4-1. S creening essential oils for the ability to activate hT R PM 8 but not hT R PA 1 T R PA 1 is an excitatory ion channel targeted by pungent irritants such as those from mustard oil and garlic. It is thought to function in diverse sensory processes, including cold nociception and inflammatory pain. T herefore, T R PA 1 is a promising target for the identification of analgesic drugs. A natural analgesic compound that does not accelerate pain signaling is desirable for pharmaceutical or cosmetic pain relief.
T R PA 1 antagonists (ruthenium red, HC -030031, A MG5445, A 967079 and camphor) possess analgesic properties [ Nagata et al., 2005; X u et al., 2005; K lionsky et al., 2007;
E id et al., 2008; McG araughty et al., 2010; C hen et al., 2011] . Of these, camphor is the only naturall y occurring compound and is often used in cosmetics because it possesses minimal adverse effects. However, camphor is not suited for use as an analgesic compound because it causes warm and hot sensations [ Green et al., 1990] . T hese sensations are mediated through activation of T R PV 1 [ X u et al., 2005;
Vogt-E isele et al., 2007] . Moreover, T R PM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia [ D haka et al., 2007; Proudfoot et al., 2006] . A lthough menthol, the main ingredient of peppermint, is used for pain relief in daily life through T R PM8 activation [ Proudfoot et al., 2006] , its ability to activate hT R PA 1 restricts widespread use of menthol as an analgesic [ Galeotti et al., 2002].
T herefore, chemicals that activate T R PM8 and inhibit T R PA 1, but do not activate T R PV 1, would be ideal analgesic agents.
I found that the activation of hT R PA 1 (induced by several agonists with different
activation mechanisms) can be inhibited by 1,8-cineole. Moreover, 1,8-cineole activated hT R PM8 and hT R PV 3, but not hT R PA 1, hT R PV 1 or hT R PV 2. It was recentl y shown that both peripheral and central activation of T R PM8 could produce an analgesic effect that specifically reversed the sensitization of behavioral reflexes elicited by peripheral nerve injury [ G aleotti et al., 2002; Proudfoot et al., 2006; D haka et al., 2007] . F rom this point of view, 1,8-cineole appears to be an ideal natural analgesic that activates hT R PM8 and inhibits hT R PA 1.
1,8-cineole acts as an agonist of the T R PM8 channel with lower efficacy and potency (3.4 ± 0.4 mM) on T R PM8 than menthol [ McK emy et al., 2002; Vogt-E isele et al., 2007] . 1,8-cineole also activates the T R PV 3 channel in mice, but not the western clawed frog T R PV 3 [ S aito et al., 2011] . F urthermore, 1,8-cineole inhibits the chemical nociception produced by several irritants, and it has anti-inflammatory efficacy in patients with severe asthma [ J uergens et al., 2003] . T he present study suggests that the known analgesic and anti-inflammatory actions of 1,8-cineole can be attributed to its T R PM8-activating and T R PA 1-inhibiting abilities.
1,8-cineole has a fresh smell and elicits a cooling sensation when ingested or applied to the skin and is a common additive in flavorings, food, mouthwashes and cough suppressants. 1,8-cineole is often used in aromatherapy, as a stimulant in skin baths, by the pharmaceutical industry in drug formulations to enhance percutaneous penetration and as a decongestant and antitussive [ Williams et al., 1991; L aude et al., 1994; L evison et al., 1994] . E xperimental data have shown that 1,8-cineole is an analgesic and anti-inflammatory agent with beneficial effects for patients with severe asthma [ J uergens et al., 2003] . 1,8-cineole inhibits the in vitro formation of prostaglandins and cytokines by stimulated monocytes. However, the molecular
targets and mechanisms of the analgesic effect of 1,8-cineole remain unclear [ J uergens et al., 2004] .
In a human study, I examined sensitive volunteers to determine whether 1,8-cineole could inhibit sensory irritation caused by octanol and menthol. In the cosmetic research field, both menthol and octanol are well-known to cause skin irritation, and neither cinnamaldehyde nor allicin is used for human skin studies. 1,8-cineole activated T R PM8 less efficiently than did menthol, and it inhibited menthol-evoked skin irritation. T hose results clearly suggested that the inhibitory effects of 1,8-cineole are probably due to inhibition of T R PA 1 and not activation of T R PM8.
T he inhibitory effects of 1,8-cineole on menthol-induced hT R PA 1 activation was a little greater than those for A IT C - or F FA -induced hT R PA 1 activation ( F igure 6B , D , F ).
Menthol has bimodal action through transmembrane domain 5 of T R PA 1 in some species [ X iao et al., 2008] . T herefore, the similarity between the molecular structures of menthol and 1,8-cineole (F igure 4A ) suggests that 1,8-cineole could act on the same domain of T R PA 1 as menthol, although the structural basis for menthol-evoked hT R PA 1 activation is not known. F our compounds with similar structures (F igure 4A ) exhibited different effects on hT R PM8 and hT R PA 1: i) menthol and 1,4-cineole activated both hT R PM8 and hT R PA 1 [ Macpherson et al., 2006] ; ii) camphor inhibited hT R PA 1 [ X u et al., 2005] ; iii) 1,8-cineole activated hT R PM8 and inhibited hT R PA 1 (F igs. 2-4). T he fact that the four compounds exhibited differing effects on hT R PM8 and hT R PA 1 suggests that more detailed anal yses would lead to a better understanding of the structural basis for the action of these compounds on T R PM8 and T R PA 1.
4-2. S creening camphor analogs to identify more effective T R PA 1 antagonists T R PA 1 is an excitatory ion channel targeted by pungent irritants such as those from mustard oil and garlic. It is thought to function in diverse sensory processes, including nociception and inflammatory pain. T herefore, T R PA 1 is a promising target for the development of analgesic agents. A s described above, I found that 1,8-cineole is a naturally occurring hT R PA 1 antagonist. However, this natural antagonist exhibited weaker inhibitory effects on T R PA 1 activity than other antagonists such as HC -030031, A -967079, and A Z 868. T herefore, identificati on of naturall y occurring compounds with greater inhibitory potency of hT R PA 1 activation has been sought.
B orneol is a bicyclic monoterpenoid alcohol that has been used in foods as an aromatic spice. It is a valuable medical and chemical material that has been used as a folk medicine in C hina and India [ A lmeida et al., 2013] . A dditionally, borneol is a fragrant ingredient used in decorative cosmetics, fine fragrances, shampoos, and other toiletries. Previous studies have shown that borneol has a vasorelaxant effect on the rat thoracic aorta [ S ilva-F ilho et al., 2011] and neuroprotective effects [ L iu et al., 2011] . A lthough borneol has been evaluated for anti-nociceptive and anti-inflammatory activities, the molecular targets and mechanisms of its analgesic effect remain unclear.
T he fact that this monoterpene acts as an agonist of the T R PV 3 [ Vogt-E isele et al., 2007] and T R PV 1 [ X u et al., 2005] channels and specificall y inhibits nA C hR -mediated effects in a noncompetitive way [ Park et al., 2003] does not explain its anti-nociceptive effects. 2-methylisoborneol has been implicated as the cause of the muddy odor of fish from C edar L ake, Manitoba. A lgae produce 2-methylisoborneol and geosmin that are responsible for the musty odor [ Masakazu et al., 1988] . F enchyl alcohol, which is a component of several essential oils, is a fragrance ingredient used in decorative
cosmetics, fine fragrances, shampoos, and other toiletries. Moreover, this compound has an inhibitory effect on acetylcholinesterase activity [ Miyazawa et al., 2005].
A lthough borneol is the only terpene known to have anti-nociceptive effects, the two other terpenes identified in this study could have similar effects because all three monoterpenes inhibit hT R PA 1, and their derivatives might function as analgesics.
A lpizar et al. reported that camphor exhibited a bimodal effect on mT R PA 1 [ A lpizar et al., 2013] . T he authors observed the inhibition of mouse T R PA 1-mediated basal current with 1 mM camphor, and the current increased as camphor was washed out, suggesting a bimodal action for camphor on mT R PA 1. In addition, 1-5 mM camphor increased the cytosolic C a
2+
concentration upon washing out without any effect during camphor application. I examined the effect of camphor (1 mM) in a C a
2+
-imaging method, but failed to observe changes in the cytosolic C a
2+
concentration (F igure 8).
C amphor blocked the menthol-induced increase in cytosolic C a
2+
concentrations (1 mM, F igure 10E ). In addition, camphor inhibited the T R PA 1 current activated by 20 µ M A IT C in a dose-dependent manner ( F igure 13E ). A t present, I do not have an explanation for the apparent differences between the two studies, although a species difference mi ght have caused the different outcomes. T ransient enhancement of T R PA 1 current was observed after washing out of the three test compounds in F FA - or A IT C -evoked responses (F igs. 12 and 13), but not in the menthol-evoked response (F igure 12). S everal agonists have bimodal actions on mammalian T R PA 1, apparentl y involving non-covalent mechanisms. However, A lpizar et al. reported that cinnamaldehyde (a T R PA 1 activator with covalent modification) and camphor, which was thought to be a mammalian T R PA 1 antagonist, also exhibit bimodal actions on mT R PA 1, indicating that bimodal actions could be a more general phenomenon than
previously thought [ A lpizar et al., 2013] . T herefore, the transient enhancement of the hT R PA 1 current upon washing out of the antagonists (F igure 12, 13) might result from bimodal actions of the compounds. Interestingly, enhancement was not observed in the menthol-evoked T R PA 1 response ( F igure 12), possibly because menthol and hT R PA 1 inhibitors (borneol, 2-methyisoborneol and fenchyl alcohol) are all monoterpenes that act at similar sites, including ones that were identified in this study, through their cyclohexyl hydroxyl groups.
A nalogs of camphor have exhibited variable effects on hT R PA 1. B orneol, 2-methylisoborneol and fenchyl alcohol inhibited hT R PA 1. In contrast, norcamphor had no effect on hT R PA 1, and other related compounds activated hT R PA 1 (F igure 8). T he mechanisms of action with hT R PA 1 remain unclear although these monoterpenes have similar molecular structures. B orneol, which was synthesized by chemical reduction of camphor, Inhibited hT R PA 1, and the effects were greater than those of camphor. In addition, fenchyl alcohol, which was synthesized by chemical reduction of fenchone, inhibited hT R PA 1 activity, while fenchone activated hT R PA 1. C ommon structural differences between camphor and borneol and between fenchone and fenchyl alcohol are hydroxyl and carbonyl groups at the same position of their cyclohexane, which suggests that hydrogen bonding plays a pivotal role in the action of these compounds.
T his idea is supported by the results from mutation of T 874, an amino acid thought to form a hydrogen bond with menthol, resulting in a reduction in the activity of borneol.
In our study, S 873 and T 874 in T M5 and Y 812 in T M3 were found to be involved in the inhibitory effects of borneol. B ecause these two sites are somewhat distant from each other, borneol could fit separately into both sites.
4-3. T he effects of menthol on human T R PV 1
In the human study, I examined whether menthol could inhibit sensory irritation caused by V B E . In the cosmetic research field, V B E is well-known as a skin irritant, and neither capsaicin nor resiniferatoxin is used for human skin studies. S urprisingly, I found that V B E activated both hT R PV 1 and hT R PA 1. T his property could explain the marked irritation produced by V B E when used in humans. T he in vitro study showed that the IC50 value for menthol-induced hT R PV 1 inhibition was 1.2 ± 0.2 mM and that 10 mM menthol completely inhibited hT R PV 1 currents (F igure 21). However, 0.3 wt% menthol, which exhibited anti-nociceptive effects in the in vivo study (F igure 17), corresponded to 19.2 mM, which is within the attainable concentration range in clinical use [ Galeotti et al., 2002; Proudfoot et al., 2006] . Menthol is reported to cause analgesic effects through several different mechanisms, including those that are T R PM8-dependent and -independent [ Galeotti et al., 2001; Galeotti et al., 2002; McK ay et al., 2006; Proudfoot et al., 2006; D haka et al., 2007; Gaudioso et al., 2012] . Menthol also activates hT R PA 1 at higher concentrations compared with the concentrations activating hT R PM8 [ K lein et al., 2011] . T herefore, in humans, I observed the net result of V B E -hT R PV 1 interaction: (1) inhibition of V B E -evoked hT R PV 1 activation by menthol, (2) menthol-evoked hT R PA 1 activation and (3) V B E -evoked hT R PA 1 activation and other mechanisms. T his complicated mechanism could partl y explain the small inhibition of V B E -induced irritation by menthol in human subjects. Nevertheless, the observation that high concentrations of menthol (0.3%) (which could activate hT R PA 1 leading to greater irritation) inhibited V BE -induced irritation could be significant. T hus, the reduction of V B E -induced irritation by 0.3 wt% menthol in vivo might be partl y attributed to the inhibition of hT R PV 1 activity by
menthol shown in the in vitro study.
Preparations containing menthol are used topically to relieve neuralgia in traditional C hinese and E uropean medicine [ Wright et al., 1870] . In addition, mint oil has been reported to alleviate thermally elicited pain and post-herpetic neuralgia and orally applied menthol can achieve short-term analgesia [ Green et al., 2000] . F urthermore, in mice, oral or intracerebroventricular application of menthol decreased nociceptive responses in the hot-plate test and acetic acid writhing test [ L iu et al., 2013] . D espite these analgesic actions of menthol in the literature, the mechanism of action has not been fully clarified, although menthol-induced anti-nociception was reduced in T R PM8-deficint mice [ D haka et al., 2007] . Inhibition of T R PV 1 by menthol as shown in the current study could be one of the underlying mechanisms for the analgesic effects of menthol observed in rodents and humans. However, menthol could also achieve its anti-nociceptive effects through activation of T R PM8 and other mechanisms as reported [ Galeotti et al., 2002; Proudfoot et al., 2006] . T R PV 1 is activated by capsaicin as well as heat, protons and some endogenous substances known to be associated with tissue inflammation [ C aterina et al., 1997; Tominaga et al., 1998; Tominaga et al., 2004] . S ince T R PV 1 acts as an integrator of painful stimuli, T R PV 1 antagonists can be viewed as promising and novel types of analgesics [ Gavva et al., 2008; Gunthorpe et al., 2009;
Marcello et al., 2010; Marcello et al., 2013] . A number of potent, small T R PV 1 antagonists such as capsazepine, B C T C , C T PC , A MG9810 and S B -452533 [ B evan et al., 1992; V alenzano et al., 2003; R ami et al., 2004; Gavva et al., 2005; Weil et al., 2005] have advanced into clinical trials for the evaluation of their analgesic activities.
A lthough some of these antagonists reduced noxious heat sensation, hyperthermia as a serious side effect often led to their withdrawal from the clinical trials. T hus, novel
approaches to the development of anti-T R PV 1 antagonists are needed, and this study shows that derivatives of menthol could be promi sing molecules to develop for T R PV 1 antagonists.
A s discussed above, agonists of T R PV 1 and T R PM8 seem to mutually oppose one another. W hat is the physiological significance of their apparently reciprocal interaction? T R PM8 is not generall y co-expressed with T R PV 1 in primary afferent neurons [ Story et al., 2003; K obayashi et al., 2005; A be et al., 2005; F acer et al., 2007], suggesting that the information conducted by T R PM8-expressing neurons and T R PV 1-expressing neurons could influence one another. T he data presented here suggest the possibility that menthol-induced T R PM8-mediated cold sensation could be enhanced by inhibition of T R PV 1 and that capsaicin-induced T R PV 1-mediated heat sensation could be enhanced by inhibition of T R PM8. T he enhancement could work to strengthen the difference between T R PV 1 and T R PM8 activities in some specific concentration ranges. A lthough the physiological significance of T R PM8 inhibition by capsaicin is not evident, their reciprocal interactions could lead to the enhancement of T R PV 1-mediated nociceptive signals that effectively alert us to noxious conditions.