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Oral irritation by sodium chloride: sensitization, self-desensitization, and cross-sensitization to capsaicin
Physiology & Behavior 72 (2001) 317 ± 324
Oral irritation by sodium chloride: sensitization, self-desensitization, and cross-sensitization to capsaicin J.-M. Dessiriera,b, M. O'Mahonyb, M. Iodi-Carstensa, E. Yaob, E. Carstensa,* a
Section of Neurobiology, Physiology, and Behavior, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA b Department of Food Science and Technology, University of California at Davis, Davis, CA, USA Received 12 May 2000; received in revised form 27 July 2000; accepted 6 September 2000
Abstract Psychophysical methods were used to investigate the irritant sensory properties of concentrated NaCl. The first experiment investigated potential sensitization and desensitization properties. Subjects rated the intensity of the irritation elicited by 10 successive applications of 5 M NaCl on one side of the dorsal surface of the tongue. The mean irritant sensation increased significantly across trials, consistent with sensitization. To test for self- and cross-desensitization effects of unilateral sequential stimulation with NaCl followed by a 10-min rest period, either 5 M NaCl or 10 mM capsaicin was applied bilaterally. In a two-alternative forced-choice (2-AFC) test, subjects indicated which side of the tongue had a stronger irritant sensation. They also rated the intensity of irritation on each side separately. When NaCl was applied bilaterally, the side not previously receiving NaCl was chosen as stronger by a significant majority of subjects and was given significantly higher intensity ratings, consistent with self-desensitization. In contrast, when capsaicin was applied bilaterally, the side that had previously received sequential NaCl was perceived as having a significantly more intense irritation, consistent with cross-sensitization. In a second experiment, the effect of amiloride on NaCl-evoked irritation was studied. One side of the tongue was treated with 1 mM amiloride, after which 5 M NaCl was applied bilaterally and subjects performed the same 2-AFC and rating procedures. Since amiloride significantly reduced the intensity of the irritant sensation, the contribution of amiloride-sensitive ionic currents or the Na + /H + exchange pump (NHE) are suggested as possible transduction mechanisms in lingual nociceptors mediating NaCl-evoked oral irritation. D 2001 Elsevier Science Inc. All rights reserved. Keywords: NaCl; Capsaicin; Vanilloid; Oral irritation; Trigeminal; Desensitization; Psychophysics; 2-AFC
1. Introduction In addition to oral gustatory sensations, monovalent salts, such as NaCl or KCl, can stimulate the chemesthetic system to elicit sensations of irritation and pain when applied to oral mucosa [1] or skin [2]. Salts activate rat lingual afferents [3] including identified nociceptors [4], inner dentine afferents [5], and corneal polymodal nociceptors [6,7]. Intraoral or ocular application of NaCl was shown to excite nociceptive neurons in the trigeminal subnucleus caudalis (Vc) of rats in a concentration-related manner [8]. Hypertonic saline also induces abdominal constrictions when injected intraperitoneally [9] and increases luminal gastric mucosal blood flow in the stomach [10]. * Corresponding author. Tel.: +1-530-752-6640; fax: +1-530-7525582. E-mail address: [email protected] (E. Carstens).
The mechanism by which salts activate nociceptors is uncertain. Hypertonic saline in the airways induces coughing that is not reduced by the vanilloid receptor antagonist, capsazepine [11], and also evokes the release of calcitonin gene-related peptide (CGRP) from rat urinary bladder in a manner that is not blocked by another vanilloid antagonist, ruthenium red [12]. These results indicate that vanilloid receptors are not involved in salt-induced irritation, in contrast to acid-evoked irritation and pain for which vanilloid receptors do appear to play a role (see Refs. [13 ± 15]). However, salt-induced irritation and nociception are reduced by treatment with capsaicin [4,10,16,17], the pungent principle in capsicum fruits that is well known to desensitize polymodal nociceptors (e.g., Refs. [18,19]). This indicates that irritation and pain elicited by salts is due at least partly to excitation of capsaicin-sensitive nociceptors, although not directly via vanilloid receptors. NaCl given sequentially at a short interstimulus interval (ISI) induces a progressive rise in the intensity of
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irritation, called sensitization [1]. Sensitization has also been observed with capsaicin [20 ± 22], piperine [23,24], and citric acid [15]. Other irritant chemicals, such as menthol [25,26], zingerone [27,28], or nicotine [23,29], do not exhibit such a sensitization, but rather a reduction in the intensity of irritation (self-desensitization) across trials. However, if a rest period of 5 min or more is imposed following the last stimulus presentation (i.e., longer ISI imposed), subsequent application of each chemical tested to date, including those exhibiting sensitization, elicits a significantly reduced sensory intensity, also called self-desensitization. Furthermore, irritant chemicals that exhibit sensitization Ð capsaicin and piperine Ð also induce desensitization to irritation elicited by to other compounds, a phenomenon called cross-desensitization. Since it is currently unknown whether NaCl at concentrations eliciting irritation also induces self- or cross-desensitization, we have presently undertaken experiments to address this question. The first aim of this study (Experiment I) was, therefore, to reproduce Green and Gelhard's [1] finding of NaCl sensitization using a higher, 5 M concentration. In addition, we investigated whether NaCl exhibits properties of selfdesensitization and cross-desensitization to capsaicin. The second aim of this study (Experiment II) was to investigate the involvement of amiloride-sensitive ion channels in NaCl-induced irritation. 2. Materials and methods 2.1. Experiment I 2.1.1. Subjects Twenty-five healthy individuals (7 males, 18 females, age 18 ±27 years), who were students and staff at the University of California at Davis, volunteered to participate in the study. All refrained from eating or drinking at least 1 h prior to each experimental session, and were asked not to eat spicy food for 2 days prior to testing as verified by questionnaire. 2.1.2. Chemical stimuli and application procedure All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified. A 0.1% w/v capsaicin stock solution (98 ± 100% pure) was made up in 95% ethanol and a 3-ppm (10 mM) capsaicin solution was made by diluting the stock solution with dH2O. Fifteen microliters of this solution were pipetted on to small (78.5 mm2) and 40 ml on to large (176.7 mm2) filter paper disks (Whatmann, Maidstone, UK). To avoid any stimulating effect of ethanol, the filter papers were air-dried and then soaked with the same volumes of distilled water immediately prior to application. NaCl was dissolved in distilled water to a concentration of 5 M and was pipetted in the same volumes onto the small or large filter papers just prior to application.
This concentration of NaCl was chosen because pilot studies had determined that it induced an irritant sensation similar to that elicited by 3-ppm capsaicin. Throughout the experiment, a suction device (Saliva Ejector, 6-in. clear, Sullivan Dental Products, Sacramento, CA) was placed in the mouth to remove saliva and avoided spread of the chemicals. Subjects were instructed to use the suction device freely, except for the 15-s period prior to giving intensity ratings or two-alternative forced-choice (2AFC) responses so as to avoid a cooling effect. 2.1.3. Experimental design 2.1.3.1. Matching intensities evoked by capsaicin and NaCl. All subjects participated in an initial session to verify that the concentration of capsaicin yielded an intensity of irritation approximately equivalent to that of NaCl. One large filter paper disk soaked with 40 ml of the 3-ppm capsaicin solution was placed on to one side of the dorsal anterior surface of the tongue and another, soaked with 40 ml of the 5-M NaCl solution, was simultaneously placed onto the other side. The sides receiving capsaicin or NaCl were counterbalanced across subjects. After 15 s, subjects performed a 2-AFC test [30] by reporting which side of the tongue had a stronger sensation. Subjects then separately rated the intensity of irritation on each side using a category scale with the end points 0 (no sensation) and 10 (intense irritation). To assess changes in intensity over time, 2-AFC and rating tasks were repeated at 30-s intervals for a total period of 3 min during which the filter papers remained on the tongue. 2.1.3.2. Sequential stimulation and desensitization. After verifying an approximate match in intensity for capsaicin and NaCl, two additional experimental sessions followed, each separated by at least 2 days. Each session consisted of two parts. In the first part, the larger size filter paper disks containing 40 ml of 5 M NaCl were applied with forceps to one side of the anterior dorsal surface of the tongue at 1-min ISI as in previous studies [23,29]. The filter paper was applied for 30 s and then removed. Thirty seconds later, a fresh filter paper containing the same NaCl stimulus was applied in the same manner; this was repeated 10 times. For each application, subjects rated the perceived intensity of irritation 15 s after stimulus onset. Ratings were made using a bipolar category scale used in a previous study (see Ref. [23]). Subjects were asked to focus on, and rate, only the irritant (biting/ pricking/stinging/burning) quality and to ignore gustatory sensory qualities (saltiness, sourness, bitterness). After the 10 unilateral sequential stimuli, the subjects rested for 10 min quietly without speaking. Then two smallsized filter papers containing either the same NaCl (selfdesensitization) or capsaicin 3 ppm (cross-desensitization) were applied simultaneously with two forceps at corresponding sites on the side that previously received NaCl
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as well as on the opposite side. Subjects then performed the 2-AFC test and intensity ratings as described below. 2.1.4. Rating procedures 2.1.4.1. Bipolar scale. Subjects rated the intensity of successively evoked sensations using a bipolar category scale that was specifically designed to accurately measure changes in perceived intensity across trials (see Fig. 1 of Ref. [23]). The method was virtually identical to that used in our previous studies (e.g., Ref. [23]) and is only briefly described. On a single sheet of paper is printed a series of 10 identical scales, each having the category `1' in the center, and categories ranging from 2 to 15 toward the right and left of the center. The sensation elicited by the first stimulus is obligatorily assigned a rating of `1'. The sensation elicited by the second stimulus is assigned a higher number toward the right if the sensation is stronger, or a higher number toward the left if the sensation is weaker, than the initial rating. Subsequent ratings are made relative to the first stimulus in this manner, for all 10 stimuli. The subject has access to prior ratings to reduce memory errors. To avoid `cut-off' effects, extensions with categories raging from 16 to 30, and additional unnumbered categories if required, are allowed at either end of the scale. 2.1.4.2. 2-AFC. This method is identical to that employed in our earlier studies [23,29]. Thirty seconds after bilateral application of capsaicin or 15 s after NaCl, subjects were asked to choose which side of the tongue gave rise to a stronger sensation (2-AFC, Ref. [30]). The rating times were selected because pilot tests suggested that NaCl-evoked irritation grew more rapidly and was shorter-lived than that elicited by capsaicin. The use of the 2-AFC ensured sufficient sensitivity to detect small and confusable differences between the two sides of the tongue, and also enabled d 0 values to be computed as a measure of difference magnitude. However, this method is inappropriate for perfectly discriminable differences as d 0 tends toward infinity. Accordingly, subjects gave ratings of the intensity of perceived irritation on each side of the tongue separately, using the unipolar category scale described earlier. As before, 2AFC and rating tasks were performed at 30-s ISI for a total of 3 min. 2.1.5. Statistical analysis The sequential stimulation data were analyzed using analysis of variance (ANOVA) and Fisher's Least Significant Difference (LSD) post hoc tests. To perform the ANOVA, the ratings were first transformed by allocating a positive sign to category ratings on the right side of the scale (stronger sensations) and a negative sign to those on the left side of the scale (weaker sensations). Then, the scale was renumbered by restoring zero to the center of the scale [23]. A d 0 analysis [31,32] was also performed deriving a group d 0 value from the proportion of subjects
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exhibiting sensitization from the first trial to the tenth in both experimental sessions. The self- and cross-desensitization experiments were analyzed using a d 0 analysis with an additional binomial analysis for the 2-AFC results and Student's t test for the intensity ratings. For all significant differences reported, P < .05. 2.2. Experiment II 2.2.1. Subjects Twenty-five healthy individuals (6 males, 19 females, age 18 ± 23 years), who were students and staff at the University of California at Davis, volunteered to participate in the study. None had participated in Experiment I and all consented to the restrictions noted earlier. 2.2.2. Application and rating procedures Amiloride (Sigma; dissolved in d H2O to a concentration of 1 mM) was applied to one side of the tongue while d H2O was applied to the other side using cotton swabs (Puritan, Hardwood Product, Guilford, ME, USA). Each cotton swab was rolled back and forth from the tip of the tongue to the back, as well as from the midline to the sides, in three strokes repeated three times. The side of the tongue receiving amiloride was counterbalanced across subjects. Subjects then rested with their mouth closed for 1 min after which the same treatment was repeated. One minute later, two NaClsoaked filter paper disks were placed onto each side of the tongue at the corresponding locations receiving amiloride and d H2O. Subjects performed a 2-AFC test and provided intensity ratings for the two sides as above. To control for possible taste cues (e.g., bitterness) of amiloride, its effect on capsaicin-evoked irritation was also studied. The same subjects participated in a second session that was identical except that a 3-ppm capsaicin solution was used instead of the 5 M NaCl solution. The order of presentation of amiloride plus NaCl vs. capsaicin was counterbalanced across subjects. 3. Results 3.1. Experiment I 3.1.1. Matching of capsaicin- and NaCl-evoked irritation Fifteen seconds after capsaicin and NaCl were simultaneously applied to opposite sides of the tongue, 17 of 25 subjects chose the NaCl-stimulated side as yielding a stronger sensation. This was not a significant majority (binomial, P = .11). However, the mean ratings on the two sides of the tongue were significantly different (4.3 vs. 3.2; t test, P = .025). There was no significant difference 30 s or 1 min later. These results confirm the large degree of interindividual variability in perception of irritation from various compounds, and indicate that the NaCl
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and capsaicin solutions were sufficiently well matched in intensity for present purposes. 3.1.2. Sensitization to sequential NaCl stimulations Repeated application of NaCl led to a significant increase in the perceived intensity of irritation across trials, consistent with sensitization. In both sessions, mean intensity rating increased significantly from the first to the tenth application (ANOVA, P < .001). Results from the first session are presented in Fig. 1. Despite some interindividual variability in the degree of sensitization, there was good within-subject consistency with a significant majority of subjects exhibiting sensitization in both sessions. In order to obtain a measure of the magnitude of sensitization that could be readily be compared with that of the self-desensitization and cross-sensitization to capsaicin (see below), a d 0 analysis was also performed. A beta binomial analysis first revealed that overdispersion over the two experimental sessions was not significant (g = .01, P = .75). This enabled the pooling of the data into one set of 50 subjects of whom 45 exhibited sensitization from the first to the tenth trial of application. This corresponded to a significant group d 0 value of 1.81 ( P < .001). Our observation of sensitization during repeated stimulation of NaCl 5 M, thus, confirms the findings of Green and Gelhard [1] using lower (0.4 or 0.8 M) NaCl concentrations as well as that reported by Green [33] with NaCl 1.5 M. 3.1.3. NaCl self-desensitization When NaCl was applied bilaterally following the unilateral sequential application of NaCl and a 10-min rest period, the side of the tongue that had previously received NaCl was chosen as having a weaker irritant sensation by a significant majority of subjects (21/25; binomial, P = .001) that corresponded to a significant d 0 value of 1.41 ( P = .001). The effect of self-desensitization was, therefore, smaller than that of sensitization (d 0 = 1.81). In addition, the mean intensity rating on that side was significantly higher (2.6 vs. 1.7; t test, P < .001). This self-desensitization remained significant during the entire 2-min testing period (Fig. 2).
Fig. 1. Sensitization by NaCl stimulation. Graph plots mean irritant intensity reported 15 s following stimulus onset as a function of trials of NaCl (5 M) application at 1-min ISI (N = 25 subjects). Error bars: S.E. Asterisk indicates first significant increase from initial rating ( P < .05, ANOVA with LSD post hoc test).
Fig. 2. NaCl self-desensitization. Graph plots the mean irritant intensity for the NaCl pretreated (T, filled circles) and nontreated (NT, open circles) side of the tongue vs. time following bilateral application of NaCl. Note the significant self-desensitization throughout the 2-min testing period. Error bars: S.E. Asterisks indicate significant difference between T and NT ( P < .05, t test).
3.1.4. NaCl cross-sensitization to capsaicin NaCl induced a cross-sensitization, rather than crossdesensitization, of capsaicin-evoked irritation. When capsaicin was applied bilaterally following the sequential NaCl series and rest period, the side that had previously received NaCl stimulation was chosen as having a stronger irritation by a significant majority of subjects (19/25; binomial, P = .015), corresponding to a significant group d 0 of 1.0 ( P = .008). Therefore, the magnitude of the cross-sensitizing effect too was smaller than that of self-sensitization over repeated trials. The mean rating on the treated side was significantly greater (3.5 vs. 2.7; t test, P = .008). The difference remained significant for another 1.5 min (Fig. 3). 3.2. Experiment II Amiloride significantly reduced the intensity of perceived irritation induced by application of 5 M NaCl. Following bilateral application of NaCl, a significant majority of subjects (16/20; binomial, P = .012) chose the side that had not received amiloride as having a stronger irritation. This corresponded to a significant group d 0 of 1.19
Fig. 3. NaCl cross-sensitization of capsaicin-evoked irritation. Graph as in Fig. 2 plotting mean ratings for NaCl pretreated and nontreated sides following bilateral application of capsaicin. Note that capsaicin elicited significantly more intense irritation on the NaCl-pretreated side.
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4. Discussion
or more of the following consequences of increased extracellular [Na + ]. First, lingual application of highly concentrated NaCl will raise the intraepithelial extracellular [Na + ] in the vicinity of nociceptors. One speculative consequence might be a direct depolarization of nociceptor endings by passive influx of Na + into the terminals through open cation channels, some of which may be amiloride-sensitive, in a manner analogous to salt taste transduction [34]. However, there is currently no evidence for amiloride-sensitive cation channels in nociceptors. Another consequence of increased extracellular [Na + ] is an osmotic effect of hypertonicity [35], leading to cell shrinkage. It is unknown if shrinkage of nociceptor endings leads to excitation via, for example, stretch-activated ion channels. Cell shrinkage initiated by hyperosmolarity is known to activate an amiloride-sensitive Na + /H + exchange pump (NHE) [36], an isoform of which (NHE-1) is ubiquitously expressed [37]. NHE plays a key role in the regulation of cell volume by exchanging intracellular protons with extracellular Na + to increase intracellular [Na + ] and draw water back into the cell (see Ref. [38] for recent review). However, if cell shrinkage were a major factor in NaCl-induced activation of nociceptors, amiloride treatment should block cellular rehydration and, thus, enhance, rather than reduce, NaCl-evoked irritation. We presently found that amiloride reduced NaCl-evoked irritation. Furthermore, other compounds having an osmolality similar to that of concentrated salts did not excite the lingual nerve, arguing against osmotic effects as the primary mechanism underlying NaClinduced irritation [3]. In support of this, we observed informally that lingual application of 5 M dextrose (equivalent in osmolality to 2.5 M NaCl) did not elicit any irritation.
The present study confirms and extends our knowledge of NaCl as an oral irritant. Repeated application of NaCl at short ISI induces sensitization, confirming earlier studies [1]. Furthermore, we showed that NaCl induces self-desensitization consistent with all other irritant chemicals studied to date, as well as cross-sensitization to capsaicin. The latter finding indicates that NaCl-elicited irritation is mediated partly via capsaicin-sensitive lingual nociceptors. The results of Experiment II suggest that the irritant sensation induced by NaCl may be partly mediated by an amiloridesensitive transduction mechanism in lingual nociceptors. Amiloride had an opposite (enhancing) effect on capsaicin-evoked irritation. Thus, irritation elicited by both NaCl and capsaicin appears to be mediated by activation of capsaicin-sensitive trigeminal nociceptors in the oral epithelium, but by distinct cellular transduction mechanisms.
4.1.1. NaCl sensitization As noted in the Introduction, hypertonic saline excites lingual and cutaneous nociceptors [4], as well as Vc neurons [8]; but it is not known if repetitive stimulation at short ISI sensitizes their responses. The presently observed increase in irritant intensity with repeated NaCl might be attributed to an increase in extracellular [Na + ] resulting in depolarization of lingual nociceptor endings. Further increases in extracellular [Na + ] with repeated stimuli might lead to spatial recruitment of nociceptors as well as increases in their firing rates, both of which could contribute to sensitization. Besides these peripheral effects, activation of nociceptors by NaCl might also induce central sensitization of Vc neurons such that successive stimuli elicit progressively larger responses in the trigeminal nociceptive pathway.
4.1. Transduction mechanisms for NaCl-evoked irritation
4.1.2. NaCl self-desensitization and cross-sensitization to capsaicin Following repetitive unilateral application of NaCl and a rest period, subsequent bilateral application of NaCl elicited
Fig. 4. Amiloride reduction of NaCl-induced irritation. Bar graph plots mean intensity ratings on the amiloride pretreated (T, open bar) and nontreated (NT, filled bar) sides of the tongue. Note the small but significant reduction on the side receiving amiloride. Error bars: S.E. Asterisk over open bars indicates significant difference between T and NT ( P < .05, t test).
( P = .008). In addition, the difference between the mean intensity ratings on each side was significantly different (6.0 vs. 4.9; t test, P = .008; Fig. 4). In contrast to its reduction of NaCl-evoked irritation, amiloride mildly but significantly enhanced capsaicininduced irritation. Following bilateral application of capsaicin, the amiloride-treated side was chosen as having a stronger irritant intensity by a significant majority of subjects (15/20; binomial, P = .004) that corresponded to a significant group d 0 of .95 ( P = .002). However, the difference in mean intensity ratings between the amiloride- and d H2O-treated sides fell short of significance (t test, P = .07). In a separate identical study with a larger sample of different subjects [15], we recorded a significantly higher mean rating of capsaicin-evoked irritant intensity on the amiloride-pretreated side, corroborating the present results.
The transduction mechanism by which NaCl depolarizes nociceptors is not certain, but may involve one
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irritation that was significantly weaker on the side of the tongue that had previously received NaCl. NaCl can, thus, be added to the list of other irritants shown to exhibit selfdesensitization: capsaicin [20], piperine [24], nicotine [23], menthol [26], zingerone [28], and citric acid [15]. The finding that NaCl exposure led, after a 5-min hiatus, to a heightened sensitivity to capsaicin is consistent with previous findings. Lower concentrations of NaCl enhanced capsaicin irritation [39] as well as detection [40] when both were presented in mixture. Furthermore, cross-sensitization of ethanol-evoked irritation following repeated exposure to 1.5 M NaCl was reported [33]. Menthol also cross-sensitized capsaicin-evoked irritation [26], and capsaicin crosssensitized irritation elicited by zingerone [22]. The cellular mechanisms underlying NaCl self-desensitization and cross-sensitization with capsaicin are not known. It is conceivable that the extracellular NaCl concentration in the lingual epithelium may have still been elevated when capsaicin was applied, possibly enhancing capsaicin-evoked irritation by increased Na + influx through VR-1 receptor-gated cation channels [41,42]. Another possibility is that repeated exposure to NaCl-sensitized nociceptors to induce primary hyperalgesia (see Refs. [43,44] for reviews) or initiated a central sensitization of Vc neurons signaling oral irritation. While both mechanisms would explain cross-sensitization of capsaicin-evoked irritation, neither is consistent with self-desensitization to NaCl. The present data suggest that exposure to concentrated NaCl has separate, opposing effects on nociceptors, reducing their responsiveness to subsequent NaCl while enhancing their responsiveness to subsequent capsaicin. 4.1.3. Amiloride Amiloride significantly reduced NaCl-evoked irritation, in contrast to a report that amiloride did not affect the lingual whole-nerve response to 2.5 M NaCl [3]. This discrepancy might be attributable to species differences such that amiloride more readily accesses intraepithelial nociceptors in the lingual epithelium of humans compared to rats, and/or to the higher concentration (1 mM) of amiloride used in the present compared to the previous study (0.1 mM). We speculate that the amiloride reduction of NaClevoked irritation is most simply explained by a blockade of amiloride-sensitive cation channels in nociceptor terminals, similar to the transduction mechanism proposed for taste receptor cells (see Ref. [34] for review). In human studies, amiloride reduced gustatory sensations elicited by NaCl [45 ±48]. That the present results are due to the known reduction by amiloride of taste elicited by NaCl, rather than chemesthesis, is unlikely since subjects were specifically instructed to ignore taste and to focus on the distinct and easily distinguishable irritant sensory quality instead. It was recently reported that amiloride enhanced the response of cutaneous nociceptors to saturated CO2 solu-
tions having an acidic pH [49], an effect attributed to the blockade of NHE. However, we do not believe that this mechanism is likely to be relevant at neutral pH as in the present study, unless intracellular acidification resulting from the blockade of the NHE is sufficient to potentiate the action of capsaicin, as previously demonstrated in in vitro recordings of dorsal root ganglion neurons [50]. This mechanism would explain the observation made presently, and in our previous study [15], that amiloride enhanced capsaicin-evoked irritation. Chloride ions might also play a role in NaCl-induced irritation. Many cells, including neurons, have a Cl ÿ / HCO3 ÿ antiport system that exchanges extracellular Cl ÿ ions with intracellular HCO3 ÿ (see Ref. [51]) to effect a cystolic acidification that might conceivably lead to depolarization. However, it is generally thought that osmotically induced cell shrinkage primarily activates NHE, leading to changes in intracellular pH that secondarily stimulates the Cl ÿ /HCO3 ÿ anion exchanger [36]. As noted earlier, blockade of the NHE by amiloride would be expected to increase, rather than decrease, NaCl-induced activation. Finally, it was previously shown that NaCl and other monovalent chloride salts (KCl and NH4Cl) do not uniformly excite all lingual nerve fibers [4], indicating that chloride ions are not the primary determinant of saltinduced activation. Amiloride also inhibits the Na + /Ca2 + exchange system as well as electrogenic Na + channels in transport epithelia [52]. However, it is not clear how these effects might explain amiloride's reduction of NaCl-evoked irritation observed in the present study. Amiloride was reported to block acid-sensitive ion channels (ASIC) (e.g, Ref. [53]) that may be expressed in lingual nociceptor endings, a mechanism that might explain our previous finding that amiloride significantly reduced irritation induced by citric acid on the tongue [15]. It is difficult to envision the involvement of ASIC in NaCl-evoked irritation, unless the extracellular pH dropped sufficiently due to hyperosmotic activation of NHE. In that case, the blockade of ASIC by amiloride would be irrelevant since extracellular acidification would already be prevented by amiloride blockade of NHE.
Acknowledgments Supported by a grant from the California TobaccoRelated Disease Research Program (No. 6RT-0231).
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Oral irritation by sodium chloride: sensitization, self-desensitization, and cross-sensitization to capsaicin J.-M. Dessiriera,b, M. O'Mahonyb, M. Iodi-Carstensa, E. Yaob, E. Carstensa,* a
Section of Neurobiology, Physiology, and Behavior, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA b Department of Food Science and Technology, University of California at Davis, Davis, CA, USA Received 12 May 2000; received in revised form 27 July 2000; accepted 6 September 2000
Abstract Psychophysical methods were used to investigate the irritant sensory properties of concentrated NaCl. The first experiment investigated potential sensitization and desensitization properties. Subjects rated the intensity of the irritation elicited by 10 successive applications of 5 M NaCl on one side of the dorsal surface of the tongue. The mean irritant sensation increased significantly across trials, consistent with sensitization. To test for self- and cross-desensitization effects of unilateral sequential stimulation with NaCl followed by a 10-min rest period, either 5 M NaCl or 10 mM capsaicin was applied bilaterally. In a two-alternative forced-choice (2-AFC) test, subjects indicated which side of the tongue had a stronger irritant sensation. They also rated the intensity of irritation on each side separately. When NaCl was applied bilaterally, the side not previously receiving NaCl was chosen as stronger by a significant majority of subjects and was given significantly higher intensity ratings, consistent with self-desensitization. In contrast, when capsaicin was applied bilaterally, the side that had previously received sequential NaCl was perceived as having a significantly more intense irritation, consistent with cross-sensitization. In a second experiment, the effect of amiloride on NaCl-evoked irritation was studied. One side of the tongue was treated with 1 mM amiloride, after which 5 M NaCl was applied bilaterally and subjects performed the same 2-AFC and rating procedures. Since amiloride significantly reduced the intensity of the irritant sensation, the contribution of amiloride-sensitive ionic currents or the Na + /H + exchange pump (NHE) are suggested as possible transduction mechanisms in lingual nociceptors mediating NaCl-evoked oral irritation. D 2001 Elsevier Science Inc. All rights reserved. Keywords: NaCl; Capsaicin; Vanilloid; Oral irritation; Trigeminal; Desensitization; Psychophysics; 2-AFC
1. Introduction In addition to oral gustatory sensations, monovalent salts, such as NaCl or KCl, can stimulate the chemesthetic system to elicit sensations of irritation and pain when applied to oral mucosa [1] or skin [2]. Salts activate rat lingual afferents [3] including identified nociceptors [4], inner dentine afferents [5], and corneal polymodal nociceptors [6,7]. Intraoral or ocular application of NaCl was shown to excite nociceptive neurons in the trigeminal subnucleus caudalis (Vc) of rats in a concentration-related manner [8]. Hypertonic saline also induces abdominal constrictions when injected intraperitoneally [9] and increases luminal gastric mucosal blood flow in the stomach [10]. * Corresponding author. Tel.: +1-530-752-6640; fax: +1-530-7525582. E-mail address: [email protected] (E. Carstens).
The mechanism by which salts activate nociceptors is uncertain. Hypertonic saline in the airways induces coughing that is not reduced by the vanilloid receptor antagonist, capsazepine [11], and also evokes the release of calcitonin gene-related peptide (CGRP) from rat urinary bladder in a manner that is not blocked by another vanilloid antagonist, ruthenium red [12]. These results indicate that vanilloid receptors are not involved in salt-induced irritation, in contrast to acid-evoked irritation and pain for which vanilloid receptors do appear to play a role (see Refs. [13 ± 15]). However, salt-induced irritation and nociception are reduced by treatment with capsaicin [4,10,16,17], the pungent principle in capsicum fruits that is well known to desensitize polymodal nociceptors (e.g., Refs. [18,19]). This indicates that irritation and pain elicited by salts is due at least partly to excitation of capsaicin-sensitive nociceptors, although not directly via vanilloid receptors. NaCl given sequentially at a short interstimulus interval (ISI) induces a progressive rise in the intensity of
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irritation, called sensitization [1]. Sensitization has also been observed with capsaicin [20 ± 22], piperine [23,24], and citric acid [15]. Other irritant chemicals, such as menthol [25,26], zingerone [27,28], or nicotine [23,29], do not exhibit such a sensitization, but rather a reduction in the intensity of irritation (self-desensitization) across trials. However, if a rest period of 5 min or more is imposed following the last stimulus presentation (i.e., longer ISI imposed), subsequent application of each chemical tested to date, including those exhibiting sensitization, elicits a significantly reduced sensory intensity, also called self-desensitization. Furthermore, irritant chemicals that exhibit sensitization Ð capsaicin and piperine Ð also induce desensitization to irritation elicited by to other compounds, a phenomenon called cross-desensitization. Since it is currently unknown whether NaCl at concentrations eliciting irritation also induces self- or cross-desensitization, we have presently undertaken experiments to address this question. The first aim of this study (Experiment I) was, therefore, to reproduce Green and Gelhard's [1] finding of NaCl sensitization using a higher, 5 M concentration. In addition, we investigated whether NaCl exhibits properties of selfdesensitization and cross-desensitization to capsaicin. The second aim of this study (Experiment II) was to investigate the involvement of amiloride-sensitive ion channels in NaCl-induced irritation. 2. Materials and methods 2.1. Experiment I 2.1.1. Subjects Twenty-five healthy individuals (7 males, 18 females, age 18 ±27 years), who were students and staff at the University of California at Davis, volunteered to participate in the study. All refrained from eating or drinking at least 1 h prior to each experimental session, and were asked not to eat spicy food for 2 days prior to testing as verified by questionnaire. 2.1.2. Chemical stimuli and application procedure All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified. A 0.1% w/v capsaicin stock solution (98 ± 100% pure) was made up in 95% ethanol and a 3-ppm (10 mM) capsaicin solution was made by diluting the stock solution with dH2O. Fifteen microliters of this solution were pipetted on to small (78.5 mm2) and 40 ml on to large (176.7 mm2) filter paper disks (Whatmann, Maidstone, UK). To avoid any stimulating effect of ethanol, the filter papers were air-dried and then soaked with the same volumes of distilled water immediately prior to application. NaCl was dissolved in distilled water to a concentration of 5 M and was pipetted in the same volumes onto the small or large filter papers just prior to application.
This concentration of NaCl was chosen because pilot studies had determined that it induced an irritant sensation similar to that elicited by 3-ppm capsaicin. Throughout the experiment, a suction device (Saliva Ejector, 6-in. clear, Sullivan Dental Products, Sacramento, CA) was placed in the mouth to remove saliva and avoided spread of the chemicals. Subjects were instructed to use the suction device freely, except for the 15-s period prior to giving intensity ratings or two-alternative forced-choice (2AFC) responses so as to avoid a cooling effect. 2.1.3. Experimental design 2.1.3.1. Matching intensities evoked by capsaicin and NaCl. All subjects participated in an initial session to verify that the concentration of capsaicin yielded an intensity of irritation approximately equivalent to that of NaCl. One large filter paper disk soaked with 40 ml of the 3-ppm capsaicin solution was placed on to one side of the dorsal anterior surface of the tongue and another, soaked with 40 ml of the 5-M NaCl solution, was simultaneously placed onto the other side. The sides receiving capsaicin or NaCl were counterbalanced across subjects. After 15 s, subjects performed a 2-AFC test [30] by reporting which side of the tongue had a stronger sensation. Subjects then separately rated the intensity of irritation on each side using a category scale with the end points 0 (no sensation) and 10 (intense irritation). To assess changes in intensity over time, 2-AFC and rating tasks were repeated at 30-s intervals for a total period of 3 min during which the filter papers remained on the tongue. 2.1.3.2. Sequential stimulation and desensitization. After verifying an approximate match in intensity for capsaicin and NaCl, two additional experimental sessions followed, each separated by at least 2 days. Each session consisted of two parts. In the first part, the larger size filter paper disks containing 40 ml of 5 M NaCl were applied with forceps to one side of the anterior dorsal surface of the tongue at 1-min ISI as in previous studies [23,29]. The filter paper was applied for 30 s and then removed. Thirty seconds later, a fresh filter paper containing the same NaCl stimulus was applied in the same manner; this was repeated 10 times. For each application, subjects rated the perceived intensity of irritation 15 s after stimulus onset. Ratings were made using a bipolar category scale used in a previous study (see Ref. [23]). Subjects were asked to focus on, and rate, only the irritant (biting/ pricking/stinging/burning) quality and to ignore gustatory sensory qualities (saltiness, sourness, bitterness). After the 10 unilateral sequential stimuli, the subjects rested for 10 min quietly without speaking. Then two smallsized filter papers containing either the same NaCl (selfdesensitization) or capsaicin 3 ppm (cross-desensitization) were applied simultaneously with two forceps at corresponding sites on the side that previously received NaCl
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as well as on the opposite side. Subjects then performed the 2-AFC test and intensity ratings as described below. 2.1.4. Rating procedures 2.1.4.1. Bipolar scale. Subjects rated the intensity of successively evoked sensations using a bipolar category scale that was specifically designed to accurately measure changes in perceived intensity across trials (see Fig. 1 of Ref. [23]). The method was virtually identical to that used in our previous studies (e.g., Ref. [23]) and is only briefly described. On a single sheet of paper is printed a series of 10 identical scales, each having the category `1' in the center, and categories ranging from 2 to 15 toward the right and left of the center. The sensation elicited by the first stimulus is obligatorily assigned a rating of `1'. The sensation elicited by the second stimulus is assigned a higher number toward the right if the sensation is stronger, or a higher number toward the left if the sensation is weaker, than the initial rating. Subsequent ratings are made relative to the first stimulus in this manner, for all 10 stimuli. The subject has access to prior ratings to reduce memory errors. To avoid `cut-off' effects, extensions with categories raging from 16 to 30, and additional unnumbered categories if required, are allowed at either end of the scale. 2.1.4.2. 2-AFC. This method is identical to that employed in our earlier studies [23,29]. Thirty seconds after bilateral application of capsaicin or 15 s after NaCl, subjects were asked to choose which side of the tongue gave rise to a stronger sensation (2-AFC, Ref. [30]). The rating times were selected because pilot tests suggested that NaCl-evoked irritation grew more rapidly and was shorter-lived than that elicited by capsaicin. The use of the 2-AFC ensured sufficient sensitivity to detect small and confusable differences between the two sides of the tongue, and also enabled d 0 values to be computed as a measure of difference magnitude. However, this method is inappropriate for perfectly discriminable differences as d 0 tends toward infinity. Accordingly, subjects gave ratings of the intensity of perceived irritation on each side of the tongue separately, using the unipolar category scale described earlier. As before, 2AFC and rating tasks were performed at 30-s ISI for a total of 3 min. 2.1.5. Statistical analysis The sequential stimulation data were analyzed using analysis of variance (ANOVA) and Fisher's Least Significant Difference (LSD) post hoc tests. To perform the ANOVA, the ratings were first transformed by allocating a positive sign to category ratings on the right side of the scale (stronger sensations) and a negative sign to those on the left side of the scale (weaker sensations). Then, the scale was renumbered by restoring zero to the center of the scale [23]. A d 0 analysis [31,32] was also performed deriving a group d 0 value from the proportion of subjects
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exhibiting sensitization from the first trial to the tenth in both experimental sessions. The self- and cross-desensitization experiments were analyzed using a d 0 analysis with an additional binomial analysis for the 2-AFC results and Student's t test for the intensity ratings. For all significant differences reported, P < .05. 2.2. Experiment II 2.2.1. Subjects Twenty-five healthy individuals (6 males, 19 females, age 18 ± 23 years), who were students and staff at the University of California at Davis, volunteered to participate in the study. None had participated in Experiment I and all consented to the restrictions noted earlier. 2.2.2. Application and rating procedures Amiloride (Sigma; dissolved in d H2O to a concentration of 1 mM) was applied to one side of the tongue while d H2O was applied to the other side using cotton swabs (Puritan, Hardwood Product, Guilford, ME, USA). Each cotton swab was rolled back and forth from the tip of the tongue to the back, as well as from the midline to the sides, in three strokes repeated three times. The side of the tongue receiving amiloride was counterbalanced across subjects. Subjects then rested with their mouth closed for 1 min after which the same treatment was repeated. One minute later, two NaClsoaked filter paper disks were placed onto each side of the tongue at the corresponding locations receiving amiloride and d H2O. Subjects performed a 2-AFC test and provided intensity ratings for the two sides as above. To control for possible taste cues (e.g., bitterness) of amiloride, its effect on capsaicin-evoked irritation was also studied. The same subjects participated in a second session that was identical except that a 3-ppm capsaicin solution was used instead of the 5 M NaCl solution. The order of presentation of amiloride plus NaCl vs. capsaicin was counterbalanced across subjects. 3. Results 3.1. Experiment I 3.1.1. Matching of capsaicin- and NaCl-evoked irritation Fifteen seconds after capsaicin and NaCl were simultaneously applied to opposite sides of the tongue, 17 of 25 subjects chose the NaCl-stimulated side as yielding a stronger sensation. This was not a significant majority (binomial, P = .11). However, the mean ratings on the two sides of the tongue were significantly different (4.3 vs. 3.2; t test, P = .025). There was no significant difference 30 s or 1 min later. These results confirm the large degree of interindividual variability in perception of irritation from various compounds, and indicate that the NaCl
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and capsaicin solutions were sufficiently well matched in intensity for present purposes. 3.1.2. Sensitization to sequential NaCl stimulations Repeated application of NaCl led to a significant increase in the perceived intensity of irritation across trials, consistent with sensitization. In both sessions, mean intensity rating increased significantly from the first to the tenth application (ANOVA, P < .001). Results from the first session are presented in Fig. 1. Despite some interindividual variability in the degree of sensitization, there was good within-subject consistency with a significant majority of subjects exhibiting sensitization in both sessions. In order to obtain a measure of the magnitude of sensitization that could be readily be compared with that of the self-desensitization and cross-sensitization to capsaicin (see below), a d 0 analysis was also performed. A beta binomial analysis first revealed that overdispersion over the two experimental sessions was not significant (g = .01, P = .75). This enabled the pooling of the data into one set of 50 subjects of whom 45 exhibited sensitization from the first to the tenth trial of application. This corresponded to a significant group d 0 value of 1.81 ( P < .001). Our observation of sensitization during repeated stimulation of NaCl 5 M, thus, confirms the findings of Green and Gelhard [1] using lower (0.4 or 0.8 M) NaCl concentrations as well as that reported by Green [33] with NaCl 1.5 M. 3.1.3. NaCl self-desensitization When NaCl was applied bilaterally following the unilateral sequential application of NaCl and a 10-min rest period, the side of the tongue that had previously received NaCl was chosen as having a weaker irritant sensation by a significant majority of subjects (21/25; binomial, P = .001) that corresponded to a significant d 0 value of 1.41 ( P = .001). The effect of self-desensitization was, therefore, smaller than that of sensitization (d 0 = 1.81). In addition, the mean intensity rating on that side was significantly higher (2.6 vs. 1.7; t test, P < .001). This self-desensitization remained significant during the entire 2-min testing period (Fig. 2).
Fig. 1. Sensitization by NaCl stimulation. Graph plots mean irritant intensity reported 15 s following stimulus onset as a function of trials of NaCl (5 M) application at 1-min ISI (N = 25 subjects). Error bars: S.E. Asterisk indicates first significant increase from initial rating ( P < .05, ANOVA with LSD post hoc test).
Fig. 2. NaCl self-desensitization. Graph plots the mean irritant intensity for the NaCl pretreated (T, filled circles) and nontreated (NT, open circles) side of the tongue vs. time following bilateral application of NaCl. Note the significant self-desensitization throughout the 2-min testing period. Error bars: S.E. Asterisks indicate significant difference between T and NT ( P < .05, t test).
3.1.4. NaCl cross-sensitization to capsaicin NaCl induced a cross-sensitization, rather than crossdesensitization, of capsaicin-evoked irritation. When capsaicin was applied bilaterally following the sequential NaCl series and rest period, the side that had previously received NaCl stimulation was chosen as having a stronger irritation by a significant majority of subjects (19/25; binomial, P = .015), corresponding to a significant group d 0 of 1.0 ( P = .008). Therefore, the magnitude of the cross-sensitizing effect too was smaller than that of self-sensitization over repeated trials. The mean rating on the treated side was significantly greater (3.5 vs. 2.7; t test, P = .008). The difference remained significant for another 1.5 min (Fig. 3). 3.2. Experiment II Amiloride significantly reduced the intensity of perceived irritation induced by application of 5 M NaCl. Following bilateral application of NaCl, a significant majority of subjects (16/20; binomial, P = .012) chose the side that had not received amiloride as having a stronger irritation. This corresponded to a significant group d 0 of 1.19
Fig. 3. NaCl cross-sensitization of capsaicin-evoked irritation. Graph as in Fig. 2 plotting mean ratings for NaCl pretreated and nontreated sides following bilateral application of capsaicin. Note that capsaicin elicited significantly more intense irritation on the NaCl-pretreated side.
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4. Discussion
or more of the following consequences of increased extracellular [Na + ]. First, lingual application of highly concentrated NaCl will raise the intraepithelial extracellular [Na + ] in the vicinity of nociceptors. One speculative consequence might be a direct depolarization of nociceptor endings by passive influx of Na + into the terminals through open cation channels, some of which may be amiloride-sensitive, in a manner analogous to salt taste transduction [34]. However, there is currently no evidence for amiloride-sensitive cation channels in nociceptors. Another consequence of increased extracellular [Na + ] is an osmotic effect of hypertonicity [35], leading to cell shrinkage. It is unknown if shrinkage of nociceptor endings leads to excitation via, for example, stretch-activated ion channels. Cell shrinkage initiated by hyperosmolarity is known to activate an amiloride-sensitive Na + /H + exchange pump (NHE) [36], an isoform of which (NHE-1) is ubiquitously expressed [37]. NHE plays a key role in the regulation of cell volume by exchanging intracellular protons with extracellular Na + to increase intracellular [Na + ] and draw water back into the cell (see Ref. [38] for recent review). However, if cell shrinkage were a major factor in NaCl-induced activation of nociceptors, amiloride treatment should block cellular rehydration and, thus, enhance, rather than reduce, NaCl-evoked irritation. We presently found that amiloride reduced NaCl-evoked irritation. Furthermore, other compounds having an osmolality similar to that of concentrated salts did not excite the lingual nerve, arguing against osmotic effects as the primary mechanism underlying NaClinduced irritation [3]. In support of this, we observed informally that lingual application of 5 M dextrose (equivalent in osmolality to 2.5 M NaCl) did not elicit any irritation.
The present study confirms and extends our knowledge of NaCl as an oral irritant. Repeated application of NaCl at short ISI induces sensitization, confirming earlier studies [1]. Furthermore, we showed that NaCl induces self-desensitization consistent with all other irritant chemicals studied to date, as well as cross-sensitization to capsaicin. The latter finding indicates that NaCl-elicited irritation is mediated partly via capsaicin-sensitive lingual nociceptors. The results of Experiment II suggest that the irritant sensation induced by NaCl may be partly mediated by an amiloridesensitive transduction mechanism in lingual nociceptors. Amiloride had an opposite (enhancing) effect on capsaicin-evoked irritation. Thus, irritation elicited by both NaCl and capsaicin appears to be mediated by activation of capsaicin-sensitive trigeminal nociceptors in the oral epithelium, but by distinct cellular transduction mechanisms.
4.1.1. NaCl sensitization As noted in the Introduction, hypertonic saline excites lingual and cutaneous nociceptors [4], as well as Vc neurons [8]; but it is not known if repetitive stimulation at short ISI sensitizes their responses. The presently observed increase in irritant intensity with repeated NaCl might be attributed to an increase in extracellular [Na + ] resulting in depolarization of lingual nociceptor endings. Further increases in extracellular [Na + ] with repeated stimuli might lead to spatial recruitment of nociceptors as well as increases in their firing rates, both of which could contribute to sensitization. Besides these peripheral effects, activation of nociceptors by NaCl might also induce central sensitization of Vc neurons such that successive stimuli elicit progressively larger responses in the trigeminal nociceptive pathway.
4.1. Transduction mechanisms for NaCl-evoked irritation
4.1.2. NaCl self-desensitization and cross-sensitization to capsaicin Following repetitive unilateral application of NaCl and a rest period, subsequent bilateral application of NaCl elicited
Fig. 4. Amiloride reduction of NaCl-induced irritation. Bar graph plots mean intensity ratings on the amiloride pretreated (T, open bar) and nontreated (NT, filled bar) sides of the tongue. Note the small but significant reduction on the side receiving amiloride. Error bars: S.E. Asterisk over open bars indicates significant difference between T and NT ( P < .05, t test).
( P = .008). In addition, the difference between the mean intensity ratings on each side was significantly different (6.0 vs. 4.9; t test, P = .008; Fig. 4). In contrast to its reduction of NaCl-evoked irritation, amiloride mildly but significantly enhanced capsaicininduced irritation. Following bilateral application of capsaicin, the amiloride-treated side was chosen as having a stronger irritant intensity by a significant majority of subjects (15/20; binomial, P = .004) that corresponded to a significant group d 0 of .95 ( P = .002). However, the difference in mean intensity ratings between the amiloride- and d H2O-treated sides fell short of significance (t test, P = .07). In a separate identical study with a larger sample of different subjects [15], we recorded a significantly higher mean rating of capsaicin-evoked irritant intensity on the amiloride-pretreated side, corroborating the present results.
The transduction mechanism by which NaCl depolarizes nociceptors is not certain, but may involve one
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irritation that was significantly weaker on the side of the tongue that had previously received NaCl. NaCl can, thus, be added to the list of other irritants shown to exhibit selfdesensitization: capsaicin [20], piperine [24], nicotine [23], menthol [26], zingerone [28], and citric acid [15]. The finding that NaCl exposure led, after a 5-min hiatus, to a heightened sensitivity to capsaicin is consistent with previous findings. Lower concentrations of NaCl enhanced capsaicin irritation [39] as well as detection [40] when both were presented in mixture. Furthermore, cross-sensitization of ethanol-evoked irritation following repeated exposure to 1.5 M NaCl was reported [33]. Menthol also cross-sensitized capsaicin-evoked irritation [26], and capsaicin crosssensitized irritation elicited by zingerone [22]. The cellular mechanisms underlying NaCl self-desensitization and cross-sensitization with capsaicin are not known. It is conceivable that the extracellular NaCl concentration in the lingual epithelium may have still been elevated when capsaicin was applied, possibly enhancing capsaicin-evoked irritation by increased Na + influx through VR-1 receptor-gated cation channels [41,42]. Another possibility is that repeated exposure to NaCl-sensitized nociceptors to induce primary hyperalgesia (see Refs. [43,44] for reviews) or initiated a central sensitization of Vc neurons signaling oral irritation. While both mechanisms would explain cross-sensitization of capsaicin-evoked irritation, neither is consistent with self-desensitization to NaCl. The present data suggest that exposure to concentrated NaCl has separate, opposing effects on nociceptors, reducing their responsiveness to subsequent NaCl while enhancing their responsiveness to subsequent capsaicin. 4.1.3. Amiloride Amiloride significantly reduced NaCl-evoked irritation, in contrast to a report that amiloride did not affect the lingual whole-nerve response to 2.5 M NaCl [3]. This discrepancy might be attributable to species differences such that amiloride more readily accesses intraepithelial nociceptors in the lingual epithelium of humans compared to rats, and/or to the higher concentration (1 mM) of amiloride used in the present compared to the previous study (0.1 mM). We speculate that the amiloride reduction of NaClevoked irritation is most simply explained by a blockade of amiloride-sensitive cation channels in nociceptor terminals, similar to the transduction mechanism proposed for taste receptor cells (see Ref. [34] for review). In human studies, amiloride reduced gustatory sensations elicited by NaCl [45 ±48]. That the present results are due to the known reduction by amiloride of taste elicited by NaCl, rather than chemesthesis, is unlikely since subjects were specifically instructed to ignore taste and to focus on the distinct and easily distinguishable irritant sensory quality instead. It was recently reported that amiloride enhanced the response of cutaneous nociceptors to saturated CO2 solu-
tions having an acidic pH [49], an effect attributed to the blockade of NHE. However, we do not believe that this mechanism is likely to be relevant at neutral pH as in the present study, unless intracellular acidification resulting from the blockade of the NHE is sufficient to potentiate the action of capsaicin, as previously demonstrated in in vitro recordings of dorsal root ganglion neurons [50]. This mechanism would explain the observation made presently, and in our previous study [15], that amiloride enhanced capsaicin-evoked irritation. Chloride ions might also play a role in NaCl-induced irritation. Many cells, including neurons, have a Cl ÿ / HCO3 ÿ antiport system that exchanges extracellular Cl ÿ ions with intracellular HCO3 ÿ (see Ref. [51]) to effect a cystolic acidification that might conceivably lead to depolarization. However, it is generally thought that osmotically induced cell shrinkage primarily activates NHE, leading to changes in intracellular pH that secondarily stimulates the Cl ÿ /HCO3 ÿ anion exchanger [36]. As noted earlier, blockade of the NHE by amiloride would be expected to increase, rather than decrease, NaCl-induced activation. Finally, it was previously shown that NaCl and other monovalent chloride salts (KCl and NH4Cl) do not uniformly excite all lingual nerve fibers [4], indicating that chloride ions are not the primary determinant of saltinduced activation. Amiloride also inhibits the Na + /Ca2 + exchange system as well as electrogenic Na + channels in transport epithelia [52]. However, it is not clear how these effects might explain amiloride's reduction of NaCl-evoked irritation observed in the present study. Amiloride was reported to block acid-sensitive ion channels (ASIC) (e.g, Ref. [53]) that may be expressed in lingual nociceptor endings, a mechanism that might explain our previous finding that amiloride significantly reduced irritation induced by citric acid on the tongue [15]. It is difficult to envision the involvement of ASIC in NaCl-evoked irritation, unless the extracellular pH dropped sufficiently due to hyperosmotic activation of NHE. In that case, the blockade of ASIC by amiloride would be irrelevant since extracellular acidification would already be prevented by amiloride blockade of NHE.
Acknowledgments Supported by a grant from the California TobaccoRelated Disease Research Program (No. 6RT-0231).
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