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

Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

doi:10.1038/srep17969

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Published: 10 December 2015

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

Anne Virsolvy¹,
Charlotte Farah²,
Nolwenn Pertuit¹,
Lingyan Kong¹,
Alain Lacampagne¹,
Cyril Reboul²,
Franck Aimond¹ &
…
Sylvain Richard¹

Scientific Reports volume 5, Article number: 17969 (2016) Cite this article

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Abstract

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 (I_Na) 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 (Na_v) 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 I_Na. We observed that in the presence of the Na_v channel agonist veratridine, ranolazine inhibited I_Na and intracellular Ca²⁺ calcium increase in SMC and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Na_v 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 Na_v channels could be involved in the vascular effects of ranolazine.

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Introduction

Ranolazine is a potent antianginal drug approved for the treatment of inadequately controlled chronic stable angina in adult patients ineligible for coronary revascularization and intolerant to first-line therapies (nitrates, β-blockers, Ca²⁺ antagonists). Clinical trials have shown that ranolazine reduces the symptoms of angina and improves exercise tolerance in patients with coronary heart disease^1,2. Unlike conventional antianginal drugs that reduce heart rate or blood pressure, ranolazine acts on ventricular cardiomyocytes^3,4. Reduction of electrical and mechanical dysfunction by ranolazine is thought to occur via the inhibition of the persistent Na⁺ current (I_Na)^5,6,7,8 that is enhanced during ischemia⁹. Through the preferential blockade of the persistent I_Na, ranolazine prevents the Na⁺-induced Ca²⁺ overload that occurs during ischemia, ultimately protecting the myocardium and attenuating ischemia^10,11. The electrophysiological consequences of ranolazine and its pharmacological effects on action potential duration and intracellular Na⁺ and Ca²⁺ homeostasis are critical for its therapeutic effects¹².

Voltage-gated Na⁺ currents have been described in vascular smooth muscle cells (SMCs)^13,14,15,16. In human coronary SMCs, I_Na has been recorded and has been shown to regulate intracellular Na⁺ and Ca²⁺ levels^13,17. Vascular voltage-gated sodium channels (Na_v) are sensitive to small changes in membrane potential and provide SMCs with an effective mechanism to elevate intracellular sodium [Na⁺]_i, and, thereby, calcium [Ca²⁺]_i via the Na⁺-dependent activation of the reverse mode of the Na⁺/Ca²⁺exchanger (NCX)^18,19. In rat arteries, it has been evidenced that Na_v channels contribute to the contractile response of SMCs^18,19.

In addition to protecting the heart from the consequences of ischemia, recent evidence suggests that ranolazine also improves regional coronary blood flow and exerts a vasorelaxant effect comparable to that of nitroglycerin in magnitude, but more persistent²⁰. Vasorelaxant responses to ranolazine have also been described in ex vivo and in vivo animal models and could combine the blockade of α₁-adrenergic receptors^21,22,23 and voltage-gated Ca²⁺ channels antagonism (Ca_v)^24,25. However, the precise molecular mechanisms implicated have not been studied. It 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_Nawas 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 μM), 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 μM TTX, all currents were blocked (Fig. 1A). Ranolazine (20 μM) 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 Ca²⁺ in rat aortic myocytes

In primary cultured rat aortic SMCs, veratridine (100 μM) induced a transient and reproducible increase in [Ca²⁺]_i (Fig. 2). Ranolazine (20 μM) and TTX (1 μM) similarly inhibited the veratridine-induced [Ca²⁺]_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 [Ca²⁺]_i suggesting that Na_v channels were not activated at rest.

Ranolazine inhibited Na_v channel-dependent aortic contraction

In aortic rings, veratridine (100 μM) triggered an increase in tension corresponding to 44 ± 3% of the maximal contraction induced by phenylephrine (Phe, 10 μM) 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 μM, both in aortic rings with an intact endothelium (IC₅₀ 2.5 ± 0.9 μM, n = 6) and in endothelium-free preparations (IC₅₀ 2.9 ± 1.3 μM, n = 6) (Fig. 3A). Prior incubation with ranolazine (20 μM) 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 function¹⁸. 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 α₁-adrenergic receptor blockade with prazosin (10 μM). We observed that ranolazine (20 μM) prevented the contraction induced by low KCl concentrations (less than 10 mM and below EC₅₀ 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 EC₅₀ 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 μM), the KCl response was rightward shifted for concentrations below 10 mM, reflecting Na_v channel inhibition. The same effect was obtained with ranolazine (20 μM). There was no additional inhibition of ranolazine in the presence of TTX (Fig. 3C). The same inhibitory profiles were obtained with KB-R7943 (10 μM), a blocker of the reverse mode of the NCX²⁶. 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 (1μM), a Ca²⁺ channel blocker.

Ranolazine inhibited α₁-adrenergic-dependent rat aortic contraction

Since antagonistic effects of ranolazine on the α₁-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 (IC₅₀ 8.4 ± 1.3 μM; n = 6) of rat aorta previously contracted with a non-maximally active concentration of Phe (1 μM) (Fig. 4A). In the presence of ranolazine (20 μM), 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 α₁-adrenergic receptor with ranolazine was confirmed on [Ca²⁺]_i levels in cultured SMCs (Fig. 4C) and on the binding of a α₁-adrenergic agonist in situ on rat aortic SMCs (Fig. 4D). We observed that Phe induced a transient and reproducible increase in [Ca²⁺]_i (Fig. 4C). This response was antagonized by ranolazine (20 μM), suppressed by the positive control prazosin (10 μM) and insensitive to TTX (1 μM) both in absence and presence of ranolazine (Fig. 4C). Prazosin binds the α₁-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 μM) prevented the contractile response to low KCl concentrations (less than 30 mM and below EC₅₀ 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 EC₅₀ 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 μM) (Fig. 5B-a). The effect of ranolazine was dose-dependent with an IC₅₀ value of 2.5 ± 0.5 μM consistent with therapeutic concentrations. In the presence of ranolazine (20 μM), the dose-dependent response to Phe was significantly shifted to the right, reflecting competitive inhibition of the α₁-adrenergic receptor (Fig. 5B-b) whereas no effect was observed on the maximal contractile response to Phe (Fig. 5B-b-inset).

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 α₁-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 mechanisms¹⁸ (Fig. 6). One mechanism involves Na_v channels isoforms from the vascular myocytes. Na⁺ entry through the SMCs Na_v channels triggers Ca²⁺ 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 α₁-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 Ca²⁺ 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 [Ca²⁺]_i via a cascade of pathways which elicits contraction^18,19 and involves the NCX reverse mode^13,28, Ca²⁺-activated Cl^- channels and voltage-activated Ca²⁺ channels²⁹. The effects of ranolazine on Na⁺ influx and Ca²⁺ homeostasis evidenced here in vascular myocytes are very similar to those described in cardiac myocytes²⁷. 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 mechanism³⁰. 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 myocytes³¹. 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_vchannels when activated.

Voltage-gated Ca²⁺ (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 events¹⁷. 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 μM), 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 α₁-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 Ca²⁺ in aortic SMCs induced by Phe. The stimulation of α₁-adrenergic receptors regulates arterial blood pressure in the rat aorta^33,34 and modulates vasoconstriction in coronary arteries. There is little α₁-adrenergic coronary vasomotor tone at rest but α₁-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 α₁-adrenergic tone at rest and vasorelaxation was achieved only when the α₁-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 Ca²⁺ overload in ischemic cardiomyocytes³⁹. Improvement of regional coronary perfusion was also suggested but no molecular mechanism has been proposed²⁰.

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 Ca²⁺ 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 arteries¹⁹. 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 blockers⁴⁰. Hypoxic conditions mimic pathological situations such as angina; ranolazine through vascular Na_v channels inhibition could regulate vascular tone in these circumstances.

The α₁-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 conditions⁴¹. 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 see³⁶). We hypothesize that dynamic coronary stenosis could be reversed by ranolazine through an antagonistic action on the α₁-adrenergic mediated vasoconstriction⁴².

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 α₁-adrenergic receptor antagonists used to treat hypertension have side effects such as orthostatic hypotension or tachycardia⁴⁷. 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 IC₅₀ values that we determined for both Na_v channels and α₁-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 α₁-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 α₁-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 Ca²⁺ overload in cardiomyocytes.

Methods

Preparation of vascular tissue and myocytes

Investigations on animal tissue conformed to the guidelines for the Care and Use of Laboratory Animals (NIH, N°.85–23, revised 1996) and European directives (2010/63/EU)) and were approved by the committee for Animal Care of Montpellier-Languedoc-Roussillon (N° CEEA-LR-12075). Experiments were performed on male Sprague-Dawley rats (22–25 weeks) anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg). The human tissues used in this study were considered as surgical waste in accordance with French ethics laws (L.1211-3 – L.1211-9) and their use was approved by the national ethics Committee and the French Ministry of Research (DC-2008-488). Specimens of uterine arteries were obtained after written consent from non-pregnant women (aged 40–60 years) undergoing hysterectomy for benign gynaecological disorders.

Arterial tissues (rat thoracic aorta and human uterine arteries) were immersed in a physiological saline solution (PSS, in mM: 140 NaCl, 5 KCl, 1 MgCl₂, 0.5 KH₂PO₄, 0.5 Na₂HPO₄, 2.5 CaCl₂, 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.

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 MgCl₂, 11 EGTA, 10 HEPES, 1 CaCl₂, 5 ATP-Na₂ and 10 TEA-Cl (pH 7.3 with CsOH). The bath solution contained (in mM): 135 NaCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, 2 NiCl₂(pH 7.4 with CsOH) and 0.1 veratridine. Our experimental conditions were optimized to record only voltage activated I_Na. We used NiCl₂ (2 mM in bath solution) to block Ca_v channels⁴⁸. 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.

Measurement of intracellular Ca²⁺ variations

Intracellular Ca²⁺ variations ([Ca²⁺]_i) in cultured SMCs were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2 as previously described^49,50. SMCs sub-cultured for 4 days in Lab-Tek II® chambers (Nunc, USA) were loaded with 2.5 μM Fura-2AM plus 0.02% Pluronic F-127. Cells rinsed with PSS were maintained in basal buffer during a 15-min waiting period for the 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 [Ca²⁺]_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.

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 O₂. Changes in isometric tension were measured as previously described¹⁸ 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 μM phenylephrine (Phe). In some experiments, the successful removal of the endothelium was confirmed by the inability of acetylcholine (Ach, 1 μM) 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 μM), tetrodotoxin (1 μM, TTX), KB-R7943 (10 μM, KBR) and nifedipine (1 μM) were used to block α₁-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 α₁-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 others⁵¹. 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 μM), Phe (10 μM) and ranolazine (100 μM). 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 μm intervals from the internal elastic lamina through the media, yielding z-series in stacks of approximately 20–50 μm 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.

Additional Information

How to cite this article: Virsolvy, A. et al. Antagonism of Na_v channels and a₁-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine. Sci. Rep. 5, 17969; doi: 10.1038/srep17969 (2015).

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Authors and Affiliations

PhyMedExp, INSERM U1046, UMR CNRS 9214, Université de Montpellier, Montpellier, F-34295, France
Anne Virsolvy, Nolwenn Pertuit, Lingyan Kong, Alain Lacampagne, Franck Aimond & Sylvain Richard
Avignon Université, LAPEC, EA4278, F-84000, Avignon, France
Charlotte Farah & Cyril Reboul

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Anne Virsolvy
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Charlotte Farah
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Nolwenn Pertuit
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Lingyan Kong
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Alain Lacampagne
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Cyril Reboul
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Franck Aimond
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Sylvain Richard
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A.V., C.F., N.P., L.K. and F.R. performed the experiments; A.L. helped with confocal images acquisition and analysis; C.R. helped with experimental design; A.V., F.R. and S.R. analyzed the data; A.V. and S.R. conceived and designed the study and wrote the manuscript.

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Virsolvy, A., Farah, C., Pertuit, N. et al. Antagonism of Na_v channels and α₁-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine. Sci Rep 5, 17969 (2016). https://doi.org/10.1038/srep17969

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Received: 07 August 2015
Accepted: 10 November 2015
Published: 10 December 2015
DOI: https://doi.org/10.1038/srep17969

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