Oral irritant properties of menthol: sensitizing and desensitizing effects of repeated application and cross-desensitization to nicotine

The irritant properties of menthol and its interactions with nicotine were investigated psychophysically in human subjects. In the first experiment, 0.3% L-menthol was applied successively to one side of the tongue 10 times at a 1-min interval (30-s interstimulus interval, ISI), and subjects rated the intensity of the perceived irritation. The intensity of irritation progressively decreased across trials, consistent with desensitization. To test for cross-desensitization of nicotine-evoked irritation by menthol, nicotine (0.6%) was applied to both sides of the tongue simultaneously, 5 min after the conclusion of menthol application. Using both a two-alternative forced choice (2-AFC) paradigm, and also by obtaining independent ratings of the irritant intensity on each side of the tongue, it was found that nicotine-evoked irritation was significantly weaker on the menthol-pretreated side. To control for a possible confounding effect of cooling, nicotine was applied bilaterally only after the cooling sensation of menthol had subsided. Nicotine-induced irritation was still significantly weaker on the menthol-pretreated side, consistent with cross-desensitization of nicotine-evoked irritation by menthol. In a final experiment, menthol was repeatedly applied to one side of the tongue at a shorter (20 s) interval (5-s ISI), and elicited a rapid increase in irritant sensation over the initial trials, consistent with sensitization, followed in subsequent trials by a progressive reduction in irritation (desensitization). After a 5-min rest period, self-desensitization was confirmed. Repeated application of menthol at the same short ISI was then resumed, and resulted in a significant mean increase in irritant intensity consistent with stimulus-induced recovery (SIR).     

Sensitization, desensitization and stimulus-induced recovery of trigeminal neuronal responses to oral capsaicin and nicotine

Repeated application of capsaicin at a 1-min interstimulus interval (ISI) to the tongue induces a progressively increasing irritant sensation (sensitization), followed after a rest period by reduced sensitivity to further capsaicin (desensitization). Sequential reapplication of capsaicin induces irritation that eventually increases to initial levels: stimulus-induced recovery (SIR). In contrast, repeated application of nicotine elicits a declining irritant sensation across trials. To investigate possible neural correlates of these phenomena, we recorded from single units in superficial laminae of the dorsomedial trigeminal subnucleus caudalis (Vc) that responded to noxious thermal (54 degrees C) and chemical (1 M pentanoic acid) stimulation of the tongue of anesthetized rats. We then recorded responses to either capsaicin (330 microM) or nicotine (0.6 M), delivered either once, repeatedly at 1-min ISI, or continually by constant flow. After the initial capsaicin application and a rest period, the capsaicin was reapplied in the identical manner to test for SIR. The mean response of 14 Vc units to sequential application of pentanoic acid did not vary significantly across trials, indicating lack of tachyphylaxis or sensitization. The averaged response of 11 Vc units to repeated capsaicin increased significantly across the first eight trials and then plateaued. Following the rest period, spontaneous firing had returned to the precapsaicin level. With capsaicin reapplication, the averaged response increased again after a significant delay (due to desensitization), but did not reattain the peak firing rate achieved in the initial series (partial SIR). Constant-flow application of capsaicin induced an identical sensitization followed by nearly complete SIR. A single application of capsaicin induced a significant rise in firing in eight other units, but the rate of rise and maximal firing rate were both much lower compared with repetitive or constant-flow capsaicin. When capsaicin was reapplied once after the rest period, there was no change in firing rate indicating absence of SIR. These results indicate that maintenance of the capsaicin concentration induces a progressive increase in neuronal response that parallels sensitization. With recurrent capsaicin application, desensitization can be overcome to result in a delayed recovery of Vc responses similar to SIR. In contrast, the averaged response of 17 Vc units to repeated or constant-flow application of nicotine increased only over the first 3 min, and then decreased to spontaneous levels even as nicotine was still being applied. These results are consistent with the decrease in the perceived irritation elicited by sequential application of nicotine in humans.     

Enhancement of responses to sequential presentation of oral chemical irritants

The irritative flavor compounds, capsaicin and piperine, were presented in sequence to the oral cavity of human subjects and rated for perceived intensity of irritation. Marked increases in perceived irritation were reported when the second-presented irritant differed from the first, as compared with control conditions in which the same irritant was repeated. This cross-enhancement is explained most simply by recruitment of additional receptors or fiber types, and implies a greater degree of specificity (narrowness of tuning) of trigeminal receptors than previously suggested by desensitization studies.     

Capsaicin as a probe of the relationship between bitter taste and chemesthesis

Previous studies have indicated that capsaicin, traditionally considered to be a pure chemesthetic stimulus, can evoke a bitter taste and might also cross-desensitize the tastes of some bitter and sour tastants. The purpose of the present study was to investigate further the scope and nature of capsaicin's effects on bitter taste. In Experiment 1, subjects rated the taste and burning/stinging of QSO4 (0.32 and 1.0 mM), saccharin (1.0 and 3.2 mM), urea (3.2 and 10 M), MgCl2, (0.18 and 0.56 M), PROP (0.32 mM), and sucrose (0.32 and 1.0 M) applied to the tongue tip with cotton swabs before and after 10 applications of 300 microM capsaicin. Capsaicin initially evoked a weak bitterness in some subjects that quickly diminished over repeated exposures. Following capsaicin treatment, the bitterness of QSO4, urea, MgCl2, and PROP was reduced, as was the burning sensation produced by MgCl2 and urea. In Experiment 2, we tested 29 subjects in the circumvallate (CV) region of the tongue using the same general procedure. Capsaicin induced a weak but persistent bitterness in a subset of subjects but failed to desensitize its own bitterness or that of any other tastant. Overall, the results confirm that capsaicin can both stimulate and desensitize bitter taste, but in amounts that vary for different bitter stimuli and between the front and back of the tongue. Possible reasons for these regional and stimulus-dependent differences are discussed.     

Desensitization to Oral Zingerone Irritation: Effects of Stimulus Parameters

In humans, repeated oral stimulation with the irritant capsaicin produces sensitization or desensitization, depending on the temporal relationship and, to a lesser extent, the intensity of the stimuli. We have previously shown that zingerone, an irritant present in ginger, shows only desensitization across repeated samples, as well as following a hiatus in stimulation. Because the time-course of zingerone irritation differs from that of capsaicin, it is likely that optimal temporal and other stimulation parameters may also be different. In Experiment 1, we examined the effects of stimulus intensity (0.5%, 1.0%, 2.0% zingerone) and the number of successive stimuli in a series on psychophysical responses to zingerone irritation within the series and following a 5-min hiatus. Experiment 2 examined the effect of the duration of this hiatus on desensitization and recovery. Desensitization was apparent across the initial series of stimuli in both experiments and, irrespective of zingerone concentration, in Experiment 1. Desensitization also occurred following the 5-min hiatus, evident primarily at the higher concentrations. Preceding the hiatus with 5 or 10 stimuli produced the greatest posthiatus desensitization, but a decrease in rated intensity was also evident following a single stimulus. Experiment 2 showed that the optimal hiatus for demonstrating desensitization was 5 min and that, by 15 min, recovery had begun. In both experiments, individual differences in response were marked, with some subjects showing sensitization and others little change in response across repeated zingerone stimuli. The origin of these differences is unclear but were shown to be relatively stable across multiple sessions.     

The generalizability of capsaicin sensitization and desensitization.

Studies using capsaicin-saturated filter papers have shown that the intensity of oral irritation tends to grow over successive samples, a phenomenon known as sensitization. If a hiatus of 5-15 min is then introduced, the intensity of irritation produced by a subsequent capsaicin stimulus is much reduced, and desensitization is said to have occurred. The use of other methodologies such as whole-mouth rinses, either with capsaicin or the irritants menthol or zingerone, have not consistently shown this response pattern, casting doubt on the extent to which sensitization and desensitization are general phenomena. Experiment 1 addressed this issue by comparing responses to whole-mouth rinses of 0.6 and 3 ppm capsaicin with those to 3 ppm capsaicin filter papers. Over an initial series of 10 samples, sensitization was evident but only for the 3 ppm capsaicin stimuli. Following a 10-min hiatus, desensitization was observed for all stimulus types. An examination of the data of individual subjects revealed considerable variability in response patterns over the initial 10 samples between subjects and, within subjects, between the filter paper and rinse stimuli, and between the test replications. Desensitization to the posthiatus stimuli was more consistent. A second experiment examined whether the capsaicin sensitization and desensitization shown by stimulation with filter papers or solutions also occurred in the context of the consumption of foods containing capsaicin. Two foods--soup and chili con carne--were consumed under conditions in which the rate of consumption was timed, as in Experiment 1, or was self-paced. In neither of the two conditions or foods was there strong evidence of sensitization, although, again, desensitization following a hiatus was evident. Substantial individual variability in response patterns was again apparent. There is thus no evidence for sensitization occurring during normal food consumption.     

Chemesthesis and taste: evidence of independent processing of sensation intensity

The ability to perceive taste from temperature alone ("thermal taste") was recently shown to predict higher perceptual responsiveness to gustatory and olfactory stimuli. This relationship was hypothesized to be due in part to individual differences in CNS processes involved in flavor perception. Here we report three experiments that tested whether subjects who differ in responsiveness to thermal taste and/or chemical taste also differ in responsiveness to oral chemesthesis. In experiment 1, subjects identified as 'thermal tasters' (TTs) or 'thermal non-tasters' (TnTs) used the general Labeled Magnitude Scale to rate the intensity of sensations produced on the tongue tip by capsaicin, menthol, sucrose, NaCl, citric acid, and QSO4. TTs rated all four taste stimuli higher than did TnTs, whereas sensations of burning/stinging/pricking and temperature from capsaicin and menthol did not differ significantly between groups. In experiment 2, testing with capsaicin on both the front and back of the tongue confirmed there was no difference in ratings of burning/stinging/pricking when subjects were grouped according to the ability to perceive thermal taste. In experiment 3, subjects were classified as high- or low-tasters according to their ratings of sucrose sweetness rather than thermal taste. No group difference was found for perception of capsaicin even when presented in mixture with sucrose or NaCl. The results are discussed in the context of previous evidence of an association between chemesthesis and sensitivity to the bitter tastant PROP, and in terms of the various peripheral and central neural processes that may underlie intensity perception in taste and chemesthesis.     

Human psychophysical studies of saltiness suppression by amiloride.

Two studies were performed to assess the effects of amiloride on the saltiness of NaCl. In the first study, four highly trained subjects were asked to report the first appearance of saltiness as NaCl microdrops were rapidly applied to an increasing number of fungiform papillae along the front, dorsal surface of the tongue. For two subjects, amiloride, a sodium channel blocker, caused a large and significant increase in the number of papillae needed to report a salty taste. The other two subjects were unaffected, as measured by this procedure. In the second study, four different subjects were trained in the same procedure as in the previous study. For testing, however, they were stimulated on a fixed number of papillae, which was two times the number self-selected during training as sufficient for perception of saltiness with NaCl, and sourness with citric acid. They reported the quality of the experience as salty, sour, or neither, with and without amiloride treatment of the papillae set. Two of the four subjects had a loss of saltiness of NaCl, but not sourness of citric acid, when the papillae set was treated with amiloride.     

Bretylium tosylate enhances salt taste

Bretylium tosylate (BT), an antifibrillary drug, was found to potentiate the taste of NaCl and LiCl in both humans and rats. Application of 1 mM BT (pH 6.3) to the human tongue statistically potentiated the taste of 0.2 M NaCl and 0.2 M LiCl by 33.5% and 12.5% respectively. Electrophysiological taste responses from nucleus tractus solitarius (NTS) in rat for both hyposmotic and hyperosmotic concentrations of NaCl and 0.1 M LiCl were also increased by 30 to 40% after application of 1 mM BT. This potentiation induced by BT was reduced by amiloride in both humans and rats. Furthermore, amiloride became ineffective in inhibiting taste responses to NaCl in the presence of BT.     

Cannabinoid agonist, CP 55,940, prevents capsaicin-induced sensitization of spinal cord dorsal horn neurons

Low doses of cannabinoids applied intrathecally attenuate capsaicin-evoked heat and mechanical hyperalgesia via CB1 receptors. Although cannabinoids produce antinociception, in part, by attenuating responses of nociceptive neurons in the spinal cord, few studies have examined the effect of cannabinoids on sensitization of spinal neurons. We therefore investigated whether a cannabinoid receptor agonist, CP 55,940, attenuated excitation and sensitization of spinal nociceptive neurons produced by intraplantar injection of 0.1% capsaicin (10 microl). In rats, wide-dynamic-range (WDR) and high-threshold (HT) neurons were classified according to responses evoked by mechanical stimuli of varying intensity. CP 55,940 (10 microg in 50 microl) or vehicle was applied directly to the spinal cord and responses to mechanical (von Frey monofilament) and heat stimuli were recorded 10 min after drug treatment. CP 55,940 alone did not alter responses to mechanical stimuli; however the enhanced responses to mechanical stimuli after injection of capsaicin into the receptive field were dose dependently attenuated in both HT and WDR neurons. Vehicle-treated neurons increased their response to 300.6 +/- 52.1% of baseline after capsaicin, whereas CP 55,940-treated neurons responded at 153.0 +/- 27.1% of baseline. The effects of CP 55,940 on sensitization to heat were less pronounced; however, CP 55,940 attenuated the capsaicin-evoked decrease in heat threshold in HT neurons. The attenuation by CP 55,940 of sensitization to mechanical stimuli was blocked by pretreatment of the spinal cord with the CB1 receptor antagonist, SR141716A. These studies demonstrate that cannabinoid application to the spinal cord prevents central sensitization.     

The addition of glyceryltrinitrate to capsaicin cream reduces the thermal allodynia associated with the application of capsaicin alone in humans

The aim of this study was to determine whether topical application of capsaicin cream causes thermal allodynia and the extent to which this is attenuated by the addition of glyceryltrinitrate (GTN). This was a double blind placebo controlled study of 40 consenting adult subjects. Each of four cream combinations (GTN, capsaicin, GTN/capsaicin and vehicle) were applied to the subjects with at least a 1 day interval between each application. Water at a known temperature was applied to the standard area of skin where cream had been applied. Subjects rated the resulting thermal allodynia using a 0-10 Likhert score. Thermal allodynia is usually apparent when warm water is applied to skin containing capsaicin. The thermal allodynia caused by the topical application of capsaicin was significantly reduced by the addition of GTN. The addition of GTN to capsaicin cream significantly reduces the thermal allodynia associated with the application of capsaicin cream alone.     

Sensitization of the transient receptor potential vanilloid type 1 ion channel by isoflurane or sevoflurane does not result in extracellular signal-regulated kinase 1/2 activation in rat spinal dorsal horn neurons

Clinically relevant concentrations of isoflurane or sevoflurane sensitize transient receptor potential vanilloid type 1 to several of its activators, including capsaicin. It has, moreover, been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to post-operative pain. To investigate this suggestion, we have studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons (which is a recognized marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetized with pentobarbital (which of itself does not activate extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked extracellular signal-regulated kinase 1/2 phosphorylation in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced extracellular signal-regulated kinase 1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipliateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetized animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetized animals reduced extracellular signal-regulated kinase 1/2 phosphorylation induced by the gases alone on both sides. These findings do not support the suggestion that isoflurane-, or sevoflurane-, induced sensitization of transient receptor potential vanilloid type 1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.     

Adaptation to capsaicin burn: effects of concentration and individual differences

Human subjects rated the time course of the burn produced by three concentrations of capsaicin applied to the tongue via filter papers. Data were fit to a dynamic model composed of a level detector, a change detector, and a double integrator. These three processes responded differently to concentration. 6-n-Propyl-2-thiouracil (PROP) taster status correlated positively with the integrator process. Although a minority of subjects showed evidence of the integrator process, any subject with an integrator process at a given concentration also showed it at any higher concentration.     

Mineral intake independent from gastric irritation or pica by cell-dehydrated rats

Gavage of 2 M NaCl (IG 2 M NaCl), a procedure to induce cell-dehydration—and water and 0.15 M NaCl intake in a two-bottle choice test—is also a potential gastric irritant. In this study, we assessed whether mineral intake induced by IG 2 M NaCl is associated with gastric irritation or production of pica in the rat. We first determined the amount of mineral solution (0.15 M NaCl, 0.15 M NaHCO₃, 0.01 M KCl and 0.05 mM CaCl₂) and water ingested in response to IG 2 M NaCl in a five-bottle test. Then, we used mineral solutions (0.01 M KCl and 0.15 M NaHCO₃), whose intakes were significantly increased compared to controls, and water in three-bottle tests to test the gastric irritation hypothesis. The IG 2 M NaCl induced KCl and NaHCO₃ intake that was not inhibited by gavage with gastric protectors Al(OH)₃ or NaHCO₃. IG 2 M NaCl or gavage of 0.6 N acetic acid induced mild irritation, hyperemia, of the glandular part of the stomach. A gavage of 50% ethanol induced strong irritation seen as pinpoint ulcerations. Neither ethanol nor acetic acid induced any fluid intake. Neither IG 2 M NaCl nor acetic acid induced kaolin intake, a marker of pica in laboratory rats. Ethanol did induce kaolin intake. These results suggest that IG 2 M NaCl induced a mineral fluid intake not selective for sodium and independent from gastric irritation or pica.     

Capsaicin sensitization and desensitization on the tongue produced by brief exposures to a low concentration

The intensity of sensations of burning and stinging produced by repeated exposures to capsaicin (at a nominal concentration of 3 ppm) was measured on a localized area of the tongue as a function of both the number of exposures and the time between them. It was discovered in the initial experiment that stimulation at the rate of 1/min (for up to 25 min) resulted in a monotonic increase in the intensity of burning sensations in a manner consistent with the phenomenon of sensitization. However, the insertion of a 15-min delay in stimulation resulted in a reduction in the intensity of the sensations produced by further stimulation, i.e., desensitization occurred. Desensitization was statistically significant even after exposure to as few as 5 stimuli prior to the delay. A subsequent experiment established that the minimum delay necessary to produce desensitization was between 2.5 and 5 min. Hence, paradoxically, the sensory response to capsaicin on the tongue increased as stimulation continued, then decreased after stimulation had ceased.     

Magnitude estimation of adaptation to salt using a flow chamber for stimulus delivery

Adaptation course to NaCl was estimated using a flow chamber for stimulus delivery and changes in the perceived taste magnitude as the criterion. The adapting stimulus of different durations was applied to one side of the tongue. Magnitude estimates were made by comparing the intensity of taste at the end of each adaptation time with the perceived intensity of the same stimulus applied briefly to the unadapted side of the tongue. The course of adaptation followed a negatively accelerated decreasing function. The relationship between taste magnitude and the duration of the adapting stimulus can be approximated by a logarithmic equation and, somewhat less accurately, also by an exponential equation, which can be related to the Beidler theory of taste stimulation. Some advantages of the closed flow technique and comparison of taste magnitudes on the adapted and unadapted sides of the tongue are discussed.     

Lack of amiloride sensitivity in SHR and WKY glossopharyngeal taste responses to NaCl.

To explore possible functional strain differences in taste receptors located on the posterior tongue, we recorded electrophysiological taste responses from the glossopharyngeal nerve of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Multifiber responses to a concentration series (0.5 M to 2.0 M) of NaCl, KCl and NH4Cl were recorded before and after lingual application of the epithelial sodium transport blocker, amiloride. Responses to a concentration series (0.0025 M to 0.1 M) of quinine hydrochloride were also recorded. When expressed relative to the 0.5-M NH4Cl response, responses to the monochloride salts were equivalent between SHR and WKY. Surprisingly, NaCl responses were not suppressed by the sodium transport blocker, amiloride. This is in direct contrast to the dramatic suppression observed in the chorda tympani. Also, relative responses to quinine were greater in the glossopharyngeal nerve of SHR than WKY. These results indicate that taste receptors innervated by the glossopharyngeal nerve lack amiloride sensitivity and that posterior taste receptor function to monochloride salts is equivalent between SHR and WKY.     

Temporal gustatory and salivary responses to capsaicin upon repeated stimulation

Repeated oral stimulation with solutions of 2 ppm capsaicin increased time to maximum intensity, but yielded a decrease in sequential mouth-burn intensity due to a rising minimum irritation rating between stimuli. Two response patterns were observed: for 18 subjects mouth-burn increased, while 14 subjects showed depression upon repeated stimulation. Because of across-subject variability, there was little correlation between parotid salivary response and perceived sensory responses. Capsaicin-stimulated salivary flow was significantly higher than flow stimulated by distilled water in 9 high-flow subjects, but not in 14 low-flow subjects. Oral adaptation to capsaicin may attenuate parotid salivary response, although eaters and noneaters of chili peppers did not differ significantly in their perception of mouth-burn or in salivary flow.     

The effect of amiloride analogs on taste responses in gerbil

Amiloride analogs that were designed to inhibit three types of Na+ transport systems (the epithelial Na+ channel, the Na+/H+ antiporter, and the Na+/Ca++ exchanger) were applied to the tongue of the gerbil to determine their effects of electrophysiological taste responses to NaCl, CaCl2, sucrose, and glutamic acid. The pattern of responses from the chorda tympani nerve indicates that the taste of NaCl is almost totally accounted for by the epithelial Na+ channel. Phenamil, an amiloride analog which specifically blocks the epithelial Na+ channel at low concentrations, suppressed the taste responses to 0.03 M NaCl by 97%. The pattern of responses also indicates that the Na+/H+ antiporter and the Na+/Ca2+ exchanger do not mediate salt taste in the gerbil. None of the amiloride analogs blocked taste responses to CaCl2, sucrose, or glutamic acid. It is concluded that the salty taste of NaCl in the gerbil is almost totally mediated by the epithelial Na+ channel, and the kinetics of this channel are identical to amiloride-sensitive sodium channels in other systems.