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Eugenol Inhibits Sodium Currents in Dental Afferent Neurons
1 Department of Physiology and Correspondence: * corresponding author, odolbae{at}snu.ac.kr
Although eugenol is widely used in dentistry, little is known about the molecular mechanisms responsible for its anesthetic properties. In addition to calcium channels, recently demonstrated by our group, there could be another molecular target for eugenol. Using a whole-cell patch-clamp technique, we investigated the effect of eugenol on voltage-gated sodium channel currents (INa) in rat dental primary afferent neurons identified by retrograde labeling with a fluorescent dye in maxillary molars. Eugenol inhibited action potentials and INa in both capsaicin-sensitive and capsaicin-insensitive neurons. The pre-treatment with capsazepine, a competitive antagonist of transient receptor potential vanilloid 1 (TRPV1), failed to block the inhibitory effect of eugenol on INa, suggesting no involvement of TRPV1. Two types of INa, tetrodotoxin (TTX)-resistant and TTX-sensitive INa, were inhibited by eugenol. Our results demonstrated that eugenol inhibits INa in a TRPV1-independent manner. We suggest that INa inhibition by eugenol contributes to its analgesic effect.
Key Words: eugenol • trigeminal ganglion neurons • voltage-gated sodium channels
Eugenol is extensively used in dentistry, due to its ability to allay tooth pain. However, little is known about the molecular mechanisms underlying its anesthetic properties. It has been shown that eugenol irreversibly blocked the conduction of action potential in the frog sciatic nerve (Kozam, 1977). Eugenol slowed nerve conduction in crayfish neurons and made them less excitable (Ozeki, 1975). Eugenol inhibited intradental nerve activity in cats (Trowbridge et al., 1982), as well as compound action potentials in the rat phrenic nerve in a reversible manner, like local anesthetics (Brodin and Roed, 1984). All of these in vivo and ex vivo results suggest that eugenol has a direct effect on ion channels expressed by peripheral sensory nerve fibers. Voltage-gated sodium channels are critical elements of action potential initiation and propagation in excitable cells, including sensory neurons, because they are responsible for the initial depolarization of the membrane (Hodgkin and Huxley, 1952). Therefore, it is possible that voltage-gated sodium channels are a molecular target for eugenol to elicit these effects. Eugenol and capsaicin share the vanilloid moiety in their chemical structures (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999). Like capsaicin (Petersen et al., 1989; Bleakman et al., 1990), eugenol has inhibitory effects on voltage-gated calcium channel currents (ICa), and these effects might contribute to the analgesic effect of eugenol (Lee et al., 2005). Interestingly, capsaicin requires transient receptor potential vanilloid 1 (TRPV1) for its inhibitory effect on voltage-gated calcium currents (Wu et al., 2005), but eugenol does not (Lee et al., 2005). Capsaicin also inhibits voltage-gated sodium channel currents (INa) only in capsaicin-sensitive trigeminal ganglion neurons (Liu et al., 2001). Likewise, it is possible that eugenol might regulate INa. In the present study, we investigated the effect of eugenol on INa and its mode of action in rat dental primary afferent neurons, using a whole-cell patch-clamp technique.
All procedures for animal use were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Dentistry, Seoul National University.
Preparation of Dental Primary Afferent Neurons
Electrophysiological Recordings
Drugs
Statistical Analysis
Eugenol Inhibited Action Potential in Both Capsaicin-sensitive and Capsaicin-insensitive Dental Primary Afferent Neurons Dental primary afferent neurons recorded in the study ranged from 15 to 55 µm in diameter, and the resting membrane potential was –50 ± 10 mV (n = 72). Analogous to characteristics previously described in DRG neurons (Cardenas et al., 1995), we distinguished two types of dental primary afferent neurons on the basis of their action potential duration, action potential shape, and their capsaicin sensitivity (Fig. 1  ). Type I neurons, mostly small (< 25 µm) and medium-sized neurons (25 ~ 35 µm), were characterized by long action potential duration (APD50 = 5.7 ± 0.5 ms), with a prominent shoulder in the falling phase (Fig. 1B). Type II neurons, mostly found in medium and large neurons (> 35 µm), had significantly shorter action potential durations (APD50 = 4.5 ± 0.6 ms) than did type I neurons (Fig. 1C). These type II neurons exhibited a small shoulder in the falling phase. A 1-µM concentration of capsaicin inhibited the generation of action potentials in type I neurons (n = 15), but not in type II neurons (n = 15). In contrast, 1 mM eugenol inhibited the generation of action potentials in both capsaicin-sensitive type I (n = 15) and capsaicin-insensitive type II dental primary afferent neurons (n = 15).
Action Potential Blockade Produced by Eugenol is Due to its INa Inhibition Because voltage-gated sodium channels are responsible for the initial depolarization phase involved in the generation of action potential (Lai et al., 2004), we explored the effect of eugenol on INa. A 1-mM concentration of eugenol inhibited INa in both capsaicin-sensitive neurons and capsaicin-insensitive neurons (Figs. 2A, 2B ). The magnitude of INa inhibition by 1 mM eugenol was similar in capsaicin-insensitive neurons (77 ± 4%, n = 30) (Fig. 2Ab) and capsaicin-sensitive neurons (78 ± 3%, n = 20) (Figs. 2Bb, 2Da). INa inhibition by eugenol was dose-dependent (Fig. 2C, IC50 = 600 ± 75 µM). To test whether TRPV1 activation mediates the inhibitory effect of eugenol on INa, we examined the effect of eugenol (1 mM) in the presence of capsazepine (10 µM), a competitive TRPV1 antagonist (n = 6). When we applied 10 µM capsazepine for 5 min, it did not produce any effect on the voltage-gated sodium channels, which was consistent with the previous report (Liu et al., 2001). The INa inhibition by the combined application of eugenol and capsazepine (73 ± 5%, n = 15) was not significantly different from that of eugenol (75 ± 4%, n = 20) (Fig. 2Db).
Both TTX-s INa and TTX-r INa are Blocked by Eugenol Two types of sodium channels, TTX-s and TTX-r, are expressed in sensory neurons (Wu and Pan, 2004). Consistent with this report, TTX-s INa and TTX-r INa were differentially distributed in the dental primary afferent neurons tested. Most of the INa was TTX-r INa in small neurons (< 25 µm), whereas TTX-s INa was predominant in large neurons (> 35 µm). TTX-s INa is the difference current of total INa and TTX-r INa (Fig. 3Aa ). Eugenol significantly inhibited both TTX-r INa (Fig. 3Ab) and TTX-s INa (Fig. 3B). The magnitude of TTX-r INa inhibition by 1 mM eugenol (72 ± 4%, n = 7) (Fig. 3Ab) was similar to that of TTX-s INa (80 ± 3%, n = 5) (Figs. 3B, 3C).
Recently, we demonstrated that eugenol inhibits voltage-gated calcium currents in dental primary afferent neurons in a TRPV1-independent manner (Lee et al., 2005). It was thought that the inhibition of voltage-gated calcium channels might contribute to the analgesic effect of eugenol. In the present study, we further investigated whether eugenol would inhibit INa and its mode of action. We found that eugenol elicited an inhibitory effect on INa, thereby blocking the generation of action potential in dental primary afferent neurons, and this effect was observed in both capsaicin-sensitive and -insensitive neurons. We found two types of action potentials in dental primary afferent neurons, as in other sensory neurons (Mirnics and Koerber, 1997; Lawson, 2002). Capsaicin-sensitive type I neurons, which have a relatively wide action potential with a shoulder on the falling phase, are likely to be nociceptive neurons. Capsaicin-insensitive type II neurons, exhibiting a narrow action potential but having no shoulder on the falling phase, could be non-nociceptive neurons. We observed that eugenol inhibited action potentials in both nociceptive and non-nociceptive neurons, regardless of their sensitivity to capsaicin. The extent of INa by eugenol in capsaicin-sensitive neurons was comparable with that in capsaicin-insensitive neurons, and capsazepine failed to block the eugenol-induced INa inhibition in dental primary afferent neurons. These findings suggest that TRPV1 activation might not be involved in the inhibitory effect of eugenol on INa. In contrast, capsaicin inhibits INa only in capsaicin-sensitive trigeminal ganglion neurons, and capsazepine, a competitive TRPV1 antagonist, inhibits capsaicin-induced blockade of INa in rat trigeminal ganglion neurons (Liu et al., 2001). These observations are reminiscent of the effects of eugenol and capsaicin on ICa. There are two general classes of sodium currents in sensory neurons: One is blocked by TTX (TTX-sensitive or TTX-s INa), and the other is insensitive to TTX (TTX-resistant or TTX-r INa). We found that eugenol inhibited both TTX-s INa and TTX-r INa in dental primary afferent neurons. Because TTX application to distal axons completely blocks conduction of action potentials (Ritter and Mendell, 1992; Brock et al., 1998), it is clear that TTX-s INa mediate action potential conduction along both myelinated and unmyelinated axons. TTX-r INa were also found to mediate action potential initiation in polymodal nociceptive afferents (Brock et al., 2001). In addition, TTX-r INa may contribute to the release of transmitter from the central terminals of nociceptive afferents (Gu and MacDermott, 1997). Thus, the inhibitory effect of eugenol on both TTX-s and TTX-r INa may contribute to its analgesic effect. We have recently reported that eugenol inhibited voltage-gated calcium currents (ICa) in dental primary afferent neurons (Lee et al., 2005). The ranges of eugenol concentrations that produced inhibitory effects on INa (10–4 to ~10–3 M) were lower than those on ICa (~ 5 x 10–4 to ~ 10–3 M), indicating that voltage-gated sodium channels are more sensitive to eugenol than are voltage-gated calcium channels. Therefore, inhibition of action potential generation and propagation is likely to be a major molecular mechanism for the analgesic effect of eugenol. It is interesting to note that, although eugenol and capsaicin share a vanillyl-like moiety in their chemical structure (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999), the mechanisms underlying inhibitory effects between eugenol and capsaicin on voltage-gated sodium channels and voltage-gated calcium channels are different: One is TRPV1-independent, the other is TRPV1-dependent. These findings suggest that the vanillyl-moiety, which is known to be important for capsaicin-like action of vanilloid compound (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999), might not be the critical factor for determining mechanisms of voltage-gated channel regulation by vanilloid compounds, such as capsaicin and eugenol. In summary, we demonstrate that eugenol produces an inhibitory effect on INa in dental primary afferent neurons, and that TRPV1 activation is not a prerequisite for the inhibitory effect of eugenol on INa. The inhibition of INa, in addition to ICa, is likely to be one of the molecular mechanisms underlying the analgesic effect of eugenol.
This research was supported by a grant (R01-2004-000-10384-0) from the Basic Research Program of the Korea Science & Engineering Foundation, a grant (M103KV010009-05K2201-00930) from the Brain Research Center of the 21st Century Frontier Research Program, funded by the Ministry of Science and Technology, and a Korea Research Foundation Grant (KRF-042-2003-E00124), Republic of Korea..
 authors contributing equally to this work  Received for publication October 14, 2005. Revision received June 5, 2006. Accepted for publication June 7, 2006.
Journal of Dental Research, Vol. 85, No. 10,
900-904 (2006)
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ABSTRACT
INTRODUCTION
. The pipette solution for current-clamp experiments was composed of (mM): K-gluconate 145, MgCl2 2, CaCl2 1, EGTA 10, HEPES 5, and K2ATP 5, adjusted to pH 7.2 ~ 7.3 with KOH. Extracellular solution for current-clamp experiments contained (mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, and glucose 10, adjusted to pH 7.4 with NaOH. In current-clamp experiments, the resting potential was adjusted to –65 mV at the beginning of the experiment. Action potentials were evoked with 5-ms depolarizing current pulses with increasing amplitude (0.2 ~ 1 nA in 50.0-pA steps). Action potential duration was measured at 50% repolarization (APD50). The pipette solution for INa was composed of (mM): CsCl 100, sodium L-glutamic acid 5, TEACl 30, CaCl2 0.1, MgCl2 2, EGTA 11, and HEPES 10, adjusted to pH 7.4 with CsOH. Extracellular solution for INa contained (mM): NaCl 90, choline chloride 30, TEACl 20, CaCl2 0.1, MgCl2·5, CoCl2 5, HEPES 10, and glucose 10, adjusted to pH 7.4 with NaOH. In voltage-clamp experiments, the INa was evoked by a test pulse to +0 mV from the holding potential, –80 mV every 10 sec. INa was classified into tetrodotoxin-sensitive (TTX-s) and tetrodotoxin-resistant (TTX-r) INa. For TTX-r INa, 1 µM TTX (Sigma, St. Louis, MO, USA) was used to block TTX-s INa. We obtained TTX-s INa by subtracting TTX-r INa from the total INa. In both voltage- and current-clamp experiments, series resistance was compensated for (> 80%), and leak subtraction was performed. Data were lowpass-filtered at 2 kHz, and sampled at 10 kHz. The pClamp8 (Axon Instruments) software was used during experiments and analysis. All the experiments were performed at room temperature. 
