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Eugenol Inhibits Calcium Currents in Dental Afferent Neurons
1 Department of Physiology, and Correspondence: * corresponding author, odolbae{at}snu.ac.kr
Eugenol is a topical analgesic agent widely used in the dental clinic. To elucidate the molecular mechanism underlying its analgesic action, we investigated the effect of eugenol on high-voltage-activated calcium channel (HVACC) currents in dental primary afferent neurons, and with a heterologous expression system. Dental primary afferent neurons were identified by retrograde labeling with a fluorescent dye, DiI. Eugenol inhibited HVACC currents in both capsaicin-sensitive and capsaicin-insensitive dental primary afferent neurons. The HVACC inhibition by eugenol was not blocked by capsazepine, a competitive transient receptor potential vanilloid 1 (TRPV1) antagonist. Eugenol inhibited N-type calcium currents in the cell line C2D7, stably expressing the human N-type calcium channels, where TRPV1 was not endogenously expressed. Our results suggest that the HVACC inhibition by eugenol in dental primary afferent neurons, which is not mediated by TRPV1 activation, might contribute to eugenol’s analgesic effect. Abbreviations: high-voltage-activated calcium channel, HVACC; transient receptor potential vanilloid 1, TRPV1; trigeminal ganglion, TG; dorsal root ganglion, DRG; capsazepine, CZP.
Key Words: eugenol • transient receptor potential vanilloid 1 • trigeminal ganglion • C2D7 cells • high-voltage-activated calcium currents
Eugenol has been widely used in dental clinics to relieve pain (Markowitz et al., 1992). Recently, we have demonstrated that the influx of cations through the activation of transient receptor potential vanilloid 1 (TRPV1), thereby inducing depolarization of the membrane potential to evoke action potentials, could be one of the major molecular mechanisms by which eugenol produces irritant actions (Yang et al., 2003). However, because response patterns of eugenol were distinct from those of capsaicin, we suggested that TRPV1-independent mechanisms also appear to be involved in the pharmacological actions of eugenol (Yang et al., 2003). In spite of its widespread therapeutic use as a topical analgesic in dentistry, little is known about the mechanisms underlying the analgesic properties of eugenol. It has been reported that beneficial effects of eugenol might be due to inhibition of the cyclo-oxygenase enzyme (Thompson and Eling, 1989; Dohi et al., 1991). However, the inhibitory effect of eugenol on nerve transmission (Trowbridge et al., 1982; Brodin and Roed, 1984) implies its potential actions on ion channels. However, to date, it remains unclear whether eugenol regulates ion channels in sensory neurons, including dental primary afferent neurons. Voltage-activated calcium channels are key mediators in sensory neurons and play a crucial role in the transmission of pain signals at the spinal/medullary dorsal horn level (Altier and Zamponi, 2004). Capsaicin has been demonstrated to inhibit high-voltage-activated calcium channel (HVACC) currents (Petersen et al., 1989; Bleakman et al., 1990). Since the chemical structure of eugenol is similar to that of capsaicin (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999), there is a possibility that eugenol modulates HVACC as capsaicin does in sensory neurons (Petersen et al., 1989; Bleakman et al., 1990). In the present study, we investigated if eugenol would regulate HVACC in rat dental primary afferent neurons, and with a heterologous expression system.
All procedures for animal use were reviewed and approved by the Animal Care and Use Committee of the Seoul National University prior to the experiments.
Labeling of Dental Primary Afferent Neurons and Cell Preparation
mRNA Preparation and Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
Electrophysiological Recordings
Drugs
Statistical Analysis
Eugenol Inhibited HVACC Currents in Dental Primary Afferent Neurons When the acutely isolated TG neurons were visualized under fluorescent microscopy after 3 wks of DiI crystal placement into both sides of maxillary molar, DiI-labeled neurons, ~ 40 to 60 TG neurons per animal, were clearly detectable (Fig. 1A  ). Using these dental primary afferent neurons, we examined whether eugenol would regulate HVACC currents. Whole-cell recordings of Ca2+ currents, with 5 mM Ba2+ as the charge carrier (IBa), were performed under experimental conditions that suppressed other voltage-dependent currents, such as Na+ and K+ currents. Membrane currents did not change during the application of 1 mM eugenol (n = 13) (Fig. 1B). However, when dental primary afferent neurons, which ranged from 15 to 50 µm in diameter, were exposed to eugenol (1 mM), the peak IBa was readily inhibited in a reversible manner (Fig. 1C). In only 12% of neurons tested (n = 5/43) was the IBa inhibition not recovered to the basal level, and these data were excluded from the results.
Eugenol Inhibited Ca Currents in Both Capsaicin-sensitive and Capsaicin-insensitive Dental Primary Afferent Neurons We next explored whether the inhibitory effect of eugenol on HVACC would be specific only in capsaicin-sensitive dental primary afferent neurons. We found that eugenol inhibited IBa in both capsaicin-sensitive neurons and capsaicin-insensitive neurons (Figs. 2A , 2B). Moreover, eugenol also inhibited IBa in other TG neurons, as well as dental primary afferent neurons (data not shown). The magnitude of IBa inhibition by 1 mM eugenol in capsaicin-insensitive dental primary afferent neurons (45 ± 5%) (n = 5) (Fig. 2B) was similar to that obtained in capsaicin-sensitive dental primary afferent neurons (42 ± 3%, n = 20) (Figs. 2A, 2D). The inhibition of the peak IBa in capsaicin-sensitive dental primary afferent neurons was dose-dependent (Fig. 2C). To determine whether the inhibitory effect of eugenol on HVACC was mediated by TRPV1, we examined the effects of eugenol in the presence of 10 µM capsazepine (CZP) (Chaudhary et al., 2001). When we applied 10 µM CZP, a competitive TRPV1 antagonist, for 5 min, it produced inhibitory effects on HVACC (n = 7) (Fig. 2D), as previously reported (Docherty et al., 1997). However, the extent of TRPV1-independent CZP-induced IBa inhibition (15 ± 2%) was smaller than that in DRG neurons (Docherty et al., 1997). The application of 10 µM CZP together with 1 mM eugenol inhibited IBa by 43 ± 4% (n = 6), which was not significantly different from the inhibition by eugenol alone (42 ± 3%) (n = 20) (Fig. 2D).
Inhibition of Ca Currents by Eugenol Does Not Require VR1 Activation We then tested if the IBa inhibition by eugenol was mediated by the activation of TRPV1 in a heterologous system—a cell line, C2D7, that stably expresses human N-type calcium channels. RT-PCR analysis indicated that TRPV1 was not endogenously expressed in C2D7 cells, whereas DRG and TG expressed TRPV1 (Fig. 3A ). In these C2D7 cells (n = 5), 1 mM eugenol still produced an inhibitory effect on N-type calcium currents (Figs. 3B, 3C). Interestingly, the IBa inhibition was significantly bigger than that in TG neurons (Fig. 3D).
The analgesic pharmacological action elicited by eugenol is the basis for its wide use in dental clinics as a sedative and anodyne agent. In the present study, we investigated whether eugenol would modulate HVACC currents. We found that eugenol elicited an inhibitory effect on HVACC in all TG neurons, including dental primary afferent neurons (Fig. 1  ), and these effects were not restricted to only capsaicin-sensitive dental primary afferent neurons (Fig. 2). Analysis of these findings implies that, although eugenol and capsaicin share vanillyl-like moiety in their chemical structures (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999), the mechanisms underlying inhibitory effects between eugenol and capsaicin on HVACC might be different. Capsaicin reduces HVACC currents of rat DRG neurons through the mediation of capsaicin receptor activation (Bleakman et al., 1990), which indicates the involvement of TRPV1 in the inhibitory effects of capsaicin on calcium channels. We also found, using TG neurons and a heterologous expression system, that TRPV1 activation is required for the inhibition of calcium current by capsaicin (unpublished data). Moreover, CZP, a competitive TRPV1 antagonist, failed to block the inhibitory effects of eugenol on HVACC completely (Fig. 2D). The extent of HVACC inhibition by eugenol was comparable with the combined application of eugenol and CZP, showing that the inhibitory effect on HVACC produced by eugenol was not mediated by TRPV1 activation (Fig. 2D). Taken together, the activation of TRPV1 in dental primary afferent neurons is not likely to be involved in the eugenol-induced inhibitory effects on HVACC.
TRPV1-independency to eugenol action was further confirmed with a heterologous system. In C2D7 cells that do not contain endogenous TRPV1 (Fig. 3A The concentration of eugenol in the cavity preparation below ZOE has been reported to be 10–2 ~ 10–4 M (Craig and Powers, 2002). This supports our contention that the range of eugenol concentration (~ 10–3 to 10–4 M), which produced inhibitory effects on HVACC in our study, corresponds to that used in dental therapeutic applications. Our findings that the inhibitory effects of eugenol on HVACC currents were readily reversible and dose-dependent argue against the inhibitory effects on Ca channels being due to the non-specific effects by rather high concentrations of eugenol. The inhibition of prostaglandin and leukotriene synthesis may also contribute to the analgesic effects of eugenol (Hirafuji, 1984; Dohi et al., 1991). It has been demonstrated that eugenol given intrathecally produced anti-nociceptive effects via the capsaicin receptors located on sensory terminals in the spinal cord (Ohkubo and Shibata, 1997). Although our data are not consistent with this report in terms of TRPV1 involvement, the inhibition of HVACC—thereby decreasing Ca-dependent neurotransmitter release from the pre-synaptic terminals of the neurons in the medullary dorsal horn—could be one of the mechanisms by which eugenol produces its analgesic effects. In summary, we demonstrate that eugenol produces inhibitory effects on HVACC currents in dental primary afferent neurons, and these inhibitory actions could contribute to the analgesic effect produced by eugenol. However, TRPV1 activation is not a prerequisite for the inhibitory effects of eugenol on HVACC.
This research was supported by a Grant-in-aid for scientific research from the Ministry of Health and Welfare, 03-PJ1-PG3-21300-0007, and a grant (M103KV01000 9-04K2201-00930) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.
 authors contributing equally to this work;  Received for publication February 1, 2004. Revision received May 16, 2005. Accepted for publication May 24, 2005.
Journal of Dental Research, Vol. 84, No. 9,
848-851 (2005)
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ABSTRACT
INTRODUCTION
1B-a, β1b, 
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. Series resistance was compensated for (> 80%), and leak subtraction was performed. Data were low-pass-filtered at 2 kHz, and sampled at 10 kHz. The pClamp8 (Axon Instruments) software was used during experiments and analysis. The pipette solution for IBa was composed of (mM): CsCl 100, MgCl2 1, HEPES 10, BAPTA 10, Mg-ATP 3.6, phosphocreatine 14, GTP 0.1, and creatine phosphokinase 50 units/mL, adjusted to pH 7.4 with CsOH. The extracellular solution for IBa contained (mM): tetraethylammonium chloride (TEACl) 151, HEPES 10, BaCl2 5, MgCl2 1, and glucose 10, adjusted to pH 7.4 with TEAOH. The IBa was evoked by a test pulse to +0 mV from the holding potential, –80 mV every 10 sec. 
