Electrophysiological properties of the tongue epithelium of the toad Bufo marinus

The landmark studies of DeSimone et al.(1981, 1984) demonstrated that isolated canine lingual epithelium, mounted an Ussing-type chamber, develops a short-circuit current (I_sc) like that of the frog skin,which is the result of the active transport of Na⁺ from the outer(mucosal) to the inner (serosal) surface of the tissue(Ussing and Zerahn, 1951). The I_sc could be inhibited by the pyrazine diuretic amiloride,which blocks epithelial Na⁺ channels in the apical membrane of the epithelial cells, including the taste cells. The neural response of the chorda tympani nerve to NaCl solutions applied to the tongue was similarly reduced by the application of amiloride to the mucosal surface of the tongue in situ, and it was suggested that the transport of Na⁺ across the lingual epithelium is a primary mechanism for Na⁺ taste transduction in mammals. More recent studies on mammalian (rat and hamster)tongue indicate that, in addition to transcellular currents across specific taste cells, transcellular and paracellular transport of salts across the lingual epithelium also serves a role in salt taste transduction(Ye et al., 1991; Gilbertson and Zhang, 1998). Gilbertson and Zhang (1998)suggest that `...the whole lingual tissue may be viewed as a taste organ involving both taste receptor cells and non-taste epithelium in parallel'.

Among other vertebrate classes, the taste response of the amphibian tongue was studied by Pumphry (1935),who demonstrated that the application of salty or acidic solutions to the lingual surface stimulated the glossopharyngeal nerve of frogs. Sato(1976) has summarized the historical development of studies on the chemosensory responses of the amphibian tongue, and the morphology of the tongue has been described by Jaeger and Hillman (1976). Experiments to date with the isolated lingual epithelium of amphibians have not demonstrated a transepithelial voltage difference when the tissue was bathed on both sides with identical Ringer's solutions(Soeda and Sakudo, 1985). Detailed studies of acutely dissociated taste cells from frog tongue have been conducted by Avenet and Lindemann(1988), who utilized whole-cell patch-clamp methodology to demonstrate a cationic current across these cells. This current was inhibited by amiloride, suggesting that, like taste cells from mammalian tongue (DeSimone et al., 1981, 1984), epithelial Na⁺ channels play a role in salt taste transduction. In the present study, we tested the hypothesis that the transepithelial current across the lingual epithelium of the toad Bufo marinus is similar to that of mammals. We observed that, in contrast to the mammalian tongue, the short-circuit current across the lingual epithelium of Bufo marinusis outwardly directed and is carried by active transport of K⁺ from the serosal to the mucosal surface of the tissue. Fluctuation analysis (for a review, see Van Driessche,1994) of the short-circuit current demonstrates the presence of spontaneously fluctuating channels in the apical membrane of the epithelium that may be involved in K⁺ secretion.

Bufo marinus L. were obtained commercially and kept at 21-23°C on a 12h:12h L:D photoperiod in a terrarium that contained moist soil, covered burrows and standing water. The toads were fed crickets twice per week. After doubly pithing the animals, the tongue was removed and pinned in a dish containing frog Ringer's solution (115 mmol l^-1 NaCl, 2.5 mmol l^-1 KHCO₃, 1 mmol l^-1 CaCl₂; NaCl Ringer). All solutions had a pH of 8.0 at the ambient temperature(21-23°C). The ventral epithelium and large muscles underlying the dorsal surface were trimmed, and the dorsal surface was mounted in an Ussing-type chamber that minimized edge damage and allowed continuous perfusion of both the mucosal and serosal epithelia (De Wolf and Van Driessche, 1986). The chamber had a cross-sectional area of 0.5 cm² and a volume of 1.5 ml. The perfusion rate of approximately 5 ml min^-1 in the present experiments was regulated by the height of the perfusion reservoirs. Short-circuit current was applied with a low-noise voltage clamp (Van Driessche and Lindemann, 1978). This voltage-clamp and electrode arrangement has been successfully used to record I_sc and current fluctuations arising from K⁺ transport across the isolated skin of the frog Rana temporaria(De Wolf and Van Driessche,1986) in addition to the standard Ussing conditions with identical NaCl Ringer on both sides of the tissue(Baker and Hillyard, 1992).

The Lorentzian function includes S_o, which is the power density of the Lorentzian plateau at low frequencies, and the corner frequency(f_c), which is the frequency at which the power density has relaxed to half that of S_o. The presence of a Lorentzian function in a power spectrum is the result of changes in the conductance state of individual ion channels and is taken as evidence for a spontaneously fluctuating population of ion channels contributing to the I_sc (Conti et al.,1975; Lindemann and Van Driessche, 1977; Van Driessche, 1994). Equations 2-5 (see Discussion) were developed in detail by Van Driessche(1994). They are presented in this paper to facilitate the interpretation of the fluctuation analysis measurements in terms of models that describe the kinetic properties of ion channels.

I_sc measurements were begun with NaCl Ringer's solution bathing both mucosal and serosal surfaces of the tissue and with the command voltage set to 0 mV. Under these conditions, there are no voltage or concentration gradients across the tissue, and any currents that are passed by the voltage clamp represent the active transport of ions(Ussing and Zerahn, 1951). Four sets of experiments were performed: cation exchange, anion exchange,mucosal Ba²⁺ treatment and mucosal quinine treatment.

The cation exchange experiments involved replacing NaCl Ringer with KCl Ringer (112.5 mmol l^-1 KCl, 2.5 mmol l^-1KHCO₃, 1 mmol l^-1 CaCl₂) on the mucosal surface, re-equilibration with NaCl in the mucosal and serosal solutions and then replacement with KCl in the serosal solution. Under these conditions,passive concentration gradients for K⁺ are established across the tissue, and the changes in current and fluctuation analysis parameters can be compared with those obtained in the absence of a K⁺ gradient.

For the anion exchange experiments, the tissue was initially bathed in NaCl Ringer. The first step was to change both mucosal and serosal solutions to Na₂SO₄ Ringer (57.5 mmol l^-1Na₂SO₄, 2.5 mmol l^-1 KHCO₃, 1 mmol l^-1 CaCl₂). When a stable I_sc had been obtained, a power spectrum was recorded. The mucosal and then the serosal solutions were exchanged with K₂SO₄ Ringer (57.5 mmol l^-1 K₂SO₄, 2.5 mmol l^-1KHCO₃, 1 mmol l^-1 CaCl₂) to duplicate the sequence of cation substitutions performed above with KCl. Chloride ions are known to be transported across many epithelia(Larsen, 1991). If the I_sc across the toad tongue is the result of Cl^-transport, substitution of Cl^- should eliminate that current.

Ba²⁺ and quinine are known blockers of K⁺ channels,so the inhibition of I_sc by these substances would support the hypothesis that the I_sc is the result of K⁺transport. The effects of Ba²⁺ on the I_sc were first recorded with NaCl Ringer bathing the mucosal and serosal surfaces of the tissue. The mucosal Ringer's solution contained, sequentially, 0.5, 1, 2,5 and 10 mmol l^-1 BaCl₂. After maximal inhibition of I_sc had been observed, the mucosal solution was replaced with Ringer containing no Ba²⁺. In a second set of experiments, the inhibition of I_sc by mucosal Ba²⁺ was recorded from tissues bathed at the serosal surface with KCl Ringer and at the mucosal surface with NaCl Ringer.

Quinine experiments were performed with mucosal applications of 10 μmol l^-1 followed by 100 μmol l^-1 quinine with NaCl Ringer as the mucosal and serosal solutions. When the quinine solution had been washed out, 10 mmol l^-1 BaCl₂ was added to the mucosal side and then washed out. I_sc was continuously recorded on a chart recorder, and noise analysis was conducted upon equilibration of I_sc after each solution change described above.

All data are reported as means ±1 S.E.M.

The initial I_sc with identical NaCl Ringer bathing both sides of the tissue was -10.71±0.82 μA cm^-2(N=24). The chart tracing from a typical experiment is shown if Fig. 1. Negative values for I_sc represent outward movement (serosa to mucosa) of positive charge, which could arise either from the outward transport of a cation (K⁺) or from the inward transport of an anion(Cl^-). In 10 preparations, the tissue resistance was calculated by recording the current change associated with a 10 mV current pulse and was found to be 615±152 Ω cm². The resistance values were highly variable among preparations and following ion substitutions during a given experiment. We report here the initial resistance values obtained with NaCl Ringer in the mucosal and serosal solutions for comparisons with other species. In most experiments, and as seen in Fig. 1, the current started near -20 μA cm^-2 before decreasing to a stable value. A representative power spectrum is shown in Fig. 2A. In all three panels of Fig. 2, the arrows represent the range selected for fitting according to equation 1. Initial values for S_o and f_c were 18.16×10^-20±2.41×10^-20 A²s cm^-2 and 10.19±1.09 Hz, respectively (N=24). Values for I_sc, S_o and f_c for the cation substitution experiments are presented in Table 1.

Replacement of mucosal NaCl Ringer's solution with KCl Ringer resulted in significant changes in I_sc, S_o and f_c: I_sc became positive, S_o declined and f_c shifted to a higher frequency. Statistical comparisons, unless otherwise specified, were with Fisher's Protected Least Significant Difference (PLSD) test using StatView Version 4.5 for Windows. A representative power spectrum obtained with KCl Ringer as the mucosal solution is shown in Fig. 2B. The spectrum in Fig. 2B is from the same preparation as that of Fig. 2Aand illustrates the decline in S_o and the increase in f_c presented in Table 1. All these effects of mucosal KCl were reversed by changing the solution back to NaCl Ringer.

When the serosal solution was changed from NaCl to KCl, I_sc became significantly more negative(Fig. 1; Table 1). This change in current was reversed by switching the serosal solution back to NaCl. With Cl^- as the anion, power spectra obtained with a serosa-to-mucosa K⁺ gradient displayed two Lorentzian components in six of seven experiments (Table 1). One Lorentzian component had an f_c value that was somewhat smaller than that obtained with NaCl Ringer on both sides of the tissue, and S_o was increased but was highly variable among preparations and not significantly greater than with NaCl solutions on both sides of the tissue (Table 1). The second Lorentzian component had a higher f_c and lower S_o than the first. A representative power spectrum fitted with two Lorentzian components is shown in Fig. 2C.

Substitution of NaCl Ringer's solutions with Na₂SO₄Ringer in both the mucosal and serosal baths produced no significant change in any of the three variables (Table 2). Substitution of Na₂SO₄ Ringer on both sides of the tissue with mucosal K₂SO₄ Ringer's solution produced results similar to those seen with mucosal KCl: I_sc increased to positive values, S_odeclined and f_c increased, all significantly(Table 2). As with the addition of mucosal KCl, these results were reversed by changing the mucosal solution back to Na₂SO₄. When the serosal solution was changed from Na₂SO₄ to K₂SO₄, with Na₂SO₄ Ringer bathing the mucosal surface, I_sc became significantly more negative. Thus, the general pattern of I_sc changes depicted in Fig. 1 also apply to experiments with SO₄^2- as the anion. Three of the seven power spectra obtained under these condition could be fitted with either one or with two Lorentzian components like that shown in Fig. 2C. The values for the single Lorentzian fits are presented in Table 2, with the understanding that the higher-frequency Lorentzian component, when present, is less prominent than that observed with Cl^- as the anion.

With NaCl Ringer in the mucosal and serosal bath solutions, the addition of increasing concentrations of BaCl₂ to the mucosal surface resulted in a dose-dependent inhibition of the negative I_sc from an initial value of -13.00±2.22 μA cm^-2 (N=6)(Fig. 3). Typically, the inhibition of I_sc was rapid, and a stable value for each concentration was achieved within 2-3 min. Upon removal of the BaCl₂ solution from the mucosal surface, the I_sc recovered to a level not significantly different from its pre-treatment value, -11.87±0.82 μA cm^-2(N=6). Statistical analysis using a non-parametric paired sign test shows the inhibition of I_sc to be significant at each concentration change (P=0.0313). The pattern of Ba²⁺inhibition was further analyzed with a direct linear plot(Eisenthal and Cornish-Bowden,1979). This is a modification of the Michaelis—Menten equation for a pseudo-first-order inhibitor. Graphically, a family of lines is drawn between the points representing the inhibitor concentrations, as negative values, on the abcissa and the decrement in I_scfor each blocker concentration on the ordinate(Fig. 4A). If the inhibitor is binding to a single inhibitory site, the lines, extended to positive x axis values, should intersect at a common point that describes the inhibitory constant (K_i) as the x coordinate and the amount of inhibitor-sensitive current as the y coordinate. It can be seen in Fig. 4A that the lines for inhibition by 0.5, 1 and 2 mmol l^-1 Ba²⁺intersect near a common point but that the lines for 5 and 10 mmol l^-1 Ba²⁺ intersect at points that indicate a higher binding constant and thus a lower binding affinity.

The inhibition of I_sc by Ba²⁺ was also examined with KCl as the serosal solution. In these experiments(N=4), the I_sc was -30.37±5.14 μA cm^-2, and a similar pattern of inhibition was seen when Ba²⁺ was added to the mucosal solution. Specifically, the lines of the direct linear plot intersect at a common point at lower Ba²⁺concentrations of 0.5-5 mmol l^-1, but the point of intersection at 10 mmol l^-1 Ba²⁺ indicates a lower-affinity blockage(Fig. 4B). If the amount of Ba²⁺-sensitive I_sc is compared between the mean values for 10 mmol l^-1 Ba²⁺ data points in Fig. 4A and B, it can be seen that -8.46 of -13.00 μA cm^-2 (65 %) is inhibited with no K⁺ gradient across the tissue while 11.72 of 30.37 μA cm^-2 (38.6 %) is inhibited in the presence of a serosa-to-mucosa K⁺ gradient, i.e. the increased negative I_sc is largely blocker-insensitive.

The fluctuation analysis results obtained for Ba²⁺ inhibition of I_sc with NaCl Ringer on both sides of the tissue showed no significant changes in either f_c or S_oas I_sc was inhibited(Fig. 3).

The addition of 10 μmol l^-1 quinine, a bitter tastant as well as a K⁺ channel blocker, to the mucosal surface consistently produced a decrease in the mean I_sc value from -14.53 to-11.62 μA cm^-2 (Fig. 5). Analysis of these data with a non-parametric paired sign test showed this to be significant (P=0.0313). Further inhibition of the current is seen with 100 μmol l^-1 quinine (P=0.0022). Wash-out of the quinine does not produce a recovery of the initial current. Addition of 10 mmol l^-1 Ba²⁺ to the mucosa, after the quinine wash-out, causes a further inhibition of the I_sc(P<0.0001), which is reversible upon its removal.

The fluctuation analysis experiments with quinine and Ba²⁺showed that S_o values declined as I_scwas inhibited and increased when Ba²⁺ was washed out. The changes in f_c were very small, however, and did not correlate with the changes in I_sc or S_o(Fig. 5).

Similar values for the outwardly directed I_sc were observed with either Cl^- or SO₄^2- as the anion, and the I_sc could be reversibly inhibited by the addition of Ba²⁺ to the mucosal bath. Both these properties suggest that the I_sc is the result of active K⁺secretion that involves K⁺ channels in the apical membrane of cells of the lingual epithelium. This pattern of transport differs qualitatively from that of the mammalian tongue, in which the I_screcorded in Ussing chamber preparations is mucosa-negative as the result of Na⁺ absorption that can be inhibited by amiloride(DeSimone et al., 1981; Gilbertson and Zhang, 1998). The qualitative difference between ionic currents across the toad and mammalian tongue epithelium thus provides an interesting model for comparative studies.

As noted above, Soeda and Sakudo(1985) conducted Ussing chamber experiments with the isolated lingual epithelium of bullfrogs but detected no spontaneous potential difference across the tissue bathed with identical Ringer on both sides. They measured a resistance of 720±180Ω cm², which is very similar to the value of 615±152Ω cm² for B. marinus tongue (present study) and 576±127 Ω cm² for dog tongue(DeSimone et al., 1981). It is generally assumed that the lingual epithelium of mammals is a low-resistance tissue with both transcellular and paracellular pathways for ion transport and sensory transduction (Ye et al.,1991; Gilbertson and Zhang,1998). Given the similar low resistance values for frog and toad tongue, it appears that these epithelia also have paracellular as well as transcellular transport pathways. Soeda and Sakudo(1985) also observed that the addition of 200 mmol l^-1 NaCl, 5 mmol l^-1 acetic acid or 5 mmol l^-1 quinine to the mucosal bath increased the potential across the bullfrog tongue, and they suggested that the change in transepithelial potential might be associated with a chemosensory function. We have conducted preliminary experiments with the lingual epithelium of the frog Rana pipiens and have noted a similar pattern of K⁺secretion as was noted above for the toad tongue, i.e. an outwardly directed I_sc, Ba²⁺-sensitivity and a Lorentzian component in power spectra. These observations suggest a mucosa-positive I_sc that is mediated by K⁺ secretion to be the pattern for the lingual epithelium of at least the bufonid and ranid families of the Anura. It is not clear why Soeda and Sakudo(1985) did not observe this pattern of transport.

We observed no effect of 10 μmol l^-1 amiloride on I_sc across the toad tongue bathed with NaCl Ringer on both sides of the tissue. The sensory processes of taste cells make up a small percentage of the membrane area of the taste discs and an even smaller percentage of the membrane area of the lingual epithelium(Jaeger and Hillman, 1976). It is possible that a small amiloride-sensitive component to the measured I_sc that might be carried by inward Na⁺transport across taste cells would not be resolved at the microampere level. Avenet and Lindemann (1988)estimated the Na⁺ current per taste cell to vary between 10 and 700 pA when clamped to -80 mV. The minimum number of cells required to produce 1μA of current is thus greater than 1000, assuming that toad and frog taste cells have a similar Na⁺ transport capability and that the in situ membrane potentials are of the order used in the patch-clamp study. Without this information and a detailed histological examination of the tissue used in specific experiments, it is not possible to speculate further. It is noteworthy that Kitada and Mitoh(1997) observed no effect of amiloride on the activity of the glossopharyngeal nerve of the frog during exposure of the tongue to NaCl concentrations between 0.1 and 0.5 mol l^-1. DeSimone et al.(1981) imposed inwardly directed Na⁺ gradients across the canine lingual epithelium to obtain larger amiloride-sensitive I_sc values. This procedure might produce a sufficiently large Na⁺ current in toad tongue to be detected at the microampere level. We have conducted a few measurements of the in situ potential across the lingual epithelium of pithed toads, and the values we obtained are similar to that observed during the initial open-circuit voltage in the Ussing chamber experiments,approximately 10-15 mV. Thus, the electrical properties of the tongue in situ appear to be similar to those of the isolated tissue.

Avenet et al. (1988) have also used patch-clamp methodology to demonstrate the presence of three types of K⁺ channel in isolated frog taste cells. The direct linear plots and the presence of two Lorentzian functions in power spectra from the present study are consistent with the presence of multiple K⁺ channels in the apical membrane of the lingual epithelium which could include both taste and non-taste cells working in parallel.

The addition of K⁺ Ringer to the serosal bath augmented I_sc and resulted in the appearance of a second Lorentzian function in the power spectra in 10 of 14 experiments. This observation suggests the presence either of two types of K⁺ channel or of multiple conductance states with different kinetics and affinity for Ba²⁺ inhibition. This procedure should depolarize the basolateral membrane, which would allow a voltage clamp of the apical membrane(Lindemann and Van Driessche,1977). The Ba²⁺-sensitive I_sc with a serosa-to-mucosa K⁺ gradient amounted to 38.6 % of the total compared with 65 % for K⁺ secretion in the absence of a concentration gradient. Thus, the bulk of the enhanced current is Ba²⁺-insensitive and may be paracellular. For this reason, there may not be substantially larger values for I or i via a transcellular pathway and, from equation 2, S_o would not be larger in these preparations relative to the control condition with NaCl Ringer on both sides of the tissue. In experiments with double Lorentzian components, the variability in f_c was large, and we have not analyzed them further.

Quinine treatment produced a decrease in I_sc that did not wash out. Van Driessche and Hillyard(1985) showed that quinidine,a stereoisomer of quinine, inhibited K⁺ channels in the basolateral membrane of larval frog skin but only when applied to the mucosal side of the tissue. The inhibition was not readily reversible, and it was suggested that inhibition occurred after the blocker had diffused into the cell and become ionized at the lower pH of the cytosol and, thus, could not be entirely washed out, as had been suggested by Yeh and Narahashi(1976) for experiments with K⁺ channels in squid axon membranes. From these experiments, it would appear that inhibition occurred as the result of blockage at the cytosolic face of the membrane, in contrast with Ba²⁺ which binds at the extracytoplasmic face of the channels and is readily washed out. We cannot rule out the possibility that quinine might be blocking channels in the basolateral as well as the apical membranes of the lingual epithelium.

The reduction in S_o during quinine treatment roughly paralleled the inhibition of I_sc and persisted after quinine had been washed out, as would be expected from equation 5. The addition of Ba²⁺ further reduced I_sc but not S_o, which is consistent with the results in Fig. 3 that also show Ba²⁺ to inhibit I_sc without lowering S_o. S_o did increase in concert with an increase in I_sc when Ba²⁺ was washed out. The changes in f_c were small and not consistent with the two-state model described by equation 4. This also suggests that the Lorenzian function that is fitted in the power spectra represents a spontaneously fluctuating K⁺ channel that is blocker-insensitive. As noted above,the analysis of blocker-induced current fluctuations that are not compatible with a two-state model is beyond the scope of the present study.

Given the complex cellular structure of the toad tongue, it is premature to describe a cellular model for K⁺ secretion. The simplest model would be one like that of the distal nephron or colon in which K⁺is transported into the epithelial cells by the Na⁺/K⁺pump in the basolateral membrane and expelled from the cells through apical K⁺ channels (Berne and Levy,1988). The role of the Na⁺/K⁺ pump in K⁺ secretion has been difficult to study because this enzyme in bufonid anurans is resistant to ouabain inhibition(Jaisser et al., 1992),presumably to protect the enzyme from alkaloid substances in the skin secretions and body fluids that are used for protection from predators(Butler et al., 1996).

Kinnamon (1992) has suggested that K⁺ channels serve a chemosensory function in the taste cells of both mammalian and amphibian tongue epithelia. Bitter tastants block K⁺ secretion and depolarize the taste cells. As noted above,the proportion of the I_sc carried by K⁺transport across taste cells versus other epithelial cells cannot be resolved from our measurements. Another source for K⁺ secretion across the lingual epithelium is salivary and mucus secretion(Jaeger and Hillman, 1976). The secretion of both mucus and saliva in mammalian digestive systems is accompanied by an elevated K⁺ concentration in the secretion(Berne and Levy, 1988). A coating of mucus on the tongue is required for the capture of insects, which raises the question of the function of taste modalities in anurans that feed in this manner. Specifically, insects are captured on a thick mucous layer at the dorsal surface of the tongue, held briefly in the mouth and then swallowed whole. Taste buds are thought to provide sensory input about the quality of food being ingested and, as pointed out by Lindemann(1996), chemosensory cells respond to relatively high concentrations of chemical tastants.

Many anuran species, including ranids and bufonids, swallow prey, primarily insects, whole, and most nutritive components are contained within an exoskeleton that is only partially degraded after ingestion(Larsen, 1992). Thus, the taste buds on the tongue would be unable to assess nutritional value. It is possible that tastants on the cuticular surface might provide some measure of prey selection. Mikulka et al.(1981) were able to show that coating prey insects with lithium chloride elicited an aversion to feeding. However, Larsen (1992)observed anecdotally that toads will swallow glass beads placed in the mouth. The thick layer of mucus required to capture the prey and the short time that prey are retained in the mouth would allow for minimal diffusion of tastant molecules to taste cells and appears to be an unreliable mechanism for evaluating food quality. Takeuchi et al.(1994) observed a rejection of gelatine capsules containing 1 mmoll^-1 quinine by the urodele amphibian Ambystoma mexicanum and that the presence of 100 mmoll^-1 NaCl in the capsules reduced the proportion rejected. It was suggested that the salamanders were able to discriminate between the taste qualities of these stimuli. The authors also noted that this salamander has a non-distensible tongue and feeds by snapping its jaws to capture food items dropped into the water. Thus, the feeding behavior does not require a layer of mucus covering the taste cells, and a limited amount of mastication occurs.

Recent studies by Sato et al.(2000, 2001) propose an interesting function for salivary secretion in chemosensory transduction. They observed that stimulation of the frog glossopharyngeal nerve elicited slow potentials at the tongue surface and in taste cells in the fungiform papillae. This was blocked by atropine, suggesting that the effect was mediated by parasympathetic nerve fibers. Depolarizing slow potentials enhanced the receptor potential response to exposure to 0.5 moll^-1 NaCl, and it was suggested that the parasympathetic stimulation had produced salivary secretions whose ionic composition modified the liquid junction potential across the lingual epithelium. Assuming that salivary secretion in toads involves K⁺ secretion, as in mammals(Berne and Levy, 1988),K⁺ secretion could contribute to the measured I_sc and also to the chemosensory function of the tongue.

This study was performed as part of an undergraduate thesis for the Honors Program at UNLV (T.K.B.) and as an REU project (K.R) supported by grant no. IBN 9215023 from the National Science Foundation to S.D.H.