverse mode 13,28 , Ca 2+ -activated Clchannels and voltage-activated Ca 2+ channels 29 . The effects of ranolazine on Na + influx and Ca 2+ homeostasis evidenced here in vascular myocytes are The contractile response to Phe was evaluated in the presence of ranolazine (20 μ M) and the dose-response curve was compared to that obtained in absence of ranolazine. The inset shows the maximal contraction (in g) induced by Phe (200 μ M) for the control and in the presence of ranolazine. Data were obtained from 6 different specimens of uterine arteries; each protocol was performed in triplicate. **p < 0.01, ***p < 0.001, two-way Anova followed by Bonferroni post-test. very similar to those described in cardiac myocytes 27 . Mechanistically, the use of an agonist (veratridine) or of a weak depolarization following addition of low KCl concentration was required to unravel ranolazine activity on the SMCs Na v channels. This is in line with previous findings that Na v channels need to be activated prior to seeing an effect of the drug and consistent with an open-state blocking mechanism 30 . The effect of ranolazine was steeply voltage-dependent with inhibition being enhanced by increasing depolarization promoting channel opening. This mechanism corresponded to the electrophysiological properties of the drug previously demonstrated in cardiac myocytes 31 . This contrasted markedly with that of TTX whose mechanism of action is to form a plug in the pore of the channel independently of voltage. In addition to vascular SMCs Na v channels, ranolazine may also target Na v channels at sympathetic perivascular nerve terminals. Although it is complex to specifically study Na v channels at the sympathetic perivascular nerve terminals, we could speculate that the inhibitory effect of ranolazine also affects these Na v channels when activated.
Voltage-gated Ca 2+ (Ca v ) channel inhibition has also been reported in the vascular effects of ranolazine 24,25 . In our study, such antagonization should be considered, especially as Ca v channels are also implicated in veratridine-induced events 17 . However, we observed that ranolazine did not mimic the effect of the Ca v channel antagonist nifedipine. Nifedipine inhibited KCl-induced contraction, particularly in the maximal contractile response. High concentrations of KCl strongly depolarize cells and Ca v channels are predominantly involved in the resulting contractile response. Absence of an inhibitory effect on that response revealed no antagonism on these channels as is the case for TTX and KBR. We observed that ranolazine, at a concentration in line with therapeutic doses (20 μ M), did not affect the response to high KCl concentration. In our conditions, Ca v channels inhibition was not substantially involved in the vasorelaxant effects of ranolazine.
Another mechanism implicated in the vasorelaxant effects of ranolazine could potentially be the antagonization of α 1 -adrenergic receptor 22,24,25,32 . Indeed, we demonstrated an inhibitory effect of ranolazine at concentrations corresponding to therapeutic doses, on both arterial contraction and intracellular rise of Ca 2+ in aortic SMCs induced by Phe. The stimulation of α 1 -adrenergic receptors regulates arterial blood pressure in the rat aorta 33,34 and modulates vasoconstriction in coronary arteries. There is little α 1 -adrenergic coronary vasomotor tone at rest but α 1 -adrenergic hyperactivity can be promoted by atherosclerosis and thereby can contribute to myocardial ischemia 29,35,36 . Consistently, we observed no effect of either ranolazine or prazosin on vascular tone at rest, in line with the absence of α 1 -adrenergic tone at rest, and vasorelaxation was achieved only when the α 1 -adrenergic system was stimulated.
Ranolazine has multiple molecular targets and is not highly specific 9,37 but it is thought to reduce electrical and mechanical cardiac dysfunctions by inhibition of persistent I Na in cardiomyocytes 27,38 . The current view of the therapeutic benefits of ranolazine in stable ischemic angina is that they arise from the normalization of cardiac Na v channel activity and, consequently, of Na + and Ca 2+ overload in ischemic cardiomyocytes 39 . Improvement of regional coronary perfusion was also suggested but no molecular mechanism has been proposed 20 .
Our results are consistent with the idea that vasorelaxant properties of ranolazine may improve myocardial perfusion under ischemic conditions. Although we had no access to human coronary arteries to assess the effect of ranolazine on their contractile activity, previous identification of Na v channels involved in intracellular Na + and Ca 2+ overload in coronary SMCs is consistent with this hypothesis 13,17 . These channels represent a contractile reserve that could significantly impact vascular tone especially in resistance arteries 19 . Although several studies have reported the functional coupling between Na v channels and arterial contraction, no pathophysiological situation involving that regulation has been clearly identified. However, it has been shown that hypoxia can induce vasoconstriction which is sensitive to Na v channels blockers 40 . Hypoxic conditions mimic pathological situations such as angina; ranolazine through vascular Na v channels inhibition could regulate vascular tone in these circumstances. The α 1 -adrenergic receptors are also critical to vasoconstriction in human coronary arteries, and are involved in enhanced vasoconstriction at both the epicardial and microcirculatory levels in atherosclerotic conditions 41 . This also further strengthens our rationale and working hypothesis for potential therapeutic benefits of ranolazine at the coronary level under ischemic conditions or following different types of coronary manipulation and intervention (for review see 36 ). We hypothesize that dynamic coronary stenosis could be reversed by ranolazine through an antagonistic action on the α 1 -adrenergic mediated vasoconstriction 42 .
Clinical trials have reported a possible association of anti-anginal properties of ranolazine and improvement of regional coronary blood flow 20,43,44 . However, ranolazine is presented as devoid of hemodynamic effects 45,46 whereas α 1 -adrenergic receptor antagonists used to treat hypertension have side effects such as orthostatic hypotension or tachycardia 47 . Interestingly, a few events of orthostatic hypotension in healthy volunteers have been reported with high doses of ranolazine (2000 mg) while no such side effect was observed at therapeutic doses (500-1000 mg) 3 . The IC 50 values that we determined for both Na v channels and α 1 -adrenergic receptors are in the range of therapeutic concentrations. At these concentrations, ranolazine induced a partial vasorelaxation and exhibited no vasodilatory effect. At higher concentrations vasorelaxation is pronounced and almost complete. This could explain the absence of hemodynamic effects and is in line with clinical observations.

Conclusion
Although the inhibition of the persistent I Na has been well-established in cardiomyocytes as the mechanism responsible for ranolazine's antianginal properties, the inhibition of persistent Na + influx through arterial Na v channels, together with an antagonization of α 1 -adrenergic system over activation, may also contribute significantly to its therapeutic action. Pharmacologically, ranolazine inhibits the activity of voltage-gated Na v channels both at the level of aortic myocytes and, potentially, at sympathetic perivascular nerve terminals thereby inhibiting catecholamine release in addition to inhibiting α 1 -adrenergic receptors which seems relevant for the antianginal effects of the drug (Fig. 6). Therefore, the therapeutic effects of ranolazine may comprise both "upstream" benefits, by preventing or stopping vasoconstriction, and down-stream therapy involving the normalization of Na + and Ca 2+ overload in cardiomyocytes. Arterial tissues (rat thoracic aorta and human uterine arteries) were immersed in a physiological saline solution (PSS, in mM: 140 NaCl, 5 KCl, 1 MgCl 2 , 0.5 KH 2 PO 4 , 0.5 Na 2 HPO 4 , 2.5 CaCl 2 , 10 HEPES and 10 glucose, pH 7.4), cleaned of fat and connective tissue, and cut into 2-3 mm-wide rings. When required by the experiment, the endothelium was removed by rubbing.

Preparation of vascular tissue and myocytes.
Isolated myocytes were obtained from the rat aorta by enzymatic dispersion of the media layer after mechanical removal of the adventitia. The tissue was incubated for 20 min at 37 °C in sterile PSS containing collagenase (1 mg/ml) and elastase (50 UI/ml). Cells harvested after mechanical dissociation were filtered through a nylon mesh,centrifuged at 250 g for 5 min and then seeded onto collagen-treated Petri dishes and cultured in specific smooth muscle growth medium (PromoCell, Heidelberg, Germany). Smooth muscle cells (SMCs) were sub-cultured once they reached 80%-90% confluence and were used between passages 2 and 5.
Electrophysiological recordings. Cellular electrophysiological recordings were performed, at room temperature (22-24 °C), on cultured arterial SMC under the whole-cell patch clamp configuration. Experiments were conducted using an Axopatch 200B amplifier (Axon Instruments), interfaced to a Dell microcomputer with a Digidata 1440A Series analog/digital interface (Axon), using pClamp 10 (Axon). Recording pipettes were filled with (in mM): 120 CsCl, 5 MgCl 2 , 11 EGTA, 10 HEPES, 1 CaCl 2 , 5 ATP-Na 2 and 10 TEA-Cl (pH 7.3 with CsOH). The bath solution contained (in mM): 135 NaCl, 1 CaCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, 2 NiCl 2 (pH 7.4 with CsOH) and 0.1 veratridine. Our experimental conditions were optimized to record only voltage activated I Na . We used NiCl 2 (2 mM in bath solution) to block Ca v channels 48 . In addition, CsCl (120 mM, instead of KCl in the recording pipette) was used to inhibit K + currents. Veratridine (100 µM) was added to promote sustained I Na inactivation. Whole-cell membrane capacitances and series resistances were compensated electronically prior to recording. Voltage errors resulting from the uncompensated series resistance were always ≤ 8 mV and were not corrected. Experimental data were filtered on-line at 10 kHz prior to digitization and storage. The presence of I Na current was revealed by the use of a ramp protocol defined as followed: from a holding potential (HP) of − 80 mV, a − 100 mV prepulse was applied for 2 sec, followed by a voltage ramp from − 100 to + 40 mV for 40 ms. Current/ voltage I Na relationship was obtained in response to 150 ms voltage steps to potentials between − 60 to + 20 mV from a HP of − 80 mV; voltage steps were applied in 5 mV increments at 1 s intervals. de-esterification of Fura-2AM and chambers were mounted on a microscope stage (Axiovert, Zeiss, Germany; 20x objective). Buffer and drugs were then applied by perfusion to the cells as indicated in the figure legends. Cells were illuminated by excitation with a dual UV light source at 340 nm and 380 nm using a lambda DG-4 excitation system (Sutter Instrument Company, CA, USA). Images were captured digitally every 0.35 seconds with a cooled CCD camera (Photometrics, Roper scientific, France) at 510 nm emission. Changes in [Ca 2+ ] i were deduced from variations in the F340/F380 ratio after correction for background and dark currents (Metafluor software, Universal Imaging Corporation, USA). Data were averaged (at least 25 cells per field chosen randomly; one field per cover glass; 4 cover glasses for each experimental condition), with n representing the number of cell cultures.

Measurement of intracellular Ca
Isometric tension recording. Arterial segments were mounted between two stainless steel hooks placed in a conventional vertical organ bath chamber filled with 5 ml of PSS, maintained at 37 °C and continuously bubbled with O 2 . Changes in isometric tension were measured as previously described 18 using an IT1-25 force transducer and an IOX computerized system (EMKA Technologies, France). Each arterial segment was subjected to a 60-min equilibration period at a basal resting tension of 2 g and its contractile function was assessed with 1 μ M phenylephrine (Phe). In some experiments, the successful removal of the endothelium was confirmed by the inability of acetylcholine (Ach, 1 μ M) to induce relaxation in Phe-contracted rings. After washout and a 20-30 min period of stabilization, protocols were followed as detailed in the legends. Concentration-response curves were generated by cumulative increases in the concentration of various agents: Phe, the depolarizing agent KCl and ranolazine. For specific protocols, prazosin (10 μ M), tetrodotoxin (1 μ M, TTX), KB-R7943 (10 μ M, KBR) and nifedipine (1 μ M) were used to block α 1 -adrenergic receptors, Na v channels, the reverse mode of NCX and Ca v channels, respectively. Rings were incubated with each compound for a 15-min period before dose responses were generated. KCl was added, at the indicated concentrations, to basal PSS containing 5.5 mM K + . Each experimental protocol was performed in duplicate (rat aorta) or triplicate (uterine artery), with n representing the number of individual.
Fluorescent ligand binding to α 1 -adrenergic receptors. Segments of rat aorta were sliced open, cleared of adventitia and incubated in the dark for one hour at room temperature with BODIPY FL-Prazosin (QAPB, 100 nM), as previously described by others 51 . Once QABP binding equilibrium was reached, the following non-fluorescent antagonists were added to the incubation media for one hour at saturating concentrations to compete for QABP binding sites in segments from the same aorta: prazosin (10 μ M), Phe (10 μ M) and ranolazine (100 μ M). Arterial segments were observed with a 40x oil-immersion objective, on an inverted Zeiss LSM Exciter laser scanning microscope (Zeiss, LePecq France). Optical images were collected at an excitation/emission of 488/515 nm for QAPB. Laser intensity, gain and offset (contrast and brightness) were kept constant for each artery and acquisition. Tissue was scanned at 1 μ m intervals from the internal elastic lamina through the media, yielding z-series in stacks of approximately 20-50 μ m in depth. Each condition was tested in triplicate on five different aortas.
Chemical reagents. TTX and KB-R7943 were obtained from Tocris Biosciences (UK) and culture medium from PromoCell (Germany). All other chemicals and compounds were purchased from Sigma-Aldrich (France). KB-R7943 was dissolved in DMSO, veratridine in 0.1N HCl and the remaining compounds in distilled water with further dilutions made from stock solutions with PSS. Data analysis. All data are expressed as means ± standard errors of the mean (SEM) with the number of experiments indicated as n. Data were analyzed using GraphPad software (USA). Statistics were performed using either the Student's t-test or two-way analysis of variance followed by Bonferroni post-test for two-group comparison or Kruskal-Wallis one-way analysis of variance followed by Dunn's test for multiple-groups comparison. P values lower than 0.05 were considered significant.