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Evidence that removal of capsaicin accelerates desensitization on the tongue
44
Neuroscience Letters, 150 (1993)44 A,8 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93l$ 06.00
NSL 09261
Evidence that removal of capsaicin accelerates desensitization on the tongue Barry G. Green Monell Chemical Senses Center, Philadelphia, PA 19104-3308 ( USA ) (Received 22 June 1992; Revised version received 19 October 1992; Accepted 19 October 1992) Key words:
Capsaicin; Desensitization; Sensitization; Zingerone; Tongue; Human; Psychophysics
It has previously been shown that repeated presentations of a moderate concentration of capsaicin could either sensitize or desensitize the tongue, depending upon the temporal pattern of stimulation. Only when stimulation was halted for 5 min or longer did sensitization begin to give way to desensitization. The apparent necessity of a hiatus in stimulation led to the hypothesis that desensitization involved an inability to re-excite quiescent fibers rather than a progressive suppression of stimulated fibers. This hypothesis was tested in the present study by measuring the perceptual response to a non-desensitizing irritant, zingerone, when it was presented at the rate ofl/min following a series ofcapsaicin conditioning stimuli. After an initial, brief period ofcross-hyper-sensitization, cross-desensitization developed at a rate equivalent to that observed when no stimulus was presented. Thus, the results disproved the hypothesis that desensitization cannot occur if capsaicin-sensitive fibers continue to be stimulated, and suggested that after an initial period of excitation, the removal of capsaicin accelerates the desensitization process. This seeming paradox is discussed in terms of the neural mechanisms that may underlie capsaicin desensitization.
It was recently reported that a small area of the tongue could be desensitized after application of as few as five capsaicin stimuli (3-30 ppm) within a 5-min period, provided no additional capsaicin was applied for at least 2.5-5.0 rain [7, 8]. Since those reports, the ability to desensitize a limited area of the tongue within minutes using a relatively low concentration ofcapsaicin has been confirmed in another laboratory [13]. However, no other studies have addressed the most perplexing aspect of the induction of lingual short-term desensitization, namely, the apparent necessity of a hiatus in stimulation. Nor has the possible influence of the temporal pattern of stimulation been systematically studied in any of the numerous biochemical, pharmacological and physiological studies of desensitization (see refs. 4, 5, 11, and 16 for recent reviews). The most parsimonious hypothesis as to why lingual desensitization failed to occur while stimulation continued (for up to 25 min) was that exposure to capsaicin could not block ongoing neural activity, but could prevent re-excitation of quiescent fibers. It was therefore hypothesized that the interruption simply allowed activity in capsaicin-sensitive (CS) fibers to fall below some Correspondence: B.G. Green, Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308, USA.
threshold level, after which they could no longer be stimulated. Although a direct test of this hypothesis would require study of individual CS fibers under appropriate conditions of capsaicin stimulation, a psychophysical experiment was designed that could determine, indirectly, whether rapid desensitization would develop while CS fibers were being stimulated. The key to the experiment was finding a chemical irritant that would stimulate, but not desensitize, CS fibers. Such a chemical could be presented after a series of capsaicin treatment stimuli to see if desensitization would ensue. If CS fibers must be quiescent for desensitization to occur, desensitization would be delayed as long as the second chemical was applied; otherwise, perceived irritation would begin to decline soon after capsaicin stimulation was terminated [7]. Based upon a behavioral study in the rat, zingerone, a pyrolytic breakdown product of ginger oleoresin [6], seemed to possess the necessary characteristics: it produced little or no self-desensitization but exhibited nearly complete cross-desensitization by capsaicin [20]. To see if zingerone was also ineffective as a desensitizing agent in humans, I tested it in the same paradigm used previously to demonstrate rapid capsaicin desensitization on the tongue [7]. A single zingerone test stimulus (1%/wt) having approximately the same perceptual impact as 10 ppm
45 capsaicin was followed by a series of 10 more zingerone conditioning stimuli (also 1%) spaced at an inter-stimulus interval (ISI) of 30 s; 15 min after the last conditioning stimulus, the test stimulus was presented again. No desensitization was found; the level of irritation reported during the zingerone post-test was 8% higher than that reported during the pretest. In contrast, when 10 ppm capsaicin was tested in the same manner on the same subjects, the post-test sensation was only 25% as strong as the pre-test sensation. Zingerone was therefore included in an experiment that had three conditions: Condition 1 was designed to assess the degree of cross-desensitization between capsaicin and zingerone. First, a 1% zingerone solution was presented via a 0.28 cm 2 filter paper disk to the tip of the tongue for 30 s. Thirty seconds later a series of ten 10ppm capsaicin stimuli was delivered to the same area of tongue. These stimuli, which were applied via 1.27 cm 2 filter paper disks, were also 30 s in duration and were separated by 30-s ISis. Ten min after the last capsaicin stimulus another 0.28 cm 2 zingerone stimulus was applied to assess cross-desensitization. Condition 2 was an attempt to replicate the earlier finding that continuing capsaicin treatment for 20 min or longer would not lead to measurable desensitization. An initial zingerone stimulus was followed by 20 rather than 10 capsaicin conditioning stimuli, and the second zingerone test stimulus was presented 30 sec, rather than 10 min, after the 20th capsaicin stimulus. Condition 3 provided a test of the main hypothesis. The initial zingerone stimulus was followed by 10 capsaicin stimuli as in Condition 1, but after the final capsaicin stimulus, 10 zingerone stimuli were applied at the same ISI on 1.27-cm 2 disks. The zingerone series was followed after 30 s by a final, 0.28-cm 2 zingerone test stimulus. The capsaicin and zingerone stimuli were prepared in a vehicle of 80% ethanol and 20% deionized water (dH20), then pipetted onto the filter paper in quantities of 6/11 (0.28 cm 2 disks) or 27.5/,tl (1.27 cm 2 disks). The papers were allowed to dry to eliminate ethanol as a confounding irritant, then wetted with dH20 immediately before application to the tongue. Reports of sensation intensity were obtained using the method of numerical magnitude estimation (modulus-free). The numerical responses were normalized across conditions and subjects by dividing each subject's mean response to the ten capsaicin conditioning stimuli in each session into an arbitrary value (20), then multiplying the raw magnitude estimates for both capsaicin and zingerone by the quotient. This practice reduced the variance produced by differences in the range of numbers chosen by individuals without affecting the ratios among ratings. The results for the three conditions are shown in Fig.
1. It can be seen in the top of the figure that when tested after a 10-min hiatus, the perceptual response to 1% zingerone was significantly reduced (t 7 = 17.7, P < 0.001) following exposure to 10-ppm capsaicin, i.e., cross-desensitization occurred. This confirms in humans what had been reported previously in rats [20]. The middle figure demonstrates again [7] that rather than leading to rapid desensitization, repeatedly stimulating the tongue with a moderate concentration of capsaicin at brief ISis tends to increase the sensory response in a manner consistent with sensitization. Furthermore, the application of zingerone 30 sec after the last capsaicin stimulus produced a slight (but statistically insignificant) increase in perceived irritation, which was suggestive of cross-sensitization between the two chemicals. The bottom graph in Fig. 1 clearly shows that rapid desensitization occurred despite the continuing presence of a non-desensitizing irritant which, as evidenced by the occurrence of both cross-sensitization and cross-desensitization, strongly stimulates CS fibers [first zingerone test vs. second zingerone test, t 7 = 3.37, P = 0.012). The development of desensitization within 10 min after the final capsaicin stimulus is all the more striking in view of the unexpected occurrence of hyper-sensitization between capsaicin and zingerone. The perceived intensity of irritation produced by the first 1.27-cm 2 zingerone stimulus was an average of 1.9 times more intense than the irritation produced by the last capsaicin stimulus (87 -- 3 . 0 0 , P = 0.02). Thus, the application of zingerone to the capsaicin-treated area confirmed capsaicin's ability to sensitize as well as to desensitize the tongue, and disproved the hypothesis that rapid desensitization could occur only if CS fibers were allowed to become quiescent. It is notable that the variance, as indicated by the standard error of the mean (SEM), increased when capsaicin stimulation continued beyond 10 min in Condition 2, and after switching to zingerone in Condition 3. In the former instance the variance reflects individual differences in the rate of sensitization, with most subjects continuing to sensitize at consistent rates throughout stimulation whereas others either reached a plateau or even exhibited a tendency toward desensitization. Differences in the time-course of the perceptual response during continuing capsaicin stimulation have also been reported for whole-mouth stimulation [19]. In the bottom of Fig. 1 the variance primarily reflects individual differences in the amount of cross-sensitization between capsaicin and zingerone. However, for every subject tested the first response to zingerone was higher than the last response to capsaicin. The hyper-sensitization to zingerone after exposure to capsaicin raised the question whether the same phenomenon would occur in the opposite direction, i.e., when cap-
46
s&in follows repeated exposures toqingerone. A group of ten subjects was subsequently testi in the manner of Condition 2. except that ten 1% zingerone stimuli were presented prior to ten IO-ppm capaaiein stimuli. all applied via 1.27-cm’ disks. The results of this second experiment are shown m Fig. 2. The abrupt increase in perceived-irritation after switching from zingerone to capsajein indicates that cross-sensitization occurred. The appearance of crosssensitization from zingerone to capsaicin is particularly notable in view of the absence OFsign&ant self-sensitization during the ten exposures to zingerone. irritation continued to grow over the first severa] presentations of capsaicin. although as the increasingly large S.E.M.s indicate. the rate of sensitizatioo varied widely across subjects. This variation is consistent with what occurred in Exp. 1 after more than IO mm of exposure to capsaicin (Fig. 1, middle graph ). The results of these two experiments provide both new insights and additional questions about capsaicin sensitization and desensitization. The occurrence of desensitization while CS fibers are being stimuIated by zingerone shows that although capsaicin triggers the desensitization process. its continuing presence can also impede it. Thus the capsaicin-initiated excitatory and desensitizing processes appear to act competitively. The notion that capsaicin’s excifatory and inhibitory elects may involve separate proccsscs is not new. It has long been known that a chemical’s ability to desensitize is not always related to its excitatory potency [20]. and it was recently shown in rats that systemic administration of Ruthenium red can attenuate the noo-specific dcseositiLing effect of capsaicin without blocking its excitatory effect [ 11.The most widely supported hypothesis as to the mechanism of non-specific desensitization is that high concentrations of capsaicin cause an excessive intlux of calcium ions that eventually disrupts ccl1 function 13. Il. 171.The present results therefore suggest that capsaicin’s excitatory effect on cutaneous nerve fibers either delays or counteracts this influx in v.way that zingerone’s excitatory effect cannot. Zingerone’s inability to self-sensitize raises the possibility that the sensitization process may play a key role in delaying desensitization. This possibility could be tested by surveying other chemicals to see if only those that produce sensitization also impede desensitization. I tested one such chemical. NaCI. in an informal experiment that yielded negative results. Even though NaCl irritation grows with repeated exposures [9]. the irritation produced on the tip of the tongue by a 5-M concentration was greatest immediately after capsaicin exposure and declined thereafter in a manner similar to what had occurred with zingerone. This single negative finding does not. of course, rule out the possibility
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Fig. I. Shown are the mean ratings of the perceived intensity of irritation as fun&on oftime of exposure for the three conditions of the first experiment. Top: the irritation produced by zingerone is significantly reduced after IO capsaicin stimuli followed by a IO-min hiatus in stimulation (cross-desensitization). Middle: capsaicin irritation continues to grow over 20 min and the sensation produced by zingerone is significantly enhanced (cross-sensitization). Note that the irritation produced by the second zingerone was higher than that produced by the last capsaicin despite the much smaller size of the zingerone stimulus. Bottom: the irritation produced by IO zingerone stimuli following 10 capsaicin stimuli of equal size first increases abruptly (hypersensitization) then declines to near zero within IO min (desensitization). The vertical bars on all three graphs denote the standard errors of the means (S.E.M.s) of the normalized data.
that sensitizing agents that are structurally more similar to capsaicin might be able to counteract desensitization. It may be that desensitization is delayed only when sensitization is caused by a particular mechanism, e.g.. by a
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Time [rain] Fig. 2. The perceived intensity of irritation is shown for a series of 10 zingerone treatment stimuli followed by 10 capsaicin stimuli of equal size, and for two zingerone test stimuli. The occurrence of hypersensitization is suggested by (a) the cross-sensitization shown by the first capsaicin stimulus despite the absence of self-sensitization by zingerone, and (b) the tendency toward stronger irritation for the final zingerone stimulus despite its small size relative to the final capsaicin stimulus (seen also in the middle graph in Fig. 1). The increase between the first (small) zingerone test stimulus and the first (larger) zingerone treatment stimulus was likely due to spatial summation. Vertical bars denote S.E.M.s.
change in m e m b r a n e c o n d u c t a n c e rather than by a buildup o f stimulus in the mucosa. In addition to the difference in ability to cause sensitization, other evidence that capsaicin and zingerone have different m o d e s o f stimulation comes f r o m the a p p a r e n t hyper-cross-sensitization that occurred between them. Whereas significant cross-desensitization means that two chemicals stimulate many, and perhaps all, o f the same nerve fibers, symmetrical hyper-sensitization suggests that they do so in part, or entirely, via different molecular receptors. T h a t is, an exaggerated sensory response m a y occur because the second chemical stimulates already excited nerve fibers at additional receptor sites, effectively p r o d u c i n g spatial s u m m a t i o n along the neural membrane. This interpretation is consistent with the idea that desensitization and sensitization owe to specific properties o f the 'capsaicin receptor' rather than to properties o f the 'capsaicin-sensitive n e u r o n ' as a whole. It is i m p o r t a n t to point out that it has been shown elsewhere that rapid capsaicin desensitization (or tachyphylaxis) can be p r o d u c e d using a perfusion technique without a hiatus in stimulation and without first inducing sensitization [2, 11, 18]. Those studies were, however, c o n d u c t e d on atrial tissue rather than on cutaneous sen-
sory fibers, and direct perfusion likely resulted in m u c h higher effective concentrations than were p r o d u c e d in o u r topical studies. A l t h o u g h this raises the possibility that the use o f significantly higher concentrations on the skin might induce desensitization without sensitization, Karrer and Bartoshuk [13] used a capsaicin concentration equivalent to the one used here as well as one 10 times higher and f o u n d that sensitization was m o r e pron o u n c e d for the higher concentration. Clearly, w h a t is needed to determine the mechanistic basis for the psychophysical results are studies o f the effects o f capsaicin concentration and ISI on isolated sensory neurons. In the meantime, the present data provide additional evidence that capsaicin desensitization in the oral mucosa depends heavily u p o n the temporal pattern o f stimulation, and new evidence that this dependency is related to the presence or absence o f capsaicin rather than to the level o f activity in CS fibers. A m o r e detailed understanding o f these factors m a y eventually help to optimize treatment strategies that take advantage o f capsaicin's ability to p r o d u c e local analgesia [4, 10, 14, 15, 21]. The a u t h o r thanks Christine D i M a r i n o for conducting the experiments and carrying out the statistical analyses reported in this paper. The research was supported in part by a grant f r o m the N a t i o n a l Institutes o f Health (DC00249). 1 Amann, R., Donnerer, J., Maggi, C.A., Giuliani, S., DelBianco, E., Weihe, E. and Lembeck, F., Capsaicin desensitization in vivo is inhibited by Ruthenium red, Eur. J. Pharmacol., 186 (1990) 169175. 2 Bernoussi, A. and Rioux, F., Effects of capsaicin desensitization on the stimulatory effects of kinins, prostaglandins, biogenic amines and various drugs in guinea-pig isolated atria, Br. J. Pharmacol., 96 (1989) 563-572. 3 Bevan,S. and Szolcsanyi, J., Sensory neuron-specific actions ofcapsaicin: mechanisms and applications, Trends Pharmacol. Sci., 11 (1990) 330-333. 4 Carter, R.B., Topical capsaicin in the treatment of cutaneous disorders, Drug Dev. Res., 22 (1991) 109-123• 5 Dickenson, A.H., Capsaicin: gaps in our knowledge start to be filled, Trends Neurosci., 14 (1991) 265-266. 6 Govindarajan, V.S., Pungency: the stimuli and their evaluation• In J.C. Boudreau (Ed.), Food Taste Chemistry: ACS Symposium Series 115, American Chemical Society, Washington, DC, 1980, pp. 53-92. 7 Green, B.G., Capsaicin sensitization and desensitization on the tongue produced by brief exposures to a low concentration, Neurosci. Lett., 107 (1989) 173-178. 8 Green, B.G., Temporal characteristics of capsaicin sensitization and desensitization on the tongue, Physiol. Behav., 49 (1991) 501505. 9 Green, B.G. and Gelhard, B., Salt as an oral irritant, Chem. Senses, 14 (1989) 259-271. 10 Hawk, R.J. and Millikan, L.E., Treatment of oral postherpetic neuralgia with topical capsaicin, Int. J. Dermatol., 27 (1988) 336.
48 11 Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143-201. 12 Hoover, D.B., Effects of capsaicin on release of substance P-like immunoreactivity and physiological parameters in isolated perfused guinea-pig heart, Eur. J. Pharmacol., 141 (1987)489~,92. 13 Karrer, T. and Bartoshuk, L., Capsaicin desensitization and recovery on the human tongue, Physiol. Behav., 49 (1991) 757 764. 14 Lacroix, J.S., Buvelot, J.M., Polla, B.S. and Lundberg, J.M., Improvement of symptoms of non-allergic chronic rhinitis by local treatment with capsaicin, Clin. Exp. Allergy, 21 (1991) 595-600. 15 Levy, D.M., Abraham, R.R. and Tomlinson, D.R., Topical capsaicin in the treatment of painful diabetic neuropathy, N. Engl. J. Med., 324 (1991) 776. 16 Maggi, C.A., Capsaicin and primary afferent neurons: from basic science to human therapy, J. Auton. Nerv. Syst., 33 (1991) 1 14. 17 Maggi, C.A. and Pierau, F.-K., Recent advances in research on
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sensory peptides and capsaicin mechanisms. A conference report, Neurosci. Lett., 122 (1991) 199-201. Miyauchi, T., Ishikawa, T., Sugishita, Y., Saito, A. and Gota, K., Involvement of calcitonin gene-related peptide in the positive chronotropic and inotropic effects of piperine and development of cross-tachyphylaxis between piperine and capsaicin in the isolated rat atria, J. Pharmacol. Exp. Ther., 248 (1989) 816-824. Nasrawi, C.W. and Pangborn, R.M,, Temporal effectiveness of mouth-rinsing on capsaicin mouth-burn, Physiol. Behav., 47 (1990) 617~23. Szolcsanyi, J. and Jancso-Gabor, A., Sensory effects of capsaicin congeners. Part II: Importance of chemical structure and pungency in desensitizing activity of capsaicin-type compounds, Arzneim. Forsch., 26 (1976) 33--37. The Capsaicin Study Group, Treatment of painful diabetic neuropathy with topical capsaicin: A multicenter, double-blind, vehicle-controlled study, Arch. Intern. Med., 151 (1991) 2225 2229.
Neuroscience Letters, 150 (1993)44 A,8 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93l$ 06.00
NSL 09261
Evidence that removal of capsaicin accelerates desensitization on the tongue Barry G. Green Monell Chemical Senses Center, Philadelphia, PA 19104-3308 ( USA ) (Received 22 June 1992; Revised version received 19 October 1992; Accepted 19 October 1992) Key words:
Capsaicin; Desensitization; Sensitization; Zingerone; Tongue; Human; Psychophysics
It has previously been shown that repeated presentations of a moderate concentration of capsaicin could either sensitize or desensitize the tongue, depending upon the temporal pattern of stimulation. Only when stimulation was halted for 5 min or longer did sensitization begin to give way to desensitization. The apparent necessity of a hiatus in stimulation led to the hypothesis that desensitization involved an inability to re-excite quiescent fibers rather than a progressive suppression of stimulated fibers. This hypothesis was tested in the present study by measuring the perceptual response to a non-desensitizing irritant, zingerone, when it was presented at the rate ofl/min following a series ofcapsaicin conditioning stimuli. After an initial, brief period ofcross-hyper-sensitization, cross-desensitization developed at a rate equivalent to that observed when no stimulus was presented. Thus, the results disproved the hypothesis that desensitization cannot occur if capsaicin-sensitive fibers continue to be stimulated, and suggested that after an initial period of excitation, the removal of capsaicin accelerates the desensitization process. This seeming paradox is discussed in terms of the neural mechanisms that may underlie capsaicin desensitization.
It was recently reported that a small area of the tongue could be desensitized after application of as few as five capsaicin stimuli (3-30 ppm) within a 5-min period, provided no additional capsaicin was applied for at least 2.5-5.0 rain [7, 8]. Since those reports, the ability to desensitize a limited area of the tongue within minutes using a relatively low concentration ofcapsaicin has been confirmed in another laboratory [13]. However, no other studies have addressed the most perplexing aspect of the induction of lingual short-term desensitization, namely, the apparent necessity of a hiatus in stimulation. Nor has the possible influence of the temporal pattern of stimulation been systematically studied in any of the numerous biochemical, pharmacological and physiological studies of desensitization (see refs. 4, 5, 11, and 16 for recent reviews). The most parsimonious hypothesis as to why lingual desensitization failed to occur while stimulation continued (for up to 25 min) was that exposure to capsaicin could not block ongoing neural activity, but could prevent re-excitation of quiescent fibers. It was therefore hypothesized that the interruption simply allowed activity in capsaicin-sensitive (CS) fibers to fall below some Correspondence: B.G. Green, Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308, USA.
threshold level, after which they could no longer be stimulated. Although a direct test of this hypothesis would require study of individual CS fibers under appropriate conditions of capsaicin stimulation, a psychophysical experiment was designed that could determine, indirectly, whether rapid desensitization would develop while CS fibers were being stimulated. The key to the experiment was finding a chemical irritant that would stimulate, but not desensitize, CS fibers. Such a chemical could be presented after a series of capsaicin treatment stimuli to see if desensitization would ensue. If CS fibers must be quiescent for desensitization to occur, desensitization would be delayed as long as the second chemical was applied; otherwise, perceived irritation would begin to decline soon after capsaicin stimulation was terminated [7]. Based upon a behavioral study in the rat, zingerone, a pyrolytic breakdown product of ginger oleoresin [6], seemed to possess the necessary characteristics: it produced little or no self-desensitization but exhibited nearly complete cross-desensitization by capsaicin [20]. To see if zingerone was also ineffective as a desensitizing agent in humans, I tested it in the same paradigm used previously to demonstrate rapid capsaicin desensitization on the tongue [7]. A single zingerone test stimulus (1%/wt) having approximately the same perceptual impact as 10 ppm
45 capsaicin was followed by a series of 10 more zingerone conditioning stimuli (also 1%) spaced at an inter-stimulus interval (ISI) of 30 s; 15 min after the last conditioning stimulus, the test stimulus was presented again. No desensitization was found; the level of irritation reported during the zingerone post-test was 8% higher than that reported during the pretest. In contrast, when 10 ppm capsaicin was tested in the same manner on the same subjects, the post-test sensation was only 25% as strong as the pre-test sensation. Zingerone was therefore included in an experiment that had three conditions: Condition 1 was designed to assess the degree of cross-desensitization between capsaicin and zingerone. First, a 1% zingerone solution was presented via a 0.28 cm 2 filter paper disk to the tip of the tongue for 30 s. Thirty seconds later a series of ten 10ppm capsaicin stimuli was delivered to the same area of tongue. These stimuli, which were applied via 1.27 cm 2 filter paper disks, were also 30 s in duration and were separated by 30-s ISis. Ten min after the last capsaicin stimulus another 0.28 cm 2 zingerone stimulus was applied to assess cross-desensitization. Condition 2 was an attempt to replicate the earlier finding that continuing capsaicin treatment for 20 min or longer would not lead to measurable desensitization. An initial zingerone stimulus was followed by 20 rather than 10 capsaicin conditioning stimuli, and the second zingerone test stimulus was presented 30 sec, rather than 10 min, after the 20th capsaicin stimulus. Condition 3 provided a test of the main hypothesis. The initial zingerone stimulus was followed by 10 capsaicin stimuli as in Condition 1, but after the final capsaicin stimulus, 10 zingerone stimuli were applied at the same ISI on 1.27-cm 2 disks. The zingerone series was followed after 30 s by a final, 0.28-cm 2 zingerone test stimulus. The capsaicin and zingerone stimuli were prepared in a vehicle of 80% ethanol and 20% deionized water (dH20), then pipetted onto the filter paper in quantities of 6/11 (0.28 cm 2 disks) or 27.5/,tl (1.27 cm 2 disks). The papers were allowed to dry to eliminate ethanol as a confounding irritant, then wetted with dH20 immediately before application to the tongue. Reports of sensation intensity were obtained using the method of numerical magnitude estimation (modulus-free). The numerical responses were normalized across conditions and subjects by dividing each subject's mean response to the ten capsaicin conditioning stimuli in each session into an arbitrary value (20), then multiplying the raw magnitude estimates for both capsaicin and zingerone by the quotient. This practice reduced the variance produced by differences in the range of numbers chosen by individuals without affecting the ratios among ratings. The results for the three conditions are shown in Fig.
1. It can be seen in the top of the figure that when tested after a 10-min hiatus, the perceptual response to 1% zingerone was significantly reduced (t 7 = 17.7, P < 0.001) following exposure to 10-ppm capsaicin, i.e., cross-desensitization occurred. This confirms in humans what had been reported previously in rats [20]. The middle figure demonstrates again [7] that rather than leading to rapid desensitization, repeatedly stimulating the tongue with a moderate concentration of capsaicin at brief ISis tends to increase the sensory response in a manner consistent with sensitization. Furthermore, the application of zingerone 30 sec after the last capsaicin stimulus produced a slight (but statistically insignificant) increase in perceived irritation, which was suggestive of cross-sensitization between the two chemicals. The bottom graph in Fig. 1 clearly shows that rapid desensitization occurred despite the continuing presence of a non-desensitizing irritant which, as evidenced by the occurrence of both cross-sensitization and cross-desensitization, strongly stimulates CS fibers [first zingerone test vs. second zingerone test, t 7 = 3.37, P = 0.012). The development of desensitization within 10 min after the final capsaicin stimulus is all the more striking in view of the unexpected occurrence of hyper-sensitization between capsaicin and zingerone. The perceived intensity of irritation produced by the first 1.27-cm 2 zingerone stimulus was an average of 1.9 times more intense than the irritation produced by the last capsaicin stimulus (87 -- 3 . 0 0 , P = 0.02). Thus, the application of zingerone to the capsaicin-treated area confirmed capsaicin's ability to sensitize as well as to desensitize the tongue, and disproved the hypothesis that rapid desensitization could occur only if CS fibers were allowed to become quiescent. It is notable that the variance, as indicated by the standard error of the mean (SEM), increased when capsaicin stimulation continued beyond 10 min in Condition 2, and after switching to zingerone in Condition 3. In the former instance the variance reflects individual differences in the rate of sensitization, with most subjects continuing to sensitize at consistent rates throughout stimulation whereas others either reached a plateau or even exhibited a tendency toward desensitization. Differences in the time-course of the perceptual response during continuing capsaicin stimulation have also been reported for whole-mouth stimulation [19]. In the bottom of Fig. 1 the variance primarily reflects individual differences in the amount of cross-sensitization between capsaicin and zingerone. However, for every subject tested the first response to zingerone was higher than the last response to capsaicin. The hyper-sensitization to zingerone after exposure to capsaicin raised the question whether the same phenomenon would occur in the opposite direction, i.e., when cap-
46
s&in follows repeated exposures toqingerone. A group of ten subjects was subsequently testi in the manner of Condition 2. except that ten 1% zingerone stimuli were presented prior to ten IO-ppm capaaiein stimuli. all applied via 1.27-cm’ disks. The results of this second experiment are shown m Fig. 2. The abrupt increase in perceived-irritation after switching from zingerone to capsajein indicates that cross-sensitization occurred. The appearance of crosssensitization from zingerone to capsaicin is particularly notable in view of the absence OFsign&ant self-sensitization during the ten exposures to zingerone. irritation continued to grow over the first severa] presentations of capsaicin. although as the increasingly large S.E.M.s indicate. the rate of sensitizatioo varied widely across subjects. This variation is consistent with what occurred in Exp. 1 after more than IO mm of exposure to capsaicin (Fig. 1, middle graph ). The results of these two experiments provide both new insights and additional questions about capsaicin sensitization and desensitization. The occurrence of desensitization while CS fibers are being stimuIated by zingerone shows that although capsaicin triggers the desensitization process. its continuing presence can also impede it. Thus the capsaicin-initiated excitatory and desensitizing processes appear to act competitively. The notion that capsaicin’s excifatory and inhibitory elects may involve separate proccsscs is not new. It has long been known that a chemical’s ability to desensitize is not always related to its excitatory potency [20]. and it was recently shown in rats that systemic administration of Ruthenium red can attenuate the noo-specific dcseositiLing effect of capsaicin without blocking its excitatory effect [ 11.The most widely supported hypothesis as to the mechanism of non-specific desensitization is that high concentrations of capsaicin cause an excessive intlux of calcium ions that eventually disrupts ccl1 function 13. Il. 171.The present results therefore suggest that capsaicin’s excitatory effect on cutaneous nerve fibers either delays or counteracts this influx in v.way that zingerone’s excitatory effect cannot. Zingerone’s inability to self-sensitize raises the possibility that the sensitization process may play a key role in delaying desensitization. This possibility could be tested by surveying other chemicals to see if only those that produce sensitization also impede desensitization. I tested one such chemical. NaCI. in an informal experiment that yielded negative results. Even though NaCl irritation grows with repeated exposures [9]. the irritation produced on the tip of the tongue by a 5-M concentration was greatest immediately after capsaicin exposure and declined thereafter in a manner similar to what had occurred with zingerone. This single negative finding does not. of course, rule out the possibility
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Fig. I. Shown are the mean ratings of the perceived intensity of irritation as fun&on oftime of exposure for the three conditions of the first experiment. Top: the irritation produced by zingerone is significantly reduced after IO capsaicin stimuli followed by a IO-min hiatus in stimulation (cross-desensitization). Middle: capsaicin irritation continues to grow over 20 min and the sensation produced by zingerone is significantly enhanced (cross-sensitization). Note that the irritation produced by the second zingerone was higher than that produced by the last capsaicin despite the much smaller size of the zingerone stimulus. Bottom: the irritation produced by IO zingerone stimuli following 10 capsaicin stimuli of equal size first increases abruptly (hypersensitization) then declines to near zero within IO min (desensitization). The vertical bars on all three graphs denote the standard errors of the means (S.E.M.s) of the normalized data.
that sensitizing agents that are structurally more similar to capsaicin might be able to counteract desensitization. It may be that desensitization is delayed only when sensitization is caused by a particular mechanism, e.g.. by a
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Time [rain] Fig. 2. The perceived intensity of irritation is shown for a series of 10 zingerone treatment stimuli followed by 10 capsaicin stimuli of equal size, and for two zingerone test stimuli. The occurrence of hypersensitization is suggested by (a) the cross-sensitization shown by the first capsaicin stimulus despite the absence of self-sensitization by zingerone, and (b) the tendency toward stronger irritation for the final zingerone stimulus despite its small size relative to the final capsaicin stimulus (seen also in the middle graph in Fig. 1). The increase between the first (small) zingerone test stimulus and the first (larger) zingerone treatment stimulus was likely due to spatial summation. Vertical bars denote S.E.M.s.
change in m e m b r a n e c o n d u c t a n c e rather than by a buildup o f stimulus in the mucosa. In addition to the difference in ability to cause sensitization, other evidence that capsaicin and zingerone have different m o d e s o f stimulation comes f r o m the a p p a r e n t hyper-cross-sensitization that occurred between them. Whereas significant cross-desensitization means that two chemicals stimulate many, and perhaps all, o f the same nerve fibers, symmetrical hyper-sensitization suggests that they do so in part, or entirely, via different molecular receptors. T h a t is, an exaggerated sensory response m a y occur because the second chemical stimulates already excited nerve fibers at additional receptor sites, effectively p r o d u c i n g spatial s u m m a t i o n along the neural membrane. This interpretation is consistent with the idea that desensitization and sensitization owe to specific properties o f the 'capsaicin receptor' rather than to properties o f the 'capsaicin-sensitive n e u r o n ' as a whole. It is i m p o r t a n t to point out that it has been shown elsewhere that rapid capsaicin desensitization (or tachyphylaxis) can be p r o d u c e d using a perfusion technique without a hiatus in stimulation and without first inducing sensitization [2, 11, 18]. Those studies were, however, c o n d u c t e d on atrial tissue rather than on cutaneous sen-
sory fibers, and direct perfusion likely resulted in m u c h higher effective concentrations than were p r o d u c e d in o u r topical studies. A l t h o u g h this raises the possibility that the use o f significantly higher concentrations on the skin might induce desensitization without sensitization, Karrer and Bartoshuk [13] used a capsaicin concentration equivalent to the one used here as well as one 10 times higher and f o u n d that sensitization was m o r e pron o u n c e d for the higher concentration. Clearly, w h a t is needed to determine the mechanistic basis for the psychophysical results are studies o f the effects o f capsaicin concentration and ISI on isolated sensory neurons. In the meantime, the present data provide additional evidence that capsaicin desensitization in the oral mucosa depends heavily u p o n the temporal pattern o f stimulation, and new evidence that this dependency is related to the presence or absence o f capsaicin rather than to the level o f activity in CS fibers. A m o r e detailed understanding o f these factors m a y eventually help to optimize treatment strategies that take advantage o f capsaicin's ability to p r o d u c e local analgesia [4, 10, 14, 15, 21]. The a u t h o r thanks Christine D i M a r i n o for conducting the experiments and carrying out the statistical analyses reported in this paper. The research was supported in part by a grant f r o m the N a t i o n a l Institutes o f Health (DC00249). 1 Amann, R., Donnerer, J., Maggi, C.A., Giuliani, S., DelBianco, E., Weihe, E. and Lembeck, F., Capsaicin desensitization in vivo is inhibited by Ruthenium red, Eur. J. Pharmacol., 186 (1990) 169175. 2 Bernoussi, A. and Rioux, F., Effects of capsaicin desensitization on the stimulatory effects of kinins, prostaglandins, biogenic amines and various drugs in guinea-pig isolated atria, Br. J. Pharmacol., 96 (1989) 563-572. 3 Bevan,S. and Szolcsanyi, J., Sensory neuron-specific actions ofcapsaicin: mechanisms and applications, Trends Pharmacol. Sci., 11 (1990) 330-333. 4 Carter, R.B., Topical capsaicin in the treatment of cutaneous disorders, Drug Dev. Res., 22 (1991) 109-123• 5 Dickenson, A.H., Capsaicin: gaps in our knowledge start to be filled, Trends Neurosci., 14 (1991) 265-266. 6 Govindarajan, V.S., Pungency: the stimuli and their evaluation• In J.C. Boudreau (Ed.), Food Taste Chemistry: ACS Symposium Series 115, American Chemical Society, Washington, DC, 1980, pp. 53-92. 7 Green, B.G., Capsaicin sensitization and desensitization on the tongue produced by brief exposures to a low concentration, Neurosci. Lett., 107 (1989) 173-178. 8 Green, B.G., Temporal characteristics of capsaicin sensitization and desensitization on the tongue, Physiol. Behav., 49 (1991) 501505. 9 Green, B.G. and Gelhard, B., Salt as an oral irritant, Chem. Senses, 14 (1989) 259-271. 10 Hawk, R.J. and Millikan, L.E., Treatment of oral postherpetic neuralgia with topical capsaicin, Int. J. Dermatol., 27 (1988) 336.
48 11 Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143-201. 12 Hoover, D.B., Effects of capsaicin on release of substance P-like immunoreactivity and physiological parameters in isolated perfused guinea-pig heart, Eur. J. Pharmacol., 141 (1987)489~,92. 13 Karrer, T. and Bartoshuk, L., Capsaicin desensitization and recovery on the human tongue, Physiol. Behav., 49 (1991) 757 764. 14 Lacroix, J.S., Buvelot, J.M., Polla, B.S. and Lundberg, J.M., Improvement of symptoms of non-allergic chronic rhinitis by local treatment with capsaicin, Clin. Exp. Allergy, 21 (1991) 595-600. 15 Levy, D.M., Abraham, R.R. and Tomlinson, D.R., Topical capsaicin in the treatment of painful diabetic neuropathy, N. Engl. J. Med., 324 (1991) 776. 16 Maggi, C.A., Capsaicin and primary afferent neurons: from basic science to human therapy, J. Auton. Nerv. Syst., 33 (1991) 1 14. 17 Maggi, C.A. and Pierau, F.-K., Recent advances in research on
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sensory peptides and capsaicin mechanisms. A conference report, Neurosci. Lett., 122 (1991) 199-201. Miyauchi, T., Ishikawa, T., Sugishita, Y., Saito, A. and Gota, K., Involvement of calcitonin gene-related peptide in the positive chronotropic and inotropic effects of piperine and development of cross-tachyphylaxis between piperine and capsaicin in the isolated rat atria, J. Pharmacol. Exp. Ther., 248 (1989) 816-824. Nasrawi, C.W. and Pangborn, R.M,, Temporal effectiveness of mouth-rinsing on capsaicin mouth-burn, Physiol. Behav., 47 (1990) 617~23. Szolcsanyi, J. and Jancso-Gabor, A., Sensory effects of capsaicin congeners. Part II: Importance of chemical structure and pungency in desensitizing activity of capsaicin-type compounds, Arzneim. Forsch., 26 (1976) 33--37. The Capsaicin Study Group, Treatment of painful diabetic neuropathy with topical capsaicin: A multicenter, double-blind, vehicle-controlled study, Arch. Intern. Med., 151 (1991) 2225 2229.