Involvement of NADPH-dependent and cAMP-PKA sensitive H+ channels in the chorda tympani nerve responses to strong acids

Abstract

To investigate if chorda tympani (CT) taste nerve responses to strong (HCl) and weak (CO(2) and acetic acid) acidic stimuli are dependent upon NADPH oxidase-linked and cAMP-sensitive proton conductances in taste cell membranes, CT responses were monitored in rats, wild-type (WT) mice, and gp91(phox) knockout (KO) mice in the absence and presence of blockers (Zn(2+) and diethyl pyrocarbonate [DEPC]) or activators (8-(4-chlorophenylthio)-cAMP; 8-CPT-cAMP) of proton channels and activators of the NADPH oxidase enzyme (phorbol 12-myristate 13-acetate [PMA], H(2)O(2), and nitrazepam). Zn(2+) and DEPC inhibited and 8-CPT-cAMP, PMA, H(2)O(2), and nitrazepam enhanced the tonic CT responses to HCl without altering responses to CO(2) and acetic acid. In KO mice, the tonic HCl CT response was reduced by 64% relative to WT mice. The residual CT response was insensitive to H(2)O(2) but was blocked by Zn(2+). Its magnitude was further enhanced by 8-CPT-cAMP treatment, and the enhancement was blocked by 8-CPT-adenosine-3'-5'-cyclic monophospho-rothioate, a protein kinase A (PKA) inhibitor. Under voltage-clamp conditions, before cAMP treatment, rat tonic HCl CT responses demonstrated voltage-dependence only at ±90 mV, suggesting the presence of H(+) channels with voltage-dependent conductances. After cAMP treatment, the tonic HCl CT response had a quasi-linear dependence on voltage, suggesting that the cAMP-dependent part of the HCl CT response has a quasi-linear voltage dependence between +60 and -60 mV, only becoming sigmoidal when approaching +90 and -90 mV. The results suggest that CT responses to HCl involve 2 proton entry pathways, an NADPH oxidase-dependent proton channel, and a cAMP-PKA sensitive proton channel.

Figure 1

Effects of Zn²⁺ and DEPC on rat CT responses to acidic stimuli. (A) Shows a representative CT recording in which the rat tongue was first superfused with a rinse solution R1 (10 mM KCl) and then with 20 mM HCl solutions containing 0, 2.5, 5.0, and 10.0 mM ZnCl₂. The arrows indicate the time period when the tongue was stimulated with different solutions. (B) The mean normalized tonic CT response in 3 rats to 20 mM HCl were plotted as a function of either Zn²⁺ (•) or DEPC (○) concentration. The tonic HCl CT responses at 10, 15, and 20 mM Zn²⁺ and 10 mM DEPC were not different from the baseline rinse values (P > 0.05). The P values were calculated with respect to the normalized HCl tonic CT response in the absence of Zn²⁺ or DEPC. The P values (*) at different Zn²⁺ concentration were 5 mM (0.0075), 10 mM (0.0023), 15 mM (0.0001), and 20 mM (0.0001) relative to control (0 Zn²⁺). The P values (*) at different DEPC concentration were 2.5 mM (0.0007), 5 mM (0.0001), and 10 mM (0.0001). (C) Shows a representative CT recording in which the rat tongue was first superfused with a rinse solution R1 (10 mM KCl) and then with acidic stimuli (20 mM HCl, 20 mM H₃PO₄, and 20 mM CH₃COOH) in the absence and presence of 10 mM ZnCl₂ (Zn²⁺). The control responses to 300 mM NH₄Cl are shown at the beginning and end of the experiment and were nearly identical. The arrows indicate the time period when the tongue was stimulated with different solutions. (D) The mean normalized tonic CT response in 3 rats to 20 mM HCl, 20 mM H₃PO₄, and 20 mM CH₃COOH are shown in the absence and presence of 10 mM ZnCl₂ (Zn²⁺). The tonic CT response to HCl + Zn²⁺ was inhibited to the baseline rinse values (**P = 0.0004). There was no difference between the tonic CT response between CH₃COOH and CH₃COOH + Zn²⁺ (P > 0.05). Zn²⁺ inhibited the tonic CT response to H₃PO₄ by 65.8% (*P = 0.0016).

Figure 2

Effects of lingual voltage clamp, cAMP, and Zn²⁺ on the rat HCl CT response. (A) Rat CT responses 20 mM HCl were monitored at applied +90, +60, +30, 0, −30, −60, and −90 mV across the receptive field under control conditions and after topical lingual application of 15 mM 8-CPT-cAMP for 20 min. The mean normalized tonic HCl CT responses in 3 rats are plotted as a function of the applied voltage before (o; Control) and after cAMP treatment (•; Post-cAMP). The cAMP-dependent mean tonic responses (▴; cAMP dependent) at each applied voltage were calculated as the difference between the HCl CT response before and after cAMP treatment. The data points were fitted to the channel model described in the Appendix. (B) The mean normalized tonic CT responses in rats to 20 mM HCl and 20 mM HCl + 20 mM ZnCl₂ are shown at +90, 0, and −90 mV applied voltage across the receptive field. cAMP significantly enhanced the HCl CT response at +90, 0, and −90 mV relative to control. Under control conditions, the P values were calculated with reference to the mean normalized tonic HCl CT response at 0 mV. The P values (*) at −90 and +90 mV were 0.0102 and 0.0158, respectively. Cyclic-AMP enhanced the HCl CT response at 0 mV relative to control (***P = 0.0001). Post-cAMP, the P values (**) at −90 and +90 mV were 0.001 and 0.0034, respectively with respect of 0 mV. Zn²⁺ inhibited the HCl CT responses at +90, 0, and −90 mV to the rinse baseline under control conditions and after cAMP treatment, (P > 0.05 with respect to zero). (C)Effect of Ca²⁺, Mg²⁺, and Zn²⁺ on the rat HCl CT response. The rat tongues were first superfused with a rinse solution R1 (10 mM KCl) and then with 20 mM HCl solutions containing 20 mM CaCl₂, 20 mM MgCl₂, or 20 mM ZnCl₂. Addition of 20 mM CaCl₂, 20 mM MgCl₂, or 20 mM ZnCl₂ to R1 did not increase the CT response above baseline relative to R1 alone (data not shown). The mean normalized tonic HCl CT responses in 3 rats are shown in the presence of Ca²⁺, Mg²⁺, and Zn²⁺. Whereas Zn²⁺ inhibited the mean normalized HCl tonic CT response to baseline (*P = 0.0001), Ca²⁺ and Mg²⁺ did not have any effect on the tonic HCl CT response (P > 0.05).

Figure 3

Effect of H₂O₂ on the rat HCl CT response. (A) The rat tongues were initially superfused with rinse solutions (R1; Table 1) containing H₂O₂ (R1 + 0, 2.5, 5.0, 10.0, and 15.0% H₂O₂) and then with 20 mM HCl solutions containing H₂O₂ (HCl + 0, 2.5, 5.0, 10.0, and 15.0% H₂O₂), respectively (see also Supplementary Figure 3). The H₂O₂-induced enhancement in the tonic HCl response at a particular H₂O₂ concentration was calculated relative to the baseline with the same H₂O₂ in R1. The mean normalized tonic HCl CT responses in 3 rats are plotted as a function of increasing H₂O₂ concentration. The P values were calculated with respect to the mean normalized HCl tonic CT response in the absence of H₂O₂. The P values (*) at different H₂O₂ concentrations were as follows: 5% (0.0019), 10% (0.0003), and 15% (0.0003). (B) CT responses were monitored while the rat tongues were superfusing with R1 and then with 20 mM HCl, R1 + 15% H₂O₂, and 20 mM HCl + 15% H₂O₂, R2 (pH 7.4) and 10% CO₂ (pH 7.4; Table 1) and R3 (pH 6.1) and 525 mM CH₃COOK + 30 mM CH₃COOH (pH 6.1; Table 1). The mean normalized tonic CT responses in 3 rats to HCl, CO₂, and CH₃COOH are shown in the absence and presence of 15% H₂O₂. H₂O₂ only enhanced CT responses to HCl (*P = 0.0003). No significant difference was observed in the normalized tonic CT response to CO₂ or CH₃COOH (P > 0.05).

Figure 4

Effect of nitrazepam and PMA on the rat CT responses to acidic stimuli. (A) Shows a representative raw neural recording in which CT responses were monitored while the rat tongue was superfusing with R1 and then with 20 mM HCl, R2 and 10% CO₂ (pH 7.4; Table 1), and R3 and 20 mM CH₃COOH (pH 6.1; Table 1) before (Control) and after topical lingual application of 0.5 mM nitrazepam (Post-nitrazepam). The control responses to 300 mM NH₄Cl are shown at the beginning and end of the experiment and were nearly identical. The arrows indicate the time period when the tongue was stimulated with different solutions. (B) The mean normalized tonic CT responses in 3 rats to HCl, CO₂, and CH₃COOH are shown before (Control) and after nitrazepam treatment (Post-nitrazepam). Nitrazepam only enhanced HCl CT responses (*P = 0.013). No effect of nitrazepam was observed on the CT responses to CO₂ or CH₃COOH relative to control (P >0.05). (C) Shows a representative CT recording in which the rat tongue was first superfused with a rinse solution R1 (10 mM KCl) and then with 20 mM HCl solution under control conditions and after topical lingual application of 0.25 mM PMA. The arrows indicate the time period when the tongue was stimulated with different solutions.

Figure 5

Effect of H₂O₂ and Zn²⁺ on HCl CT responses in WT and gp91^phox KO mice. Shows representative normalized CT responses (A) in a WT mouse and (B) a KO mouse in which the tongues were initially superfused with rinse solutions R1, R1 + 15% H₂O₂, or R1 + 10 mM Zn²⁺ and then with 20 mM HCl, HCl + 15% H₂O₂, or HCl + 10 mM Zn²⁺. The arrows indicate the time period when the tongues were stimulated with different solutions.

Figure 6

Effect of H₂O₂ on the CT responses to HCl and CO₂ in WT and gp91^phox KO mice. Shows representative normalized CT responses (A) in a WT mouse and (B) a KO mouse in which the tongues were initially superfused with rinse solutions R1, R1 + 15% H₂O₂, R2 (pH 7.4), or R2 + 15% H₂O₂ (pH 7.4; Table 1) and then with 20 mM HCl, HCl + 15% H₂O₂, 10% CO₂ (pH 7.4; Table 1), or 10% CO₂ + 15% H₂O₂ (pH 7.4; Table 1). In (B) is also shown the response to R1, R1 + 10 mM Zn²⁺, 20 mM HCl, and 20 mM HCl + 10 mM Zn²⁺. The arrows indicate the time period when the tongues were stimulated with different solutions.

Figure 7

Effect of Zn²⁺, H₂O₂, and cAMP on the CT responses to HCl and CO₂ in gp91^phox KO mice and Sprague-Dawley rats. (A) Shows a representative CT recording in a gp91^phox KO mouse in which the mouse tongue was initially superfused with R1, R1 + 10 mM Zn²⁺, R1 + 15% H₂O₂, R2 (pH 7.4), or R2 + 15% H₂O₂ (pH 7.4; Table 1) and then with 20 mM HCl, 20 mM HCl + 10 mM Zn²⁺, 20 mM HCl+15% H₂O₂, 10% CO₂ (pH 7.4; Table 1) or 10% CO₂ + 15% H₂O₂ (pH 7.4; Table 1). Also shown is the CT response to 20 mM HCl after the topical lingual application of 8-CPT-cAMP. The arrows indicate the time period when the tongue was stimulated with different solutions. (B) Shows a representative rat CT recording in which the tongue was first superfused with R1 and then with 20 mM HCl under control conditions and after topical lingual application of 15 mM 8-CPT-cAMP for 20 min. The arrows indicate the time period when the tongues were stimulated with different solutions. (C) Shows a representative rat CT recording in which the tongue was first superfused with R1 and then with 20 mM HCl under control conditions, after topical lingual application of 4.5 mM Rp-8-CPT-cAMPS for 10 min and after topical lingual application of 15 mM 8-CPT-cAMP (Table 2). The arrows indicate the time period when the tongues were stimulated with different solutions.

Figure 8

Effects of Zn²⁺, H₂O₂, and cAMP on the CT responses to acidic stimuli in WT and gp91^phox KO mice. In 3 WT mice, the P values under different conditions were calculated with respect to the mean normalized tonic HCl CT response and were as follows: HCl + Zn²⁺ (****; 0.0001), HCl + H₂O₂ (**; 0.004), 10% CO₂ (*; 0.0168), and 10% CO₂ + H₂O₂ (***; 0.0014). In 6 KO mice, the P values under different conditions were calculated with respect to the mean normalized tonic HCl CT response and were as follows: HCl + Zn²⁺ (*; 0.0276), HCl + H₂O₂ (>0.05), 10% CO₂ (*; 0.0295), 10% CO₂ + H₂O₂ (*; 0.0295) and post-cAMP HCl (****; 0.0001). The mean tonic HCl CT response in KO mice was smaller relative to its value in WT mice (****P = 0.0001).