INTRODUCTION

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Amiloride inhibits part of the chorda tympani (CT) response to NaCl, an observation that led to the identification of the epithelial sodium channel (ENaC) as a transducer in NaCl taste (Stewart et al. 1997). CT recordings to Na salts also show an amiloride-insensitive (AI) response that is anion dependent (Elliott and Simon 1990; Formaker and Hill 1988). It was proposed that anions exert their influence through modulation of transepithelial potentials set up across paracellular shunts in the taste buds (Elliott and Simon 1990), and this was proved by obtaining the CT responses to Na salts under tissue voltage clamp (Ye et al. 1994).

CT responses to potassium salts and ammonium salts show only slight amiloride sensitivity and also give CT responses that are anion dependent (Kloub et al. 1997; Ye et al. 1994). That K salt responses in mammals might be mediated by apical membrane K channels has generally not been supported by pharmacological studies (Ye et al. 1994). For these reasons, it has been proposed that the AI response to Na salts and responses to K and NH₄ salts might be mediated by basolateral membrane ion channels accessible to taste stimuli by diffusion through paracellular shunts (Kloub et al. 1997; Ye et al. 1994). A role for basolateral ENaC and basolateral K⁺ channels is supported by the observation of ENaC immunoreactivity in the basolateral domain of taste receptor cells (TRCs) (Kretz et al. 1999) and the presence of K⁺ channels on the basolateral membranes of TRCs (Furue and Yoshii 1997). However, this does not preclude the existence of additional apical membrane ion pathways (Doolin and Gilbertson 1996; Miyamoto et al. 2001).

Single-unit studies show that the amiloride-sensitive (AS) responses are associated with N fibers that respond specifically to Na salts. AI responses are associated with H fibers that respond to Na⁺, K⁺, NH, and other cations (Frank et al. 1983). N fiber responses can be ascribed to TRCs containing ENaCs. The generalist properties of H fibers suggest that TRCs might also contain an apical nonselective cation pathway that serves as the transducer for generalist cation taste responses. In pursuit of such a possibility, we have discovered that cetylpyridinium chloride (CPC) has, depending on concentration, pharmacological actions that either reversibly amplify the AI component of the CT response to NaCl or inhibit it. At inhibiting concentrations, it reversibly blocks the entire AI NaCl response, demonstrating that the NaCl CT response is composed exclusively of amiloride- and CPC-sensitive components. The effects of CPC on K⁺ and NH responses are consistent with those expected for a cation generalist transduction pathway.

	METHODS

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CT nerve recordings

Sprague-Dawley rats (150-200 g) were prepared for recording as previously described (Ye et al. 1994). Neural responses were amplified, filtered, full-wave rectified, and integrated with a time constant of 1 s. The rinse solution was 10 mM KCl. Typically stimulus solutions remained on the tongue for 2 min. Control stimuli consisting of 300 mM NaCl and 300 mM NH₄Cl, applied at the beginning and at the end of an experiment, were used to assess preparation stability. Stimuli consisted of NaCl solutions ranging from 20 to 500 mM, 300 mM KCl, and 100 mM NH₄Cl. The cetylpyridinium chloride (CPC, Sigma, St. Louis, MO) dose versus CT response relation was obtained using responses to 100 mM NaCl as baseline. CPC concentrations were (in µM): 50, 100, 250, 500, 1,000, and 2,000. The data were digitized and analyzed off-line. Responses were taken as the area under the response curve over the first minute of stimulation. The displayed CT responses in Figs. 1 and 2 are representative results from at least four separate experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 1. A: the effect of 250 µM cetylpyridinium chloride (CPC) on the integrated chorda tympani response to 300 mM NaCl and 300 mM NaCl + 100 µM amiloride. CPC enhanced the response by the same magnitude in each case. B: the effect of 2 mM CPC on the integrated chorda tympani response to 300 mM NaCl and 300 mM NaCl + 100 µM amiloride. CPC suppressed the entire amiloride-insensitive (AI) part of the response. C: the effect of increasing CPC concentration on the response to 100 mM NaCl. Rcpc is the response to 100 mM NaCl containing a given concentration of CPC, R0.1 M NaCl is the response to 100 mM NaCl. Rcpc/R0.1 M NaCl > 1 indicates an enhanced response, Rcpc/R0.1 M NaCl < 1 indicates a suppressed response. Values represent the means ± SE (n = 4)

View larger version (22K): [in this window] [in a new window]   Fig. 2. A: the enhancing effect of 250 µM CPC and suppressing effect of 2 mM CPC on the integrated chorda tympani response to 300 mM KCl. B: similar effects of CPC on the response to 100 mM NH4Cl. C: 150 mM NH4Cl causes taste receptor cells (TRCs) to acidify when placed on the apical side of a taste bud. In the presence of 2 mM CPC, NH uptake is inhibited, resulting in TRC alkalinization.

Intracellular pH measurement

To monitor the flux of NH ions across the apical membranes of polarized TRCs we used a separate in vitro preparation of a single fungiform taste bud (Lyall et al. 2001). Changes in intracellular pH (pH_i) were measured by imaging TRCs using the fluoroprobe, BCECF (Lyall et al. 2001). A decrease in pH_i indirectly indicates the apical entry of NH ions into TRCs. The TRCs were perfused on both sides with control solution containing (in mM) 150 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4. The temporal changes in pH_i were monitored following the exposure of the apical membrane to a similar solution containing 150 mM NH₄Cl at pH 7.4 in the presence and absence of 2 mM CPC.

	RESULTS

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Figure 1C shows that CPC caused the NaCl response to increase between 50 and 250 µM. Beyond 250 µM CPC, NaCl responses decreased reaching control level at ~700 µM. At 1 and 2 mM CPC, NaCl responses were less than control values. Figure 1A shows the effect of 250 µM CPC on the response to 0.3 M NaCl. When NaCl was displaced by NaCl + 250 µM CPC, the response increased rapidly to a higher level until rinsed from the tongue. Following a second NaCl stimulation, the AS response was eliminated in the presence of 100 µM amiloride. Adding 250 µM CPC in the presence of amiloride gave the same magnitude enhancement observed without amiloride, indicating that the AS and CPC-sensitive pathways are independent. Figure 1B shows the effect of 2 mM CPC on the response to 0.3 M NaCl. The presence of 2 mM CPC suppressed the response by 20%. Amiloride suppressed a second NaCl stimulation by 80%. Addition of 2 mM CPC reduced the response to baseline levels indistinguishable from the rinse response level, i.e., the NaCl response is composed entirely of AS and CPC-sensitive components.

The response to 300 mM KCl was increased by 50% in the presence of 250 µM CPC and decreased by 35% by 2 mM CPC (Fig. 2A). The response to 100 mM NH₄Cl was increased by 40% in the presence of 250 µM CPC and decreased by 30% by 2 mM CPC (Fig. 2B). In each case, a significant part of the response was CPC insensitive.

The influx of NH ions into TRCs from the apical side was observed in polarized TRCs. When the TRCs were bathed symmetrically in control solution, the mean pH_i was 7.48 (cf. Fig. 2C). The pH response to NH₄Cl depends on the relative permeability of NH and its conjugate base NH₃. Replacement of the apical control solution by the isosmotic 150 mM NH₄Cl caused pH_i to decrease, indicating that the acidic form, NH, can enter TRCs faster from the apical side than the base NH₃. Inside the TRCs NH gives up H⁺ and forms NH₃, which escapes. In contrast, when 150 mM NH₄Cl + 2 mM CPC was then placed on the apical side, pH_i increased rapidly indicating a decrease in the apical NH permeability relative to NH₃.

The CPC-enhanced NaCl response was studied over a range of NaCl concentrations (cf. Fig. 3). NaCl + 250 µM CPC was a saturating function of NaCl concentration with K_m = 185 ± 35 mM. The CPC-sensitive component, obtained with NaCl + 100 µM amiloride + 250 µM CPC, had K_m = 62 ± 11 mM. The AS component had K_m = 268 ± 71 mM.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Normalized chorda tympani (CT) response as a function of NaCl concentration for: NaCl + 250 µM CPC (), NaCl + 100 µM amiloride +250 µM CPC (), and their difference representing the AS part of the response (). In each experiment, the data were normalized to the response to 100 mM NaCl + 100 µM amiloride + 250 µM CPC. Values represent the means ± SE (n = 5). , the least-squares fit to: CTmax [NaCl]/(Km + [NaCl]).

	DISCUSSION

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Single units in the CT that respond nonselectively to various cations may imply TRCs that use nonselective cation channels as transducers. Investigation of this hypothesis has been impeded, however, by the lack of an effective pharmacological probe for such TRCs. CPC acts on the AI part of the CT response to NaCl, where it either reversibly enhances or suppresses the response. At the suppressing concentration of 2 mM, the blocking effect of CPC is additive with that of amiloride, indicating that NaCl responses are composed of two pharmacologically independent inputs. CPC has essentially similar effects on responses to KCl and NH₄Cl. In the latter case, CPC was shown to reduce the apical influx of NH into TRCs. This along with the rapidity and reversibility of CPC action suggest that the CPC-sensitive pathway is probably an apical membrane nonselective cation conductance. For KCl and NH₄Cl, there remain significant CPC-insensitive transduction pathways. We note that both pharmacological actions of CPC occur at concentrations above the CPC critical micelle concentration (Simoni and pan 1998), so the CPC actions reported here cannot be attributed to monomer-micelle transformations occurring within that concentration range.

A comparison of the parameters of the AS and CPC-sensitive parts of the NaCl response in Fig. 3 show the former to be a high-capacity, low-affinity system and the latter to be a low-capacity, high-affinity system similar respectively to N- and H-fiber types. Estimates of K_m values for N and H fibers are 220 and 81 mM, respectively (Frank et al. 1983), which compare well with 268 ± 71 and 62 ± 11 mM found here, respectively, for the AS and CPC-sensitive parts of the NaCl response. The CPC-sensitive component, like the H-fiber response, has an impact on the low concentration NaCl response and therefore has a role in determining thresholds.