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The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellnlar pH ([pH.sub.i]) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and C[O.sub.2] were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC [pH.sub.i] and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in [pH.sub.i] shifted the equilibrinin from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the [pH.sub.i]-induced decrease in TRC volume. The phasic part of the CT response to HCl or C[O.sub.2] was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 [micro]M cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The [pH.sub.i]-induced decrease in TRC volume induced a flufenamic acid-sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and C[O.sub.2]. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in phi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid-sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular [Na.sup.+] activity ([[[Na.sup.+]].sub.i]) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KC1 and NaCl were recorded in Sprague-Dawley rats, and in wild-type (WT) and vanilloid receptor-1 (VR-1) knockout mice (KO). CT responses were monitored in the presence of Bz, a specific blocker of the epithelial [Na.sup.+] channel (ENaC). CT responses were also recorded in the presence of agonists (resiniferatoxin and elevated temperature) and antagonists (capsazepine and SB-366791) of VR-1 that similarly modulate the Bz-insensitive VR-1 variant salt taste receptor. In the absence of mineral salts, ethanol induced a transient decrease in TRC volume and elicited only transient phasic CT responses. In the presence of mineral salts, ethanol increased the apical cation flux in TRCs without a change in volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a phasic component and a sustained tonic component. At concentrations 50%, those CT responses were inhibited. Resiniferatoxin and elevated temperature increased the sensitivity of the CT response to ethanol in salt-containing media, and SB-366791 inhibited the effect of ethanol, resiniferatoxin, and elevated temperature on the CT responses to mineral salts. VR-1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to ethanol. We conclude that ethanol increases salt taste sensitivity by its direct action on the Bz-insensitive VR-1 variant salt taste receptor. KEY WORDS: resiniferatoxin * SB-366791 * capsazepine * salt taste * [Na.sup.+] imaging

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular [Na.sup.+] activity ([[[Na.sup.+]].sub.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 KC1. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial [Na.sup.2+] 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. KEY WORDS: [Na.sup.+] imaging * salt taste * SB-366791 * capsazepine * chorda tympani

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH ([pH.sub.i]) and [Na.sup.+] activity ([[Na.sup.+]].sub.i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH ([pH.sub.o]). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying [pH.sub.o] to 2.0 or 10.3. At constant [pH.sub.o], buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HC[O.sub.3].sup.-/C[O.sub.2 ] inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HC[O.sub.3].sup.-/C[O.sub.2] buffer, suggesting a regulatory role for [pH.sub.i]. In polarized TRCs step changes in apical [pH.sub.o] from 10.3 to 2.0 induced a linear decrease in [pH.sub.i] that remained within the physiological range (slope = 0.035; [r.sup.2] = 0.98). At constant [pH.sub.o], perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HC[O.sub.3]-/C[O.sub.2] decreased resting TRC phi, and MK-507 or MK-417 attenuated the decrease in [pH.sub.i] in TRCs perfused with HC[O.sub.3]-/C[O.sub.2] buffer. In parallel experiments, TRC [[Na.sub.+]]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical [Na.sub.+], and (d) acid loading the cells with N[H.sub.4]Cl or sodium acetate at constant [pH.sub.o]. Diethylpyrocarbonate and [Zn.sup.2+], modification reagents for histidine residues in proteins, attenuated the C[O.sub.2]-induced inhibition of NaCl CT responses and the [pH.sub.i]-induced inhibition of apical [Na.sub.+] influx in TRCs. We conclude that TRC phi regulates [Na.sub.+]-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures. KEYWORDS: salt taste * sour taste * intracellular pH * taste mixture interaction * epithelial sodium channel

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°C or 42°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°C to 42°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. [ABSTRACT FROM AUTHOR]

Investigates mixture interactions between sour taste and salt taste modalities by monitoring chorda tympani (CT) responses to sodium chloride (NaCl) and intracellular sodium activity in polarized taste receptor cells by hydrogen ion concentration (pH). Effect of external pH, benzamil, and membrane permeable weak acids on NaCl CT responses.