Antimigraine drug, zolmitriptan, inhibits high-voltage activated calcium currents in a population of acutely dissociated rat trigeminal sensory neurons

1744-8069-2-10 1744-8069 Research Antimigraine drug, zolmitriptan, inhibits high-voltage activated calcium currents in a population of acutely dissociated rat trigeminal sensory neurons Morikawa Tomoko mrkwmane@tmd.ac.jp Matsuzawa Yoshiyasu matsuzawa_sk_hp@pop17.odn.ne.jp Makita Koshi makita.mane@tmd.ac.jp Katayama Yoshifumi kataauto@tmd.ac.jp

Department of Anesthesiology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Department of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Molecular Pain 1744-8069 2006 2 1 10 http://www.molecularpain.com/content/2/1/10 16549032 10.1186/1744-8069-2-10 19 11 2005 20 3 2006 20 3 2006 2006 Morikawa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Triptans, 5-HT_1B/IDagonists, act on peripheral and/or central terminals of trigeminal ganglion neurons (TGNs) and inhibit the release of neurotransmitters to second-order neurons, which is considered as one of key mechanisms for pain relief by triptans as antimigraine drugs. Although high-voltage activated (HVA) Ca²⁺channels contribute to the release of neurotransmitters from TGNs, electrical actions of triptans on the HVA Ca²⁺channels are not yet documented.

Results

In the present study, actions of zolmitriptan, one of triptans, were examined on the HVA Ca²⁺channels in acutely dissociated rat TGNs, by using whole-cell patch recording of Ba²⁺currents (I_Ba) passing through Ca²⁺channels. Zolmitriptan (0.1–100 μM) reduced the size of I_Bain a concentration-dependent manner. This zolmitriptan-induced inhibitory action was blocked by GR127935, a 5-HT_1B/1Dantagonist, and by overnight pretreatment with pertussis toxin (PTX). P/Q-type Ca²⁺channel blockers inhibited the inhibitory action of zolmitriptan on I_Ba, compared to N- and L-type blockers, and R-type blocker did, compared to L-type blocker, respectively (p < 0.05). All of the present results indicated that zolmitriptan inhibited HVA P/Q- and possibly R-type channels by activating the 5-HT_1B/1Dreceptor linked to G_i/opathway.

Conclusion

It is concluded that this zolmitriptan inhibition of HVA Ca²⁺channels may explain the reduction in the release of neurotransmitters including CGRP, possibly leading to antimigraine effects of zolmitriptan.

Background

It is known that the pain associated with migraine is relieved by triptans, 5HT_1B/1Dagonists, including sumatriptan, zolmitriptan, naratriptan and so on. Indeed, they are in clinical use for treatment of migraine. It is shown that trigeminal ganglion stimulation leads to the release of CGRP in humans and cats, which is antagonized by sumatriptan administration 1. Subsequently, several lines of histochemical and electrophysiological studies demonstrate the involvement of 5HT_1B/1Dagonist in neurotransmitter release from trigeminal ganglion neurons (TGNs). First, 5HT_1Band/or _1Dreceptors are localized in trigeminal vascular systems 2. 5HT_1Breceptors are demonstrated on dural arteries 2 and 5HT_1Dreceptors on trigeminal sensory neurons including peripheral and central projections 234. Second, small and medium- sized TGNs possess 5HT_1B/1Dreceptors, colocalized with CGRP and Substance P 5. Third, naratriptan inhibits neuronal activity in TGNs 6. Fourth, synaptic transmission from TGNs to central trigeminovascular neurons is blocked by activation of presynaptic 5HT_1B/1Dreceptors on central terminals of meningeal nociceptors 7. All of these studies suggest that triptans might act on 5HT_1B/1Dreceptors of TGNs and inhibit the release of neurotransmitters such as CGRP, reducing central and/or peripheral neuronal excitability.

An activation of high-voltage activated (HVA) Ca²⁺channels is known to trigger the release of neurotransmitters and to control numerous neuronal functions such as neuronal excitability. HVA Ca²⁺channels are divided into four subtypes; that is N-, P/Q-, L-, and R-type channels. All of four subtypes of HVA Ca²⁺channels are demonstrated to be expressed in TGNs 8. Recent findings indicate that the blockade of HVA Ca²⁺channels prevents CGRP release and prevents dural vessel dilation, and so HVA Ca²⁺blockade might minimize neurological inflammation 9. Although it is shown that N- and P/Q-currents are inhibited via G protein-coupled mechanisms by agonists for 5HT_1Aand _1Dreceptors in the primary spinal neurons of Xenopus larvae 1011, effects of 5HT_1B/!Dagonists on HVA Ca²⁺channels in mammalian TGNs have not yet been evaluated.

As mentioned above, involvement of triptans in modulation of CGRP release as well as neuronal activity in the trigeminal ganglion is highly plausible. This prompted us to examine whether or not triptans could act on HVA Ca²⁺channels of TGNs, leading to inhibition of the release of CGRP and neurotransmission, possibly involved in generation of migraine. In the present study, electrophysiological experiments were undertaken to analyze actions of zolmitriptan, one of triptans, on HVA Ca²⁺channels using cultured neonatal rat TGNs. This paper clarified that zolmitriptan could inhibit HVA Ca²⁺channels by activating 5HT_1B/1Dreceptor coupled to G_i/opathway.

Results

Currents carried by Ba²⁺passing through HVA Ca²⁺channels, I_Ba, were recorded from somata of neonatal rat TGNs, small to medium size of 22 to 27 μm in diameter. The peak amplitude of I_Bain control varied within the range from 230 to 1200 pA (mean ± S.E.M.; 508.5 ± 31.0 pA, n = 37).

Concentration-dependent action of zolmitriptan on I_Ba

Zolmitriptan was applied to TGNs by superfusion for two minutes. As shown in Fig. 1a, I_Bawas inhibited in the presence of zolmitriptan at 10 μM. Inhibitory actions of zolmitriptan on I_Bawere examined at concentrations between 0.1 and 100 μM (Fig. 1b, the number of cells indicated). Zolmitriptan at lower concentrations slowly started depressing the I_Baat 10 to 20 s from the onset of application. This depressing action slowly increased but could not reach its maximum in 2 min at concentrations lower than 10 μM. On the other hand, at 100 μM, the I_Bawas very rapidly inhibited within 10 s and completely abolished within one min of the drug application.

Figure 1

Inhibition of HVA I_Baby zolmitriptan

Inhibition of HVA I_Baby zolmitriptan. (a) Typical illustration of I_Baelicited in response to command pulses from -60 mV to 10 mV for 40 ms. I_Bawas inhibited by 2 min application of 10 μM zolmitriptan. (b) The average time course of I_Bainhibition by zolmitriptan at four different concentrations. Superfusing application of zolmitriptan started at t = 0 and lasted for 120 s during the period indicated by horizontal bar. Mean value of the relative amplitude of I_Bacompared to the control I_Baat t = 0 was plotted on ordinate (I_Baratio) against time on abscissa. The number of neurons examined is indicated for the respective concentrations. S.E.M. value was not indicated. (c) Concentration-inhibition relationship for zolmitriptan. Bar graph shows the relative amplitude of I_Baat two minutes after application of zolmitriptan compared to the control. (*p < 0.05 **p < 0.01).

As noticed from Fig. 1b, this inhibitory effect of zolmitriptan on I_Balasted after the end of the drug application and afterwards became more marked, attaining to its peak. Then, it should be noted that the inhibitory action of zolmitriptan on I_Bacould be hardly washed out. Therefore, the inhibitory effect of the drug was compared by using the I_Baratio (see Method and figure legend) at 2 min after the onset of the application. The I_Baratios were 0.96 ± 0.06 (0.1 μM, n = 4), 0.81 ± 0.08 (1 μM, n = 6), 0.75 ± 0.07 (5 μM, n = 6), 0.71 ± 0.06 (10 μM, n = 12), 0.40 ± 0.12 (40 μM, n = 8), and 0.00 ± 0.00 (100 μM, n = 3), and compared with the I_Baratio of control group without zolmitriptan (0.97 ± 0.03, n = 3), as summarised in Fig. 1c, showing the concentration-inhibition relationship for the action of zolmitriptan on I_Ba.

Action of zolmitriptan, inhibited by a 5HT_1B/1Dantagonist

Since triptans are known to act as 5-HT_1B/1Dagonists, we examined whether or not the zolmitriptan-induced inhibition on I_Bacould be blocked by a 5-HT_1B/1Dreceptor antagonist, GR127935. The preparations were pretreated with GR127935 for 2 min; no direct actions of the antagonist on I_Bawere observed at 0.3 μM. Following GR127935 application for more than 2 min, zolmitriptan (5 and 10 μM) was added to the superfusate. The I_Baratios with 10 μM zolmitriptan were 0.71 ± 0.06 (without GR127935, n = 12), 0.72 ± 0.10 (0.1 μM GR127935, n = 6), 1.10 ± 0.04 (0.3 μM GR127935, n = 4), as summarized in Fig. 2. It was shown that the zolmitriptan-induced inhibition of I_Bawas significantly reduced by GR127935 at 0.3 μM. On the other hand, the I_Baratios with 5 μM zolmitriptan were 0.75 ± 0.07 (without GR127935, n = 6), 0.84 ± 0.13 (0.1 μM GR127935, n = 4), showing no significant inhibition. These data suggested that zolmitriptan inhibited I_Baby activating 5-HT_1B/1Dreceptors. It should be added that GR127935 at concentrations higher than 1 μM occasionally inhibited I_Ba.

Figure 2

GR127935 modulation on zolmitriptan-sensitive I_Ba

GR127935 modulation on zolmitriptan-sensitive I_Ba. (a) GR127935, 5HT_1B/1Dantagonist, depressed the inhibition of I_Baby zolmitriptan (10 μM). Bar graph shows that the zolmitriptan-induced inhibition of I_Bawas significantly reduced by pretreatment with 0.3 μM of the antagonist GR127935 (*p < 0.05). I_Baratio is the value that I_Ba(test) was divided by I_Ba(control). Inset shows the time course of GR127935 and zolmitriptan application. I_Ba(control) and I_Ba(test) were recorded as shown in the chart. (b) Typical illustration of I_Bain control (left), in the presence of 0.3 μM GR127935 (center), and 10 μM zolmitriptan added on 0.3 μM GR127935 (right).

Action of zolmitriptan, mediated by G-protein pathway

It is widely accepted that some of 5-HT receptor subtypes are G-protein coupled. Possible involvement of G-protein pathways in the present action of zolimitriptan was tested by using pertussis toxin (PTX, an irreversible inhibitor of G_i/oproteins). When cultured TGNs were treated overnight with PTX (500 ng/ml), zolmitriptan at 10 μM could not exert an inhibitory effect on I_Ba; the amplitude of I_Bain control was almost the same as that of I_Bain the presence of zolmitriptan; that is I_Baratio = 1.0 as shown in Fig. 3. Thus, PTX pretreatment prevented the inhibition of I_Baby zolmitriptan, while I_Bawas depressed by zolmitriptan without the PTX pretreatment. This significant change induced by PTX indicated the role of G-proteins in the zolmitriptan inhibition of I_Ba.

Figure 3

PTX modulation on zolmitriptan-sensitive I_Ba

PTX modulation on zolmitriptan-sensitive I_Ba. PTX treatment prevented the inhibition of I_Baby zolmitriptan (10 μM). Bar graph shows that the zolmitriptan-induced inhibition of I_Bawas significantly reduced by overnight treatment of 500 ng/ml PTX (*p < 0.05). Recording of I_Ba(control) and I_Ba(test) in the presence of zolmitriptan were made according to the same time course shown in the inset of figure 2. I_Baratio means I_Ba(test)/I_Ba(control).

Pharmacological profile of I_Ba, sensitive to zolmitriptan

Characteristics of I_Bainhibited by zolmitriptan were pharmacologically determined by using a variety of selective Ca²⁺channel blockers. Indeed, four types of HVA Ca²⁺channels are known to be expressed in TGNs; that is, N-type, P/Q-type, R-type, and L-type channels. In the present experiments, therefore, ω-conotoxin GVIA (ω-CgTx, 1 μM), ω-agatoxin IVA (ω-Aga, 0.2 μM), SNX-482 (0.1 μM), and nicardipine (10 μM) were used to examine possible contribution of each Ca²⁺channel to the zolmitriptan-sensitive I_Ba, respectively. It is confirmed that all four Ca²⁺blockers reduced I_Ba; ratios of I_Bain the presence of Ca²⁺blockers to control I_Bawere 0.42 ± 0.05 (ω-CgTx, n = 5); 0.58 ± 0.04 (ω-Aga, n = 4); 0.84 ± 0.05 (SNX-482, n = 7); and 0.43 ± 0.08 (nicardipine, n = 4).

After pretreatment with each of blockers for 2 min, zolmitriptan (10 μM) was added to the superfusing solutions, and I_Baratios were obtained (see inset of Fig. 4). When pretreated with ω-CgTx, the I_Baratio was 0.55 ± 0.02 (n = 5); with ω-Aga, 0.89 ± 0.05 (n = 4); with SNX-482, 0.80 ± 0.03 (n = 7); and with nicardipine, 0.28 ± 0.15 (n = 4) (Fig. 4). The I_Baratios after pretreatment with ω-Aga or SNX-482 seemed to be larger than the ratio 0.71 ± 0.06 (10 μM zolmitriptan without Ca²⁺blockers) in Fig. 1c, suggesting a possibility that Ca²⁺channels sensitive to ω-Aga or SNX-482 likely contributed to the blockade of zolmitriptan I_Bainhibition. Indeed, significant difference was detected between ω-Aga and ω-CgTx or nicardipine, and also between SNX-482 and nicardipine (Fig. 4), indicating that blockade of P/Q-type and R-type Ca²⁺channels with ω-Aga and SNX-482 reduced the inhibition of I_Baby zolmitriptan. Therefore, it is likely that P/Q-type and R-type channels could be inhibited by zolmitriptan by acting on 5-HT_1B/1Dreceptors through G proteins pathways.

Figure 4

Pharmacological characteristics of zolmitriptan-sensitive I_Ba

Pharmacological characteristics of zolmitriptan-sensitive I_Ba. Bar graph shows that inhibition of I_Bawas significantly reduced with ω-Aga, compared to those with ω-CgTx and nicardipine, Aand with SNX-482, compared to that with nicardipine (*p < 0.05). I_Ba(control) after pretreatment with Ca²⁺blockers and I_Ba(test) 2 min after adding zolmitriptan were recorded as indicated in the inset. I_Baratio was obtained by I_Ba(test)/I_Ba(control).

Discussion

The present experiments demonstrated modulating actions by zolmitriptan on I_Baof the rat isolated TGNs. Zolmitriptan inhibited HVA Ca²⁺currents carried by Ba²⁺in a concentration-dependent manner within the concentration range between 0.1 μM and 100 μM by acting on 5HT_1B/1Dreceptor through G_i/oprotein-coupled pathway.

5HT receptors are divided into 7 families, 5HT_1~7receptors, on the basis of their amino acid sequences and other properties. 5HT₁receptors are further subdivided according to their physiological functions, binding affinity and other features 12. The present study showed that GR127935, a potent 5HT_1B/1Dreceptor antagonist abolished the effect of zolmitriptan, meaning that zolmitriptan acted on 5HT_1B/1Dreceptor.

5HT_1Band/or _1Dsubtypes are known as G-protein mediated receptors. In the present study, pretreatment with PTX inhibited the I_Bainhibition by zolmitriptan, indicating the involvement of G_i/oprotein coupled pathway. This observation might be compatible with the previous reports that an increase in intracellular Ca²⁺level by 5HT₁receptor is associated with activation of G_i/G_oprotein coupled pathway 1314 and that the modulation of neuronal voltage-gated Ca²⁺channel is mediated by receptors coupled to PTX-sensitive G proteins 1516. In this context, possible involvement of stimulatory of G-proteins (G_s) in the zolmitriptan action should be further investigated by using cholera toxin. A recent report shows that sumatriptan could activate the other second messenger MAPK pathway leading to changes in intracellular Ca²⁺changes 17. This possibility for the action of zolmitriptan remains to be considered in future.

It is reported that triptans, antimigraine drugs might inhibit the release of vasoactive neuropeptide from trigeminovascular nerve endings and also inhibit transmission of nociceptive impulses to second-order neurons of the trigeminocervical complex, resulting in the antimigraine effect of triptan 18. It is known that the trigeminal ganglion possesses small to medium size 5HT_1B/1Dreceptor positive peptidergic neurons 45 and furthermore that antimigraine drugs could block synaptic transmission between meningeal nociceptors and central trigeminal neurons presynaptically 7. All of these suggest that HVA Ca²⁺channels, highly responsible to neurotransmitter release from presynaptic terminal, might be involved in the antimigraine effects of triptans. Indeed the present study showed that HVA I_Baof TGNs was affected by zolmitriptan, a 5HT_1B/1Dagonist, strongly advocating the idea that triptans inhibited neurotransmitter release from peripheral or central presynaptic terminal through HVA Ca²⁺channels.

It is important to determine which subtypes of HVA Ca²⁺channels might essentially contribute to the release of different neurotransmitters from various classes of neurons. Some paper mentioned simply about HVA Ca²⁺subtype on trigeminal neurons, but there is no consensus about which subtypes mainly contribute yet. Ebersberger et al shows that discharge patterns of trigeminal second order neurons with dural input are different in the presence of each HVA Ca²⁺subtype blockade 19, On the other hand, Hong et al showed that N- and P/Q-channels are important for the release of CGRP from perivascular TGNs 20 and the release of CGRP is shown to be prevented when N-, P/Q- or L- channels are blocked on trigeminal vascular neuron 9. The present study demonstrated that the inhibition of zolmitriptan-sensitive I_Bain small-medium TGNs depended mainly on activation of P/Q- and R-type channels.

P/Q-type Ca²⁺channels are reported to locate in all brain structure 18 and also in the trigeminal ganglia 8. Furthermore, α-eudesmol, a P/Q-type channel blocker, inhibits the release of a neuropeptide from perivascular trigeminal sensory nerves 21. These observations may support our present findings that P/Q-type channels might be possible sites on which zolmitriptan could act in cultured neonatal rat TGNs. Although N-type is also known to locate in DRG neurons 222324, a few studies show the N-type channel dominance in TGNs. The present study with ω-CgTx also could not statistically demonstrate an appreciable involvement of N-type channels in the inhibition of zolmitriptan-sensitive I_Baof cultured rat TGNs.

R-type Ca²⁺channels are shown to locate presynaptically in the central nervous system, but the transmitter release mediated by R-type channels is less efficient than that by P/Q-and N-type channels 25. In the process of development, R-type channels are replaced by P/Q-type ones in the central synaptic transmission 26. There are similar results for Ca²⁺channel subtypes obtained from neonatal and adult TGNs; in neonatal 4% are provided with P/Q-type while 15% with R-type one 8; in adult 40% with P/Q-type while 5% to R-type 27. In this context, the present study, for the first time, demonstrated possible involvement of R- as well as P/Q-type channels in the actions of zolmitriptan on the cultured neonatal rat TGNs.

Although zolmitriptan (0.1~100 μM) inhibited I_Baof cultured TGNs, it is difficult to determine the effective concentration of zolmitriptan acting in vivo on the trigeminal ganglion. Sumatriptan is reported to induce discharges in dural primary afferent neurons at concentrations between 0.24 and 24 μM 28 and also cause vasocontraction in rat isolated vena portae smooth muscle at concentrations between 0.001 and 10 μM 29; these indicate that actions of two triptans could be exerted at similar concentrations.

Conclusion

Zolmitriptan inhibited I_Bain a concentration-dependent manner by acting on 5HT_1B/1Dreceptor. P/Q- and possibly R-type calcium channels contributed to the inhibition of I_Baby zolmitriptan. G_i/oprotein pathway were involved. Although this action of zolmitriptan on HVA Ca²⁺channels might explain the antimigraine effect, more detailed research of second messenger pathway would reveal the further mechanism leading to antinociceptive effect of triptans and pain pathway of migraine.

Method

Animal preparation

All procedures were carried out in accordance with the guidelines for Animal Experimentation in Tokyo Medical and Dental University (No.0060010). Wistar rats (0–7 days after birth, Saitama Experimental Animals Supply Inc., Japan) were anesthetized by pentobarbital (i.p.). After the decapitation of the rats, trigeminal ganglia were dissected and treated with papain (20.3 units/ml) in low- Ca²⁺and low-Mg²⁺Krebs' solution for 30 min at 37°C, washed with modified Krebs' solution and triturated using fire-polished Pasteur pipettes. Neurons were plated onto poly-L-lysine pretreated 35 mm dishes. The plating medium contained Dulbecco's modified Eagle's medium with10% calf serum. The TGNs were kept in culture in modified Krebs' solution saturated with 5% CO₂at 37°C for 2 hours to one day before experiment. The ionic composition of the modified Krebs' solution was (mM): NaCl, 117; KCl, 4.7; CaCl₂, 2.5; MgCl₂, 1; glucose, 11; 3-(N-morpholino) propanesulfonic acid (MOPS), 25; and pH 7.2 adjusted with NaOH. The low-Ca²⁺and low-Mg²⁺Krebs' solution was made by adding EDTA (2.5 mM) to the modified Krebs' solution.

Electrophysiological recording

Membrane currents were recorded from somata of cultured TGNs in the whole-cell voltage clamp configuration of patch clamp technique with an Axopatch 1D amplifier (Axon Instrument). Currents were filtered low-pass at 2 Hz by the built-in Bessel filter, and recorded on a chart recorder (San-ei) for later analysis. Patch pipettes were pulled from borosilicate glass capillaries (Harvard) using a puller (Narishige co.), and had input resistance of 5–10 MΩ after polishing. The ionic composition of the patch pipette solution was (mM): CsCl, 100; MOPS, 40; MgCl₂, 1; EGTA, 10; CaCl₂, 1; ATP, 2 and pH 7.2 adjusted with KOH. A series resistance of the recording system was not electrically compensated.

Currents carried by Ba²⁺passing through HVA Ca²⁺channels, I_Ba, were evoked by depolarizing voltage step command pulse to +10 mV for 40 ms from a holding potential of -60 mV every 10 s. For isolating Ba²⁺currents an external solution was used, containing (mM): TEA-Cl 140; CsCl, 2.5; BaCl₂, 2.5; MgCl₂, 1; Glu, 11; HEPES, 10 and pH 7.3 adjusted with TEA-OH. The amplitude of I_Bawas determined as the difference between the baseline and the peak inward current during each command pulse.

External solutions were applied continuously via a polyethylene tube mounted on a micromanipulator and the tip of the tube was positioned within 10 mm of the recorded neurons. External solution was kept at 37°C.The capacity of chamber was 150 μl and the flow rate of solution was 2 ml/min.

Materials

Zolmitriptan was a gift from Astrazeneca. Zolmitriptan was dissolved in dimethylsulfoxide (DMSO) and stored at -20°C. More dilute solutions were made daily dissolved in external solution before every experiment. ω-CgTx, ω-Aga and SNX-482 were purchased from Peptide Institute. Nicardipine was from Sigma. GR127935 was from Tocris.

Data analysis

All data are expressed as means ± S.E.M. I_Baratio of Fig. 1b was expressed as the relative amplitude in response to each step command pulse compared to control values, and I_Baratios shown in Fig 1c, 2, 3, 4 were expressed as the relative amplitude after 120 s zolmitriptan application compared to control values in the absence of zolmitriptan. Statistical significance was assessed with Student's t-test for simple comparisons and Bonferroni-type multiple t-test for multiple comparison. Differences of P < 0.05 were considered to be significant.

List of Abbreviation

TGN, trigeminal ganglion neuron; HVA, high-voltage activated; I_Ba, Ba²⁺currents; CGRP, calcitonin gene-related peptide; PTX, pertussis toxin; ω-Aga, ω-agatoxin IVA; ω-CgTx, ω-conotoxin GVIA; DRG, dorsal root ganglion; i.p., intraperitoneally; MOPs, 3-(N-morpholino) propanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES, 2-[4-(2-Hydroxyethyl)-1-piperadinyl] ethansulfonic acid; DMSO, dimethylsulfoxide.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

T. Morikawa conceived of the study, participated in design of the study, carried out cell-culture and electrophysiological experiments, performed the statistical analysis, and prepared the manuscript as a main investigator. Y Matsuzawa participated in experiments and discussion. K Makita participated in design of the study and did the entire summary and discussion from the viewpoint of the pain clinic. Y Katayama conceived of the study, performed in design of the study, helped to prepare the manuscript and gave financial support of the present study and approval of this version to be published. All authors read and approved the final manuscript.

Acknowledgements

This study was supported in part by Grant-in-Aid for Scientific Research (No. 13307056 to Y.K.)

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