Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux

Abstract

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular Na(+) activity ([Na(+)](i)) in polarized rat fungiform taste receptor cells (TRCs) using fluorescence imaging and by chorda tympani (CT) taste nerve recordings. CT responses were monitored during lingual stimulation with ethanol solutions containing NaCl or KCl. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial Na(+) channel [ENaC]) or the vanilloid receptor-1 (VR-1) antagonists capsazepine or SB-366791, which also block the Bz-insensitive salt taste receptor, a VR-1 variant. CT responses were recorded at 23 degrees C or 42 degrees C (a temperature at which the VR-1 variant salt taste receptor activity is maximally enhanced). In the absence of permeable cations, ethanol induced a transient decrease in TRC volume, and stimulating the tongue with ethanol solutions without added salt elicited only transient phasic CT responses that were insensitive to elevated temperature or SB-366791. Preshrinking TRCs in vivo with hypertonic mannitol (0.5 M) attenuated the magnitude of the phasic CT response, indicating that in the absence of mineral salts, transient phasic CT responses are related to the ethanol-induced osmotic shrinkage of TRCs. In the presence of mineral salts, ethanol increased the Bz-insensitive apical cation flux in TRCs without a change in cell volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a transient phasic component and a sustained tonic component. Ethanol increased the Bz-insensitive NaCl CT response. This effect was further enhanced by elevating the temperature from 23 degrees C to 42 degrees C, and was blocked by SB-366791. We conclude that in the presence of mineral salts, ethanol modulates the Bz-insensitive VR-1 variant salt taste receptor.

Figure 1.

SBFI loading and the effect of ouabain on TRC [Na⁺]_i. (A) An isolated piece of rat anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber and was perfused with Ringer's solution containing SBFI-AM. The taste bud was viewed from the basolateral side with a 60× magnification. The figure shows the transmitted image of the taste bud (Transmitted), fluorescence image of the same taste bud excited at 340 nm (340 nm), fluorescence image of the same taste bud excited at 380 nm (380 nm), and the fluorescence intensity ratio image (340 nm/380 nm) with five ROIs. Bar, 10 μm. (B) A polarized epithelial preparation was initially perfused on both sides with control Ringer's solution containing 150 mM NaCl (pH 7.4). Temporal changes in F₃₄₀/F₃₈₀ (FIR) of SBFI-loaded TRCs were monitored while the basolateral membrane was perfused with Ringer's solution containing 3 mM ouabain. Values are expressed as mean ± SEM of n, where n = number of ROIs within the taste bud.

Figure 2.

Effect of apical Na⁺, Bz, and CZP on TRC [Na⁺]_i. (A) A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NaCl (a–b), 150 mM NaCl + 25 μM Bz (b–c), and 150 mM NMDG-Cl + 25 μM Bz (c–d). (B) A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). Temporal changes in F₄₉₀ of Na-green were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NaCl (a–b), 150 mM NaCl + 25 μM Bz (b–c), and 150 mM NaCl + 25 μM Bz + 25 μM CZP (c–d). The relative changes in [Na⁺]_i are presented as percent changes in F₄₉₀ relative to bilateral 0 Na⁺ and are expressed as mean ± SEM of the of n, where n = number of ROIs within the taste bud.

Figure 3.

Effect of ethanol on TRC [Na⁺]_i. A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). (A) Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with 0 Na⁺ Ringer's solution containing 20% (a–b–c) or 10% (f–g–h) ETH and with Ringer's solution containing 150 mM NaCl + 5 μM Bz + ETH at 20% ETH (c–d–e), 10% ETH (h–i–j), and 0 ETH (k–l–m). (B) Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with 0 Na⁺ Ringer's solution containing 40% ETH (a–b–c) and with Ringer's solution containing 150 mM NaCl + 5 μM Bz + 40% ETH (c–d–e) and 0 ETH (g–h–i). The relative changes in [Na⁺]_i are presented as percent changes in F₄₉₀ relative to bilateral 0 Na⁺ and are expressed as the mean ± SEM of n, where n = number of ROIs within the taste bud. (C) Ethanol-induced changes in F₄₉₀ in different ROIs within the taste buds. Data are plotted from five individual polarized TRC preparations containing 47 ROIs. In each case, the apical membrane was first perfused with 0 Na⁺ Ringer's solution + 40% ETH and then with control Ringer's solution containing 150 mM NaCl + 5 μM Bz + 40% ETH. The histogram shows the number of ROIs that fall within a given interval corresponding to 0–10, 10–20, 20–30, and 30–40% increase in F₄₉₀.

Figure 4.

Effect of ethanol on apical Na⁺ flux. A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). (A) Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NaCl + 5 μM Bz with 40% ETH (a–b), 10% ETH (c–d), and 0 ETH (e–f). (B) Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NaCl + 5 μM Bz + ETH at 0 (a–b), 20% (b–c), and 40% (c–d) concentration. The relative changes in [Na⁺]_i are presented as percent changes in F₄₉₀ relative to bilateral 0 Na⁺ and are expressed as the mean ± SEM of n, where n = number of ROIs within the taste bud. (C) Summary of data from 17 TRC preparations. The mean percent changes in F₄₉₀ at different ethanol concentrations were normalized to F₄₉₀ values at 0 ETH. The number in parenthesis indicates the number of individual TRC preparations (N) investigated for a particular ETH concentration. All values were significantly greater (P < 0.01) than control (0 ETH).

Figure 5.

Effect of ethanol on apical Na⁺ flux. (A) A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). Temporal changes in FIR (F₃₄₀/F₃₈₀) of SBFI-loaded TRCs were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NMDG-Cl + 40% ETH (a–b–c) or with 150 mM NaCl + 5 μM Bz + 40% ETH (c–d–e) and 0 ETH (f–g–h). The relative changes in [Na⁺]_i are presented as changes in FIR relative to bilateral 0 Na⁺ and are expressed as the mean ± SEM of n, where n = number of ROIs within the taste bud. (B) A polarized epithelial preparation was initially perfused on both sides with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl (pH 7.4). Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the apical membrane was perfused with Ringer's solution containing 150 mM NaCl + 5 μM Bz + ETH at 40% (a–b–c), 10% (i–j–k), and 0 ETH (c–d–e) concentration, and with Ringer's solution containing 150 mM NaCl + 5 μM Bz + 10 μM CZP + ETH at 40% (g–h–i) or 0 (e–f–g) concentration. The relative changes in [Na⁺]_i are presented as percent changes in F₄₉₀ relative to bilateral 0 Na⁺ and are expressed as the mean ± SEM of n, where n = number of ROIs within the taste bud.

Figure 6.

Effect of ethanol on isolated TRCs. (A) Na-green loading in isolated fungiform TRCs. Isolated fungiform taste bud fragments were placed onto coverslips coated with CELL-TAK, which were attached to the bottom of the recording/perfusion chamber. Taste bud fragments were perfused with control Ringer's solution containing Na-green-AM for 2 h. The taste buds were imaged at 60× magnification. Bar, 10 μm. The figure shows the transmitted image of a taste bud fragment in an optical plane (Transmitted), and the fluorescence image of the same taste bud in the same optical plane excited at 490 nm perfused with 0 Na⁺ Ringer's solution (F₄₉₀ 0Na) and with Ringer's solution containing 150 mM Na (F₄₉₀ 150Na). Bar, 10 μm. (B) Initially, an isolated fungiform taste bud fragment with five individual TRCs was perfused with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl + 5 μM Bz + 10 μM zoniporide (pH 7.4). Zoniporide was added to block the activity of basolateral NHE-1. Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the taste bud fragment was perfused with Ringer's solution containing 150 mM NaCl + 5 μM Bz + 10 μM zoniporide + ethanol at 10 or 15% concentration. The relative changes in [Na⁺]_i in each TRC are presented as percent changes in F₄₉₀ relative to bilateral 0 Na⁺. (C) Initially an isolated fungiform taste bud fragment with three individual TRCs was perfused with 0 Na⁺ Ringer's solution containing 150 mM NMDG-Cl + 5 μM Bz + 10 μM zoniporide (pH 7.4). Temporal changes in F₄₉₀ of Na-green–loaded TRCs were monitored while the taste bud fragment was perfused with Ringer's solution containing 150 mM NaCl + 5 μM Bz + 10 μM zoniporide + 10% ethanol or with 150 mM NaCl + 5 μM Bz + 10 μM zoniporide + 10% ethanol + 1 μM SB-366791. The relative changes in [Na⁺]_i are expressed as the mean ± SEM of n, where n = number of TRCs within the taste bud.

Figure 7.

Effect of ethanol on the CT responses. Rat tongue was stimulated with 40, 60, 80, and 100% ethanol (ETH) solutions containing either 0 (A) or 0.1 μM SB-366791 (B). CT responses were recorded at 23°C (A and B) and at 42°C (C) with reference to H₂O rinse at 23°C. The time period at which the rat tongue was superfused with different solutions is indicated by arrows. Following 100% ETH stimulation, the tongue was rinsed with deionized H₂O (A; thick arrows). The tongue was rinsed with 10 mM KCl (R) and then stimulated with 300 mM NH₄Cl (NH₄Cl) to obtain a reference CT response. The data were normalized to 0.3 M NH₄Cl CT response in each animal.

Figure 8.

Effect of mannitol on the ethanol-induced transient phasic CT response. (A) CT response was monitored while the rat tongue was first rinsed with deionized H₂0 and then with 50% ethanol (50% ETH). The CT responses were also recorded when the rinse and the ethanol solutions contained 0.5 M mannitol (M). The time period at which the rat tongue was superfused with different solutions is indicated by arrows. (B) Summary of data from three such experiments. Each bar represents the mean ± SEM of the normalized peak response from three animals (N).

Figure 9.

Effect of mannitol (M), urea (U), and ethanol (ETH) on the NaCl CT response. (A) CT response was monitored while the rat tongue was first rinsed with 10 mM KCl (R) and then stimulated with 10 mM KCl + 100 mM NaCl (R + N) and with 10 mM KCl + 100 mM NaCl + 5 μM Bz (R + N + Bz). The CT responses were also recorded when the rinse and the stimulating solutions contained 1 M mannitol (M), 6.8 M urea (U), or 6.8 M ethanol (40% ETH). The arrows represent the time period when the tongue was stimulated with the various NaCl stimulating solutions. The tongue was stimulated with 300 mM NH₄Cl to obtain a reference CT response. The data were normalized to the 0.3 M NH₄Cl CT response in each animal. (B) Effect of ethanol on the transepithelial electrical resistance across the tongue. The changes in the transepithelial electrical resistance in the presence of 40% ETH were represented relative to 10 mM KCl (R) or 100 mM NaCl + 5 μM Bz (N + Bz). The values are expressed as the mean ± SEM from four animals (N). *, P < 0.05 (paired). (C) Summary of data from three individual animals. The open bars (left) represent the CT response in the presence of NaCl (N), NaCl + 1 M mannitol (N + M), NaCl + 6.8 M Urea (N + U), and NaCl + 6.8 M ethanol (N + ETH). The hatched bars (middle) represent the Bz-insensitive component of the CT response to N + Bz, N + M + Bz, N + U + Bz, and N + ETH + Bz. The crosshatched bars (right) represent the Bz-sensitive component of the CT response to N, N + M, N + U, and N + ETH. The Bz-sensitive component was obtained by subtracting the Bz-insensitive response from the corresponding CT response in the absence of Bz. The data were normalized to the 0.3 M NH₄Cl CT response in each animal. The values are expressed as the mean ± SEM from three animals (N). *, P < 0.05 (paired).

Figure 10.

Effect of SB-366791, flow rate, and temperature on the CT response to mineral salts. (A) Rat tongue was stimulated with 10 mM KCl (R), 10 mM KCl + 40% ethanol (R + ETH), or 10 mM KCl + 40% ethanol + 0.1 μM SB-366791 (R + ETH + SB). The time period at which the rat tongue was superfused with different solutions is indicated by arrows. (B) CT response was monitored while the rat tongue was first rinsed with 10 mM KCl (R) and then stimulated with 10 mM KCl + 60% ETH (R + ETH). The tongue was superfused with the rinse solution at the rate of 1 ml/s while the stimulating solutions were perfused at the rate of 1 ml/s or 0.13 ml/s. The tongue was stimulated with 300 mM NH₄Cl (1 ml/s) to obtain a reference CT response. The data were normalized to the 0.3 M NH₄Cl CT response in each animal. (C) Rat tongue was stimulated with 10 mM KCl maintained at 23°C (R_23°) and then with 10 mM KCl + 100 mM NaCl + 5 μM Bz (N + Bz) or with 10 mM KCl + 100 mM NaCl + 30% ethanol (N + Bz + ETH) maintained at 23°C or 42°C. The time period at which the rat tongue was superfused with different solutions is indicated by arrows.