- Email: [email protected]
Mecamylamine inhibits nicotine but not capsaicin irritation on the tongue: psychophysical evidence that nicotine and capsaicin activate separate molecular receptors
Neuroscience Letters 240 (1998) 65–68
Mecamylamine inhibits nicotine but not capsaicin irritation on the tongue: psychophysical evidence that nicotine and capsaicin activate separate molecular receptors Jean-Marc Dessirier a , b , c, Michael O’Mahony b, Jean-Marc Sieffermann c, E. Carstens a ,* a
Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA b Department of Food Science and Technology, University of California, Davis, CA, USA c Departement de Science de l’Aliment, ENSIA (Ecole Nationale Supe´rieure des Industries Agro-alimentaires), Massy, France Received 7 October 1997; received in revised form 20 November 1997; accepted 21 November 1997
Abstract Using a two-alternative forced-choice (2-AFC) discrimination test coupled with category intensity ratings, we investigated the effect of mecamylamine, an antagonist of neuronal nicotinic acetylcholine receptors (nAchRs), on oral irritation elicited by nicotine or capsaicin. Mecamylamine (0.075%) was first delivered to one side of the tongue with distilled H2O delivered to the other side. After 10 min either capsaicin (1 ppm) or nicotine (0.12%) was applied bilaterally to the tongue, and subjects were asked to choose which side yielded a stronger sensation (2-AFC) as well as to provide a rating of the irritation intensity difference between the two sides of the tongue. When nicotine was given after mecamylamine, a significant proportion of subjects chose the mecamylamine-untreated side as yielding stronger irritation. When capsaicin was given after mecamylamine, both sides of the tongue were chosen in equal numbers. These data indicate that mecamylamine reduced irritation elicited by nicotine but not capsaicin, and provide further evidence that nicotine oral irritation is mediated via a neuronal nAchR while capsaicin activates trigeminal fibers via a separate molecular receptor. 1998 Elsevier Science Ireland Ltd.
Keywords: Irritation; Trigeminal; Nociception; Mecamylamine; Nicotine; Capsaicin; Two-alternative forced-choice
Nicotine, the pungent principle in tobacco smoke, causes irritation in man and nocifensive responses in animals when applied to skin [10], oral [2,8], nasal [5,7,9] or ocular [9] mucosa. Nicotine excites free nerve endings in the cornea [14] and skin [17], depolarizes dorsal root [18] and trigeminal ganglion neurons [12,13], and excites second-order neurons in trigeminal subnucleus caudalis [1]. A proportion of trigeminal ganglion neurons activated by nicotine were also sensitive to capsaicin [12]. Furthermore, nicotine irritation appears to act via a neuronal nicotinic acetylcholine receptor (nAchR; see [15,16] for review), since it was reduced by pretreatment with the ganglionic blocker, mecamylamine [8], in line with observations that nicotinic antagonists block nocifensive responses [9] and depolariza-
* Corresponding author. Tel.: +1 916 7526640; fax: +1 916 7525582; e-mail: [email protected]
tion of trigeminal ganglion neurons [13] induced by nicotine. In a previous study we reported that prior application of capsaicin reduced the irritation elicited by nicotine, but that prior nicotine did not reduce capsaicin-evoked irritation [2]. This non-reciprocal cross-desensitization suggests that nicotine irritation is mediated at least partly via capsaicinsensitive fibers, but that the two compounds may act via separate molecular receptors. Capsaicin is thought to activate trigeminal neurons via a putative vanilloid receptor (see [6,19] for reviews). The aim of the present study was to test the hypothesis that nicotine and capsaicin act via separate molecular receptors by determining whether mecamylamine antagonizes the irritation produced by nicotine but not capsaicin when placed on the tongue. Twenty healthy subjects (three males, 17 females, aged 21–34 years), student and staff at the University of Califor-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00930- 0
66
J.-M. Dessirier et al. / Neuroscience Letters 240 (1998) 65–68
Fig. 1. Testing protocol: mecamylamine was applied to one side of the tongue (hatched circle in upper figurine of tongue) and water was applied to the other side (open circle). After a 10-min rest period, either nicotine (lower left figurine) or capsaicin (lower right) was applied bilaterally to the tongue (filled small circles) within the area that had previously received mecamylamine (hatched larger circle) or water (open larger circle). This was followed by a 2-AFC test in which subjects were asked to indicate which side of the tongue yielded a stronger sensation. Following bilateral nicotine, a significant majority of subjects chose the side pretreated with water as yielding greater irritation (+). Following bilateral capsaicin, subjects chose either side with equal frequency ( = ).
nia at Davis, volunteered to participate in the study. None were smokers and all had fasted at least 1 h prior to each experimental session. The experimental protocol was approved by the local Human Subjects review committee. All chemical were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified. Mecamylamine 0.0075% w/v (4.5 mM) and (−) nicotine 0.12% w/v (7.4 mM) were dissolved in distilled water. A 1% w/v capsaicin stock solution was made by dissolving capsaicin (vanillyl non-amide; 98–100%) in ethanol 95% (Quantum, Los Angeles, CA, USA) and diluted down to a 1 ppm (3.3 mM) solution with distilled water. The experimental protocol is summarized in Fig. 1. Each experimental session began by having subjects rinse their mouths three times with distilled water at room temperature. A 1.5 cm diameter filter paper disk (176.7 mm2; Whatmann, Maidstone, UK; product #1001-0155) soaked with 40 ml of mecamylamine was then placed on to one side of the dorsal surface of the tongue using forceps. Another filter paper disk
soaked with 40 ml distilled water was placed on the other side of the tongue simultaneously as a control to ensure that any effects were not due to the filter paper per se and also to reduce any expectations the subjects might have about one side of the tongue receiving a ‘treatment’. The side of the tongue receiving mecamylamine was counterbalanced across subjects. Both filter papers were left on the tongue for 1 min then removed. A 10-min rest period followed during which subjects were asked to sit quietly with the mouth closed. To prevent mecamylamine spreading from one side of the tongue to the other, subjects were instructed not to touch the roof of their mouth with the tongue and not to swallow. Additionally, saliva was removed from the mouth by a suction device (saliva ejectors, 6 inches clear; Sullivan Dental, Sacramento, CA, USA). After the 10-min waiting period, two smaller 1.0 cm diameter filter papers disks (78.5 mm2; Whatmann, Maidstone, UK; product #1001-0105) both soaked with 15 ml of either nicotine (0.12%) or capsaicin (1 ppm) were applied simultaneously onto both sides of the tongue, at locations corresponding to the ones where the larger filter papers had been applied earlier. Nicotine and capsaicin sessions were counterbalanced across subjects. Thirty seconds after application subjects were asked to perform a 2-alternative forced-choice (2AFC) test [4], in which the task was to identify the side of the tongue yielding a stronger irritant sensation. The rationale is that if mecamylamine blocks irritation, a significant majority of subjects would choose the previously untreated side. Because of the forced choice procedure, subjects were required to chose one side even if no difference was detected, so that an absence of effect of mecamylamine would result in 50% of the subjects choosing the non-pretreated side and 50% the pretreated side. After choosing, subjects were also asked to provide a rating of the difference in intensity of irritation between the two sides of the tongue using a category scale with ratings ranging from 0 (no difference) to 10 (very large difference). The 2-AFC and rating procedures were repeated 60, 90 and 120 s following bilateral application of nicotine or capsaicin. During this test period, subjects were allowed to use the suction device at any time except 15 s prior to ratings to avoid cooling the mouth. When nicotine was applied bilaterally 10 min after unilateral application of mecamylamine, a significant majority of subjects (18/20, binomial P , 0.001) chose the untreated side as yielding a stronger irritant sensation. This proportion
Table 1 Effect of mecamylamine application on the perceived irritation of nicotine: results of the 2-AFC and rating procedures Time of rating after bilateral application of capsaicin (s) Proportion of subjects choosing the untreated side as stronger Binomial test Mean rating t-test
30
60
90
120
18/20
20/20
18/20
18/20
P , 0.001 3.15 P , 0.001
P , 0.001 2.55 P , 0.001
P , 0.001 1.55 P = 0.006
P , 0.001 0.7 P = 0.031
67
J.-M. Dessirier et al. / Neuroscience Letters 240 (1998) 65–68 Table 2 Effect of mecamylamine application on the perceived irritation of capsaicin: results of the 2-AFC and rating procedures Time of rating after bilateral application of capsaicin (s) Proportion of subjects choosing the untreated side as stronger Binomial test Mean rating t-test
30 9/20
P . 0.8 0.25 P = 0.67
remained significant over the entire 2 min testing period (Table 1). The mean difference rating (for intensity of irritation on the two sides of the tongue) was 3.15, which was significantly different from zero (t-test, P , 0.001). The magnitude of the difference decreased over the 2 min testing period, but remained significantly different from zero (Table 1), indicating that mecamylamine inhibited the perceived irritation evoked by nicotine. When capsaicin was applied bilaterally following unilateral application of mecamylamine, nine subjects chose the untreated side as yielding a stronger sensation while 11 chose the mecamylamine-pretreated side (binomial, P . 0.8). This ratio remained close to 50% and non-significant throughout the 2 min testing period (Table 2). The mean difference rating between the two sides of the tongue at t = 30 s was 0.25, which was not significantly different from zero (t-test, P = 0.67). The mean ratings remained low and not significantly different from zero across the 2 min testing period (Table 2), indicating that mecamylamine did not reduce irritation evoked by capsaicin. In addition, 50% of the subjects changed their choice from one side of the tongue to the other at least once during the 2 min testing period, providing additional evidence that mecamylamine did not affect the perceived intensity of capsaicin irritation. The 2-AFC method used in the present study has the advantage of being very sensitive and hence able to detect small sensory differences [3,11]. However, it is possible that subjects may have developed a bias in choice preference due to the stronger taste of mecamylamine, which is slightly bitter/sour, compared to water. As noted above, subjects chose both sides of the tongue in equal proportions when capsaicin followed mecamylamine, arguing against this possibility. Nonetheless, a third control session was added in which 400 mM NaCl, instead of mecamylamine, was applied followed by bilateral application of 1 ppm capsaicin. The 400 mM NaCl solution was chosen to approximately match the mecamylamine in taste intensity. Approximately half of the subjects chose the untreated side as yielding a stronger sensation while the other half chose the NaCl-pretreated side, and the mean rating for intensity difference was close to zero, suggesting once again an absence of a choice preference artifact. The present results confirm, using a different method, an earlier study [8] showing a reduction in nicotine-evoked irritation by the neuronal nAchR antagonist, mecamyla-
60
90
9/20
P . 0.8 0.4 P = 0.43
11/20
P = 0.8 0 P=1
120 10/20
P . 0.8 −0.5 P = 0.35
mine. Furthermore, they also indicate that mecamylamine is selective for nicotinic receptors in that it did not reduce the perceived irritation elicited by capsaicin when placed on the same area of the tongue. These psychophysical data also confirm older studies in which a variety of nicotinic antagonists selectively prevented nocifensive responses such as blinking elicited by nicotine, but not capsaicin, instilled into the eye in rodents [9]. Although we were presently unable to demonstrate selective antagonism of capsaicin but not nicotine-evoked irritation due to the absence of a reliable capsaicin receptor antagonist for human studies, the present results nonetheless provide additional support for the idea that nicotine and capsaicin evoke irritation by acting at separate molecular receptors on polymodal nociceptors innervating the oral cavity. The present experimental protocol imposed a 10-min rest period between application of mecamylamine and subsequent application of nicotine or capsaicin. This was chosen in light of an earlier study [8] in which nicotine-evoked irritation was found to be reduced only when the nicotine was applied at least 20 min after the initial application of mecamylamine. In pilot experiments we found that irritation was not affected when nicotine was applied immediately following mecamylamine application, and that a 10-min waiting period was sufficient for the inhibitory effect of mecamylamine to be expressed. Furthermore, 12 subjects (of the 20 in this study) were additionally tested using a 5 min rest period between applications of mecamylamine and nicotine; while most exhibited an inhibition of the nicotine irritation, three did not exhibit mecamylamine antagonism of nicotine-evoked irritation with the 5-min rest period although they did with the 10-min rest. This indicates that a minimum and variable amount of time is necessary for the antagonism to take effect. This research was supported by a grant from Philip Morris Inc., USA. [1] Carstens, E., Kuenzler, N., Handwerker, H.O. and Activation of neurons in rat dorsomedial trigeminal, N., Caudalis (Vc) by application of a spectrum of irritant chemicals to the tongue, Soc. Neurosci. Abstr., 22 (1996) 863. [2] Dessirier, J.-M., O’Mahony, M. and Carstens, E., Oral irritant effects of nicotine: psychophysical evidence for decreased sensation following repeated application and lack of cross-desensitization to capsaicin, Chem. Senses, 22 (1997) 483–492.
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J.-M. Dessirier et al. / Neuroscience Letters 240 (1998) 65–68
[3] Ennis, D.M., The power of sensory discrimination methods, J. Sens. Stud., 8 (1993) 353–370. [4] Green, D.M. and Swets, J.A., Signal Detection Theory and Psychophysics, Wiley, New York, 1966, pp. 43–45. [5] Greiff, L., Wollmer, P., Andersson, M., Pipkorn, U. and Persson, C.G.A., Effects of nicotine on the human nasal mucosa, Thorax, 48 (1993) 651–655. [6] Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 49 (1991) 143–201. [7] Hummel, T., Livermore, A., Hummel, C. and Kobal, G., Chemosensory event-related potentials in man: relation to olfactory and painful sensations elicited by nicotine, Electroenceph. clin. Neurophysiol., 84 (1992) 192–195. [8] Jarvik, M.E. and Assil, K.M., Mecamylamine blocks the burning sensation of nicotine on the tongue, Chem. Senses, 13 (1988) 213–217. [9] Jansco, N., Jansco-Gabor, A. and Takats, I., Pain and inflammation induced by nicotine, acetylcholine and structurally related compounds and their prevention by desensitizing agents, Acta Physiol., 19 (1961) 113–132. [10] Keele, C.A. and Armstrong, D., Substances Producing Pain and Itch, Edward Arnold, London, 1964. [11] Kim, K.O. and O’Mahony, M., A new approach to category scales of intensity II. Use of d′ values, J. Sens. Stud., (1997), submitted.
[12] Liu, L. and Simon, S.A., Capsaicin and nicotine both activate a subset of rat trigeminal ganglion neurons, Am. J. Physiol. Cell Physiol., 39 (1996) C1807–C1814. [13] Liu, L., Pugh, W., Mam, H. and Simon, S.A., Identification of acetylcholine receptors in adult rat trigeminal ganglion neurons, Brain Res., 617 (1993) 37–42. [14] MacIver, M.B. and Tanalian, D.L., Structural and functional specialization of A-delta and C fiber free nerve endings innervating rabbit corneal epithelium, J. Neurosci., 13 (1993) 4511– 4524. [15] Ochoa, E.L.M., Li, L. and McNamee, M.G., Desensitization of central cholinergic mechanisms and neuroadaptation to nicotine, Mol. Neurobiol., 4 (1990) 251–287. [16] Sargent, P.B., The diversity of neuronal nicotinic acetylcholine receptors, Ann. Rev. Neurosci., 16 (1993) 403–443. [17] Steen, K.H. and Reeh, P.W., Actions of cholinergic agonists and antagonists on sensory nerve endings in rat skin in vitro, J. Neurophysiol., 70 (1993) 397–405. [18] Sucher, N.J., Cheng, T.P. and Lipton, S.A., Neural nicotinic acetylcholine responses in sensory neurons from postnatal rats, Brain Res., 533 (1990) 248–254. [19] Szallasi, A., The vanilloid (capsaicin) receptor: receptor types and species differences, Gen. Pharmacol., 25 (1994) 223– 243.
Mecamylamine inhibits nicotine but not capsaicin irritation on the tongue: psychophysical evidence that nicotine and capsaicin activate separate molecular receptors Jean-Marc Dessirier a , b , c, Michael O’Mahony b, Jean-Marc Sieffermann c, E. Carstens a ,* a
Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA b Department of Food Science and Technology, University of California, Davis, CA, USA c Departement de Science de l’Aliment, ENSIA (Ecole Nationale Supe´rieure des Industries Agro-alimentaires), Massy, France Received 7 October 1997; received in revised form 20 November 1997; accepted 21 November 1997
Abstract Using a two-alternative forced-choice (2-AFC) discrimination test coupled with category intensity ratings, we investigated the effect of mecamylamine, an antagonist of neuronal nicotinic acetylcholine receptors (nAchRs), on oral irritation elicited by nicotine or capsaicin. Mecamylamine (0.075%) was first delivered to one side of the tongue with distilled H2O delivered to the other side. After 10 min either capsaicin (1 ppm) or nicotine (0.12%) was applied bilaterally to the tongue, and subjects were asked to choose which side yielded a stronger sensation (2-AFC) as well as to provide a rating of the irritation intensity difference between the two sides of the tongue. When nicotine was given after mecamylamine, a significant proportion of subjects chose the mecamylamine-untreated side as yielding stronger irritation. When capsaicin was given after mecamylamine, both sides of the tongue were chosen in equal numbers. These data indicate that mecamylamine reduced irritation elicited by nicotine but not capsaicin, and provide further evidence that nicotine oral irritation is mediated via a neuronal nAchR while capsaicin activates trigeminal fibers via a separate molecular receptor. 1998 Elsevier Science Ireland Ltd.
Keywords: Irritation; Trigeminal; Nociception; Mecamylamine; Nicotine; Capsaicin; Two-alternative forced-choice
Nicotine, the pungent principle in tobacco smoke, causes irritation in man and nocifensive responses in animals when applied to skin [10], oral [2,8], nasal [5,7,9] or ocular [9] mucosa. Nicotine excites free nerve endings in the cornea [14] and skin [17], depolarizes dorsal root [18] and trigeminal ganglion neurons [12,13], and excites second-order neurons in trigeminal subnucleus caudalis [1]. A proportion of trigeminal ganglion neurons activated by nicotine were also sensitive to capsaicin [12]. Furthermore, nicotine irritation appears to act via a neuronal nicotinic acetylcholine receptor (nAchR; see [15,16] for review), since it was reduced by pretreatment with the ganglionic blocker, mecamylamine [8], in line with observations that nicotinic antagonists block nocifensive responses [9] and depolariza-
* Corresponding author. Tel.: +1 916 7526640; fax: +1 916 7525582; e-mail: [email protected]
tion of trigeminal ganglion neurons [13] induced by nicotine. In a previous study we reported that prior application of capsaicin reduced the irritation elicited by nicotine, but that prior nicotine did not reduce capsaicin-evoked irritation [2]. This non-reciprocal cross-desensitization suggests that nicotine irritation is mediated at least partly via capsaicinsensitive fibers, but that the two compounds may act via separate molecular receptors. Capsaicin is thought to activate trigeminal neurons via a putative vanilloid receptor (see [6,19] for reviews). The aim of the present study was to test the hypothesis that nicotine and capsaicin act via separate molecular receptors by determining whether mecamylamine antagonizes the irritation produced by nicotine but not capsaicin when placed on the tongue. Twenty healthy subjects (three males, 17 females, aged 21–34 years), student and staff at the University of Califor-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00930- 0
66
J.-M. Dessirier et al. / Neuroscience Letters 240 (1998) 65–68
Fig. 1. Testing protocol: mecamylamine was applied to one side of the tongue (hatched circle in upper figurine of tongue) and water was applied to the other side (open circle). After a 10-min rest period, either nicotine (lower left figurine) or capsaicin (lower right) was applied bilaterally to the tongue (filled small circles) within the area that had previously received mecamylamine (hatched larger circle) or water (open larger circle). This was followed by a 2-AFC test in which subjects were asked to indicate which side of the tongue yielded a stronger sensation. Following bilateral nicotine, a significant majority of subjects chose the side pretreated with water as yielding greater irritation (+). Following bilateral capsaicin, subjects chose either side with equal frequency ( = ).
nia at Davis, volunteered to participate in the study. None were smokers and all had fasted at least 1 h prior to each experimental session. The experimental protocol was approved by the local Human Subjects review committee. All chemical were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified. Mecamylamine 0.0075% w/v (4.5 mM) and (−) nicotine 0.12% w/v (7.4 mM) were dissolved in distilled water. A 1% w/v capsaicin stock solution was made by dissolving capsaicin (vanillyl non-amide; 98–100%) in ethanol 95% (Quantum, Los Angeles, CA, USA) and diluted down to a 1 ppm (3.3 mM) solution with distilled water. The experimental protocol is summarized in Fig. 1. Each experimental session began by having subjects rinse their mouths three times with distilled water at room temperature. A 1.5 cm diameter filter paper disk (176.7 mm2; Whatmann, Maidstone, UK; product #1001-0155) soaked with 40 ml of mecamylamine was then placed on to one side of the dorsal surface of the tongue using forceps. Another filter paper disk
soaked with 40 ml distilled water was placed on the other side of the tongue simultaneously as a control to ensure that any effects were not due to the filter paper per se and also to reduce any expectations the subjects might have about one side of the tongue receiving a ‘treatment’. The side of the tongue receiving mecamylamine was counterbalanced across subjects. Both filter papers were left on the tongue for 1 min then removed. A 10-min rest period followed during which subjects were asked to sit quietly with the mouth closed. To prevent mecamylamine spreading from one side of the tongue to the other, subjects were instructed not to touch the roof of their mouth with the tongue and not to swallow. Additionally, saliva was removed from the mouth by a suction device (saliva ejectors, 6 inches clear; Sullivan Dental, Sacramento, CA, USA). After the 10-min waiting period, two smaller 1.0 cm diameter filter papers disks (78.5 mm2; Whatmann, Maidstone, UK; product #1001-0105) both soaked with 15 ml of either nicotine (0.12%) or capsaicin (1 ppm) were applied simultaneously onto both sides of the tongue, at locations corresponding to the ones where the larger filter papers had been applied earlier. Nicotine and capsaicin sessions were counterbalanced across subjects. Thirty seconds after application subjects were asked to perform a 2-alternative forced-choice (2AFC) test [4], in which the task was to identify the side of the tongue yielding a stronger irritant sensation. The rationale is that if mecamylamine blocks irritation, a significant majority of subjects would choose the previously untreated side. Because of the forced choice procedure, subjects were required to chose one side even if no difference was detected, so that an absence of effect of mecamylamine would result in 50% of the subjects choosing the non-pretreated side and 50% the pretreated side. After choosing, subjects were also asked to provide a rating of the difference in intensity of irritation between the two sides of the tongue using a category scale with ratings ranging from 0 (no difference) to 10 (very large difference). The 2-AFC and rating procedures were repeated 60, 90 and 120 s following bilateral application of nicotine or capsaicin. During this test period, subjects were allowed to use the suction device at any time except 15 s prior to ratings to avoid cooling the mouth. When nicotine was applied bilaterally 10 min after unilateral application of mecamylamine, a significant majority of subjects (18/20, binomial P , 0.001) chose the untreated side as yielding a stronger irritant sensation. This proportion
Table 1 Effect of mecamylamine application on the perceived irritation of nicotine: results of the 2-AFC and rating procedures Time of rating after bilateral application of capsaicin (s) Proportion of subjects choosing the untreated side as stronger Binomial test Mean rating t-test
30
60
90
120
18/20
20/20
18/20
18/20
P , 0.001 3.15 P , 0.001
P , 0.001 2.55 P , 0.001
P , 0.001 1.55 P = 0.006
P , 0.001 0.7 P = 0.031
67
J.-M. Dessirier et al. / Neuroscience Letters 240 (1998) 65–68 Table 2 Effect of mecamylamine application on the perceived irritation of capsaicin: results of the 2-AFC and rating procedures Time of rating after bilateral application of capsaicin (s) Proportion of subjects choosing the untreated side as stronger Binomial test Mean rating t-test
30 9/20
P . 0.8 0.25 P = 0.67
remained significant over the entire 2 min testing period (Table 1). The mean difference rating (for intensity of irritation on the two sides of the tongue) was 3.15, which was significantly different from zero (t-test, P , 0.001). The magnitude of the difference decreased over the 2 min testing period, but remained significantly different from zero (Table 1), indicating that mecamylamine inhibited the perceived irritation evoked by nicotine. When capsaicin was applied bilaterally following unilateral application of mecamylamine, nine subjects chose the untreated side as yielding a stronger sensation while 11 chose the mecamylamine-pretreated side (binomial, P . 0.8). This ratio remained close to 50% and non-significant throughout the 2 min testing period (Table 2). The mean difference rating between the two sides of the tongue at t = 30 s was 0.25, which was not significantly different from zero (t-test, P = 0.67). The mean ratings remained low and not significantly different from zero across the 2 min testing period (Table 2), indicating that mecamylamine did not reduce irritation evoked by capsaicin. In addition, 50% of the subjects changed their choice from one side of the tongue to the other at least once during the 2 min testing period, providing additional evidence that mecamylamine did not affect the perceived intensity of capsaicin irritation. The 2-AFC method used in the present study has the advantage of being very sensitive and hence able to detect small sensory differences [3,11]. However, it is possible that subjects may have developed a bias in choice preference due to the stronger taste of mecamylamine, which is slightly bitter/sour, compared to water. As noted above, subjects chose both sides of the tongue in equal proportions when capsaicin followed mecamylamine, arguing against this possibility. Nonetheless, a third control session was added in which 400 mM NaCl, instead of mecamylamine, was applied followed by bilateral application of 1 ppm capsaicin. The 400 mM NaCl solution was chosen to approximately match the mecamylamine in taste intensity. Approximately half of the subjects chose the untreated side as yielding a stronger sensation while the other half chose the NaCl-pretreated side, and the mean rating for intensity difference was close to zero, suggesting once again an absence of a choice preference artifact. The present results confirm, using a different method, an earlier study [8] showing a reduction in nicotine-evoked irritation by the neuronal nAchR antagonist, mecamyla-
60
90
9/20
P . 0.8 0.4 P = 0.43
11/20
P = 0.8 0 P=1
120 10/20
P . 0.8 −0.5 P = 0.35
mine. Furthermore, they also indicate that mecamylamine is selective for nicotinic receptors in that it did not reduce the perceived irritation elicited by capsaicin when placed on the same area of the tongue. These psychophysical data also confirm older studies in which a variety of nicotinic antagonists selectively prevented nocifensive responses such as blinking elicited by nicotine, but not capsaicin, instilled into the eye in rodents [9]. Although we were presently unable to demonstrate selective antagonism of capsaicin but not nicotine-evoked irritation due to the absence of a reliable capsaicin receptor antagonist for human studies, the present results nonetheless provide additional support for the idea that nicotine and capsaicin evoke irritation by acting at separate molecular receptors on polymodal nociceptors innervating the oral cavity. The present experimental protocol imposed a 10-min rest period between application of mecamylamine and subsequent application of nicotine or capsaicin. This was chosen in light of an earlier study [8] in which nicotine-evoked irritation was found to be reduced only when the nicotine was applied at least 20 min after the initial application of mecamylamine. In pilot experiments we found that irritation was not affected when nicotine was applied immediately following mecamylamine application, and that a 10-min waiting period was sufficient for the inhibitory effect of mecamylamine to be expressed. Furthermore, 12 subjects (of the 20 in this study) were additionally tested using a 5 min rest period between applications of mecamylamine and nicotine; while most exhibited an inhibition of the nicotine irritation, three did not exhibit mecamylamine antagonism of nicotine-evoked irritation with the 5-min rest period although they did with the 10-min rest. This indicates that a minimum and variable amount of time is necessary for the antagonism to take effect. This research was supported by a grant from Philip Morris Inc., USA. [1] Carstens, E., Kuenzler, N., Handwerker, H.O. and Activation of neurons in rat dorsomedial trigeminal, N., Caudalis (Vc) by application of a spectrum of irritant chemicals to the tongue, Soc. Neurosci. Abstr., 22 (1996) 863. [2] Dessirier, J.-M., O’Mahony, M. and Carstens, E., Oral irritant effects of nicotine: psychophysical evidence for decreased sensation following repeated application and lack of cross-desensitization to capsaicin, Chem. Senses, 22 (1997) 483–492.
68
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[3] Ennis, D.M., The power of sensory discrimination methods, J. Sens. Stud., 8 (1993) 353–370. [4] Green, D.M. and Swets, J.A., Signal Detection Theory and Psychophysics, Wiley, New York, 1966, pp. 43–45. [5] Greiff, L., Wollmer, P., Andersson, M., Pipkorn, U. and Persson, C.G.A., Effects of nicotine on the human nasal mucosa, Thorax, 48 (1993) 651–655. [6] Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 49 (1991) 143–201. [7] Hummel, T., Livermore, A., Hummel, C. and Kobal, G., Chemosensory event-related potentials in man: relation to olfactory and painful sensations elicited by nicotine, Electroenceph. clin. Neurophysiol., 84 (1992) 192–195. [8] Jarvik, M.E. and Assil, K.M., Mecamylamine blocks the burning sensation of nicotine on the tongue, Chem. Senses, 13 (1988) 213–217. [9] Jansco, N., Jansco-Gabor, A. and Takats, I., Pain and inflammation induced by nicotine, acetylcholine and structurally related compounds and their prevention by desensitizing agents, Acta Physiol., 19 (1961) 113–132. [10] Keele, C.A. and Armstrong, D., Substances Producing Pain and Itch, Edward Arnold, London, 1964. [11] Kim, K.O. and O’Mahony, M., A new approach to category scales of intensity II. Use of d′ values, J. Sens. Stud., (1997), submitted.
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