Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats.

During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19-23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19-23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19-23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that modulates the postnatal increase in TRC ENaC and the neural and behavioral responses to NaCl.

Regulatory Effects of Ca2+ and H+ on the Rat Chorda Tympani Response to NaCl and KCl.

Modulatory effects of pHi and [Ca(2+)]i on taste receptor cell (TRC) epithelial sodium channel (ENaC) were investigated by monitoring chorda tympani (CT) responses to NaCl and KCl at various lingual voltages, before and after lingual application of ionomycin and with 0-10mM CaCl2 in the stimulus and rinse solutions adjusted to pHo 2.0-9.7. 0.1 and 0.5M KCl responses varied continuously with voltage and were fitted to an apical ion channel kinetic model using the same parameters. ENaC-dependent NaCl CT response was fitted to the same channel model but with parameters characteristic of ENaC. A graded increase in TRC [Ca(2+)]i decreased the ENaC-dependent NaCl CT response, and inhibited and ultimately eliminated its pH sensitivity. CT responses to KCl were pHi- and [Ca(2+)]i-independent. Between ±60 mV applied lingual potential, the data were well described by a linear approximation to the nonlinear channel equation and yielded 2 parameters, the open-circuit response and the negative of the slope of the line in the CT response versus voltage plot, designated the response conductance. The ENaC-dependent NaCl CT response conductance was a linear function of the open-circuit response for all pHi-[Ca(2+)]i combinations examined. Analysis of these data shows that pHi and [Ca(2+)]i regulate TRC ENaC exclusively through modulation of the maximum CT response.

Effect of ENaC modulators on rat neural responses to NaCl.

The effects of small molecule ENaC activators N,N,N-trimethyl-2-((4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanoyl)oxy)ethanaminium iodide (Compound 1) and N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide (Compound 2), were tested on the benzamil (Bz)-sensitive NaCl chorda tympani (CT) taste nerve response under open-circuit conditions and under ±60 mV applied lingual voltage-clamp, and compared with the effects of known physiological activators (8-CPT-cAMP, BAPTA-AM, and alkaline pH), and an inhibitor (ionomycin+Ca2+) of ENaC. The NaCl CT response was enhanced at -60 mV and suppressed at +60 mV. In every case the CT response (r) versus voltage (V) curve was linear. All ENaC activators increased the open-circuit response (ro) and the voltage sensitivity (κ, negative of the slope of the r versus V curve) and ionomycin+Ca2+ decreased ro and κ to zero. Compound 1 and Compound 2 expressed a sigmoidal-saturating function of concentration (0.25-1 mM) with a half-maximal response concentration (k) of 0.49 and 1.05 mM, respectively. Following treatment with 1 mM Compound 1, 8-CPT-cAMP, BAPTA-AM and pH 10.3, the Bz-sensitive NaCl CT response to 100 mM NaCl was enhanced and was equivalent to the Bz-sensitive CT response to 300 mM NaCl. Plots of κ versus ro in the absence and presence of the activators or the inhibitor were linear, suggesting that changes in the affinity of Na+ for ENaC under different conditions are fully compensated by changes in the apical membrane potential difference, and that the observed changes in the Bz-sensitive NaCl CT response arise exclusively from changes in the maximum CT response (rm). The results further suggest that the agonists enhance and ionomycin+Ca2+ decreases ENaC function by increasing or decreasing the rate of release of Na+ from its ENaC binding site to the receptor cell cytosol, respectively. Irrespective of agonist type, the Bz-sensitive NaCl CT response demonstrated a maximum response enhancement limit of about 75% over control value.

The ArrayGrid: a methodology for applying multiple samples to a single TEM specimen grid.

High throughput transmission electron microscopy (TEM) is limited by the time that it takes to prepare each specimen and insert it on the microscope. It is further impeded by the deteriorating vacuum of the microscope upon frequent specimen cycling. Nevertheless, in most cases only a small fraction of the specimen is examined and sufficient to provide hundreds of images. Here we demonstrate that microarray technology can be used to accurately position picoliter quantities of different samples in a single TEM grid, with negligible cross-contamination. Key features are a contact-mode deposition on a robust formvar-carbon support. The TEM grid containing a microarray of different samples, the ArrayGrid, can also be negatively stained. The ArrayGrid increases the efficiency of TEM grid preparation and examination by at least by one order of magnitude, and is very suitable for screening and data collection especially in experiments that generate a multiplicity of samples.

Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to salty and sour taste stimuli.

The relationship between taste receptor cell (TRC) Ca(2+) concentration ([Ca(2+)](i)) and rat chorda tympani (CT) nerve responses to salty [NaCl and NaCl+benzamil (Bz)] and sour (HCl, CO(2), and acetic acid) taste stimuli was investigated before and after lingual application of ionomycin+Ca(2+), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), U73122 (phospholipase C blocker), and thapsigargin (Ca(2+)-ATPase inhibitor) under open-circuit or lingual voltage-clamp conditions. An increase in TRC [Ca(2+)](i) attenuated the tonic Bz-sensitive NaCl CT response and the apical membrane Na(+) conductance. A decrease in TRC [Ca(2+)](i) enhanced the tonic Bz-sensitive and Bz-insensitive NaCl CT responses and apical membrane Na(+) conductance but did not affect CT responses to KCl or NH(4)Cl. An increase in TRC [Ca(2+)](i) did not alter the phasic response but attenuated the tonic CT response to acidic stimuli. A decrease in [Ca(2+)](i) did not alter the phasic response but attenuated the tonic CT response to acidic stimuli. In a subset of TRCs, a positive relationship between [H(+)](i) and [Ca(2+)](i) was obtained using in vitro imaging techniques. U73122 inhibited the tonic CT responses to NaCl, and thapsigargin inhibited the tonic CT responses to salty and sour stimuli. The results suggest that salty and sour taste qualities are transduced by [Ca(2+)](i)-dependent and [Ca(2+)](i)-independent mechanisms. Changes in TRC [Ca(2+)](i) in a BAPTA-sensitive cytosolic compartment regulate ion channels and cotransporters involved in the salty and sour taste transduction mechanisms and in neural adaptation. Changes in TRC [Ca(2+)](i) in a separate subcompartment, sensitive to inositol trisphosphate and thapsigargin but inaccessible to BAPTA, are associated with neurotransmitter release.

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

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.

Human salt taste and the lingual surface potential correlate.

We have demonstrated in humans that Na(+) evokes changes in the lingual surface potential (LSP) using a custom chamber. To assess whether a relationship exists between the Na(+)-evoked changes in the LSP and the intensity of salt taste, we measured the LSP and the intensity of salt taste simultaneously in 7 subjects using test solutions (50, 100, 300, and 1000 mM NaCl) presented in random order. The evoked LSPs and intensity scores correlated with one another well (r(2) = 0.992, P < 0.01). We then screened 14 subjects for their ability to discriminate between 100 and 300 mM NaCl using the chamber. Three subjects were consistently capable of distinguishing the salt concentrations. In these 3 subjects, an inhibitor of the epithelial sodium channel, amiloride (10 muM), blocked the ability to distinguish salt concentrations and affected the LSP. These data suggest that the LSP may be a component of the signal transduction system involved in human salt taste. In adept salt tasters, an amiloride-sensitive mechanism appears to have a role in distinguishing salt concentrations.

Effect of nicotine on chorda tympani responses to salty and sour stimuli.

The effect of nicotine on the benzamil (Bz)-insensitive (transient receptor potential vanilloid-1 variant cation channel, TRPV1t) and the Bz-sensitive (epithelial Na(+) channel, ENaC) salt taste receptors and sour taste was investigated by monitoring intracellular Na(+) and H(+) activity (pH(i)) in polarized fungiform taste receptor cells (TRCs) and the chorda tympani (CT) nerve responses to NaCl, KCl, and HCl. CT responses in Sprague-Dawley rats and both wildtype and TRPV1 knockout (KO) mice were recorded in the presence and absence of agonists [resiniferatoxin (RTX) and elevated temperature] and an antagonist (SB-366791) of TRPV1t, the ENaC blocker (Bz), and varying apical pH (pH(o)). At concentrations <0.015 M, nicotine enhanced and at >0.015 M, it inhibited CT responses to KCl and NaCl. Nicotine produced maximum enhancement in the Bz-insensitive NaCl CT response at pH(o) between 6 and 7. RTX and elevated temperature increased the sensitivity of the CT response to nicotine in salt-containing media, and SB-366791 inhibited these effects. TRPV1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to nicotine, RTX, and elevated temperature. We conclude that nicotine modulates salt responses by direct interaction with TRPV1t. At pH(o) >8, the apical membrane permeability of nicotine was increased significantly, resulting in increase in TRC pH(i) and volume, activation of ENaC, and enhancement of the Bz-sensitive NaCl CT response. At pH(o) >8, nicotine also inhibited the phasic component of the HCl CT response. We conclude that the effects of nicotine on ENaC and the phasic HCl CT response arise from increases in TRC pH(i) and volume.

A psychophysical and electrophysiological analysis of salt taste in Trpv1 null mice.

Current evidence suggests salt taste transduction involves at least two mechanisms, one that is amiloride sensitive and appears to use apically located epithelial sodium channels relatively selective for Na(+) and a second that is amiloride insensitive and uses a variant of the transient receptor potential vanilloid receptor 1 (TRPV1) that serves as a nonspecific cation channel. To provide a functional context for these findings, we trained Trpv1 knockout (KO) and wild-type (WT) C57BL/6J mice (n = 9 or 10/group) in a two-response operant discrimination procedure and measured detection thresholds to NaCl and KCl with and without amiloride. The KO and WT mice had similar detection thresholds for NaCl and KCl. Amiloride shifted the NaCl sensitivity curve to the same degree in both groups and had virtually no effect on KCl thresholds. In addition, a more detailed analysis of chorda tympani nerve (CT) responses to NaCl, with and without benzamil (Bz, an amiloride analog) treatment revealed that the tonic portion of the CT response of KO mice to NaCl + Bz was absent, but both KO and WT mice displayed some degree of a phasic response to NaCl with and without Bz. Because these transients constitute the entire CT response to NaCl + Bz in Trpv1 KO mice, it is possible that these signals are sufficient to maintain normal NaCl detectabilty in the behavioral task used here. Additionally, there may be other amiloride-insensitive salt transduction mechanisms in taste receptor fields other than the anterior tongue that maintain normal salt detection performance in the KO mice.

Intracellular pH modulates taste receptor cell volume and the phasic part of the chorda tympani response to acids.

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) 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 CO2 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 pHi 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 pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 microM 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 pHi-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 CO2. 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.

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

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.

Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. II. Effect on chorda tympani salt responses.

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular Na(+) activity ([Na(+)](i)) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KCl 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(+) 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%, ethanol enhanced responses to KCl and NaCl, while at ethanol 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.

The mammalian amiloride-insensitive non-specific salt taste receptor is a vanilloid receptor-1 variant.

The amiloride-insensitive salt taste receptor is the predominant transducer of salt taste in some mammalian species, including humans. The physiological, pharmacological and biochemical properties of the amiloride-insensitive salt taste receptor were investigated by RT-PCR, by the measurement of unilateral apical Na+ fluxes in polarized rat fungiform taste receptor cells and by chorda tympani taste nerve recordings. The chorda tympani responses to NaCl, KCl, NH4Cl and CaCl2 were recorded in Sprague-Dawley rats, and in wild-type and vanilloid receptor-1 (VR-1) knockout mice. The chorda tympani responses to mineral salts were monitored in the presence of vanilloids (resiniferatoxin and capsaicin), VR-1 antagonists (capsazepine and SB-366791), and at elevated temperatures. The results indicate that the amiloride-insensitive salt taste receptor is a constitutively active non-selective cation channel derived from the VR-1 gene. It accounts for all of the amiloride-insensitive chorda tympani taste nerve response to Na+ salts and part of the response to K+, NH4+ and Ca2+ salts. It is activated by vanilloids and temperature (> 38 degrees C), and is inhibited by VR-1 antagonists. In the presence of vanilloids, external pH and ATP lower the temperature threshold of the channel. This allows for increased salt taste sensitivity without an increase in temperature. VR-1 knockout mice demonstrate no functional amiloride-insensitive salt taste receptor and no salt taste sensitivity to vanilloids and temperature. We conclude that the mammalian non-specific salt taste receptor is a VR-1 variant.

Basolateral Na+-H+ exchanger-1 in rat taste receptor cells is involved in neural adaptation to acidic stimuli.

The role of basolateral Na(+)-H(+) exchanger isoform-1 (NHE-1) was investigated in neural adaptation of rat taste responses to acidic stimuli, by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. In TRCs perfused with CO(2)/HCO(3)(-)-free solution (pH 7.4), removal of basolateral Na(+) decreased pH(i) reversibly and zoniporide, a specific NHE-1 blocker, inhibited the Na(+)-induced changes in pH(i). The spontaneous rate of TRC pH(i) recovery from NH(4)Cl pulses was inhibited by basolateral zoniporide with a K(i) of 0.33microm. Exposure to basolateral ionomycin, reversibly increased TRC Ca(2+), resting pH(i), and the spontaneous rate of pH(i) recovery from an NH(4)Cl pulse. These effects of Ca(2+) on pH(i) were blocked by zoniporide. In in vivo experiments, topical lingual application of zoniporide increased the magnitude of the CT responses to acetic acid and CO(2), but not to HCl. Topical lingual application of ionomycin did not affect the phasic part of the CT responses to acidic stimuli, but decreased the tonic part by 50% of control over a period of about 1 min. This increased adaptation in the CT response was inhibited by zoniporide. Topical lingual application of 8-CPT-cAMP increased the CT responses to HCl, but not to CO(2), and acetic acid. In the presence of cAMP, ionomycin increased sensory adaptation to HCl, CO(2), and acetic acid. Thus, cAMP and Ca(2+) independently modulate CT responses to acidic stimuli. While cAMP enhances TRC apical H(+) entry and CT responses to strong acid, an increase in Ca(2+) activates NHE-1, and increases neural adaptation to all acidic stimuli.

Na+-H+ exchange activity in taste receptor cells.

mRNA for two Na(+)-H(+)-exchanger isoforms 1 and 3 (NHE-1 and NHE-3) was detected by RT-PCR in fungiform and circumvallate taste receptor cells (TRCs). Anti-NHE-1 antibody binding was localized to the basolateral membranes, and the anti-NHE-3 antibody was localized in the apical membranes of fungiform and circumvallate TRCs. In a subset of TRCs, NHE-3 immunoreactivity was also detected in the intracellular compartment. For functional studies, an isolated lingual epithelium containing a single fungiform papilla was mounted with apical and basolateral sides isolated and perfused with nominally CO(2)/HCO(3)(-)-free physiological media (pH 7.4). The TRCs were monitored for changes in intracellular pH (pH(i)) and Na(+) ([Na(+)](i)) using fluorescence ratio imaging. At constant external pH, 1) removal of basolateral Na(+) reversibly decreased pH(i) and [Na(+)](i); 2) HOE642, a specific blocker, and amiloride, a nonspecific blocker of basolateral NHE-1, attenuated the decrease in pH(i) and [Na(+)](i); 3) exposure of TRCs to basolateral NH(4)Cl or sodium acetate pulses induced transient decreases in pH(i) that recovered spontaneously to baseline; 4) pH(i) recovery was inhibited by basolateral amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), HOE642, and by Na(+) removal; 5) HOE642, MIA, EIPA, and amiloride inhibited pH(i) recovery with K(i) values of 0.23, 0.46, 0.84, and 29 microM, respectively; and 6) a decrease in apical or basolateral pH acidified TRC pH(i) and inhibited spontaneous pH(i) recovery. The results indicate the presence of a functional NHE-1 in the basolateral membranes of TRCs. We hypothesize that NHE-1 is involved in sour taste transduction since its activity is modulated during acid stimulation.

Salt-evoked lingual surface potential in humans.

Salt sensing in animals involves the epithelial sodium channel (ENaC). If ENaC were involved in human salt sensing, then the lingual surface potential (LSP) would hyperpolarize when exposed to sodium. We developed a chamber to measure the LSP while different solutions superfused the surface of the tongue and a technique to adjust for the junction potentials induced by varying salt concentrations. Changing the superfusion solution from rinse solution (30 mM KCl) to 300 mM NaCl (+30 mM KCl) caused the LSP to hyperpolarize by 10.1 +/- 0.7 mV (n = 13, P < 0.001). With repeated challenge the LSP response was reproducible. Increasing the Na concentration from 100 to 600 mM increased hyperpolarization by 35 +/- 4.8% (n = 9, P < 0.001). To examine whether amiloride affects the LSP, 0.1 mM amiloride was added to 300 mM NaCl; it reduced the hyperpolarization by 18.5 +/- 4.3% (P < 0.005, n = 11). However, the amiloride effect was not uniform: in six volunteers, amiloride inhibited the LSP by as much as 42%, while in five subjects, amiloride inhibited <5% of the LSP. In an amiloride sensitive volunteer, amiloride exerted 50% of its effect at 1 microM. In conclusion, we have demonstrated that the LSP can be measured in humans, that Na hyperpolarizes the LSP, that increasing the Na concentration increases LSP hyperpolarization, and that amiloride inhibits the Na evoked LSP in some humans. While ENaC is involved in sensing salt, its role appears to vary among individuals.

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH.

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](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(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(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(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 HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Excitation and adaptation in the detection of hydrogen ions by taste receptor cells: a role for cAMP and Ca(2+).

The role of intracellular cAMP and Ca(2+) in the excitation and adaptation of taste responses by HCl was investigated by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the tongue with HCl concentrations between 1 and 30 mM caused a dose-dependent increase in CT responses that were insensitive to voltage clamp of the lingual receptive field and to amiloride. At a fixed HCl concentration (20 mM) topical lingual application of 8-chlorophenylthio(CPT)-cAMP increased the magnitude of HCl-induced CT response by twofold under zero current clamp. The magnitude of the CT response increased further at -60 mV and decreased at +60 mV lingual voltage clamp but remained amiloride insensitive. In untreated polarized TRCs, apical stimulation with HCl concentrations between 1 and 30 mM HCl induced sustained decreases in TRC pH(i). The magnitude of pH(i) decrease increased with increasing HCl concentration. Following treatment of the basolateral membrane with 8-CPT-cAMP the decrease in pH(i) due to apical 1 mM HCl application was significantly increased. Treatment with cAMP alone decreased resting TRC pH(i) and inhibited the recovery of pH(i) from a basolateral NH4Cl pulse by 46%. Topical lingual application of ionomycin, a Ca(2+) ionophore, did not affect the initial CT response to 20 mM HCl +10 mM CaCl2, but the response declined rapidly to 50% of its initial level within 2 min. In polarized TRCs, basolateral exposure to ionomycin increased TRC pH(i) and activated pH(i) recovery from NH4Cl pulse by 388%. Apical HCl stimulation induced a transient decrease in resting TRC pH(i) followed by spontaneous recovery. The data suggest that cAMP enhances the sour taste of strong acids by activating a Ca(2+)- and amiloride-insensitive H(+) conductance and inhibiting pH(i) recovery in TRCs. However, an increase in [Ca(2+)]i stimulates pH(i) recovery, which, in turn, increases sensory adaptation to acids.