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.

TRPM5-dependent amiloride- and benzamil-insensitive NaCl chorda tympani taste nerve response.

Transient receptor potential (TRP) subfamily M member 5 (TRPM5) cation channel is involved in sensing sweet, bitter, umami, and fat taste stimuli, complex-tasting divalent salts, and temperature-induced changes in sweet taste. To investigate if the amiloride- and benzamil (Bz)-insensitive NaCl chorda tympani (CT) taste nerve response is also regulated in part by TRPM5, CT responses to 100 mM NaCl + 5 µM Bz (NaCl + Bz) were monitored in Sprague-Dawley rats, wild-type (WT) mice, and TRP vanilloid subfamily member 1 (TRPV1) and TRPM5 knockout (KO) mice in the presence of resiniferatoxin (RTX), a TRPV1 agonist. In rats, NaCl + Bz + RTX CT responses were also monitored in the presence of triphenylphosphine oxide, a specific TRPM5 blocker, and capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791), specific TRPV1 blockers. In rats and WT mice, RTX produced biphasic effects on the NaCl + Bz CT response, enhancing the response at 0.5-1 µM and inhibiting it at >1 µM. The NaCl + Bz + SB-366791 CT response in rats and WT mice and the NaCl + Bz CT response in TRPV1 KO mice were inhibited to baseline level and were RTX-insensitive. In rats, blocking TRPV1 by capsazepine or TRPM5 by triphenylphosphine oxide inhibited the tonic NaCl + Bz CT response and shifted the relationship between RTX concentration and the magnitude of the tonic CT response to higher RTX concentrations. TRPM5 KO mice elicited no constitutive NaCl + Bz tonic CT response. The relationship between RTX concentration and the magnitude of the tonic NaCl + Bz CT response was significantly attenuated and shifted to higher RTX concentrations. The results suggest that pharmacological or genetic alteration of TRPM5 activity modulates the Bz-insensitive NaCl CT response and its modulation by TRPV1 agonists.

N-geranyl cyclopropyl-carboximide modulates salty and umami taste in humans and animal models.

Effects of N-geranyl cyclopropyl-carboxamide (NGCC) and four structurally related compounds (N-cyclopropyl E2,Z6-nonadienamide, N-geranyl isobutanamide, N-geranyl 2-methylbutanamide, and allyl N-geranyl carbamate) were evaluated on the chorda tympani (CT) nerve response to NaCl and monosodium glutamate (MSG) in rats and wild-type (WT) and TRPV1 knockout (KO) mice and on human salty and umami taste intensity. NGCC enhanced the rat CT response to 100 mM NaCl + 5 µM benzamil (Bz; an epithelial Na(+) channel blocker) between 1 and 2.5 µM and inhibited it above 5 µM. N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791, a TRPV1t blocker) inhibited the NaCl+Bz CT response in the absence and presence of NGCC. Unlike the WT mice, no NaCl+Bz CT response was observed in TRPV1 KO mice in the absence or presence of NGCC. NGCC enhanced human salt taste intensity of fish soup stock containing 60 mM NaCl at 5 and 10 µM and decreased it at 25 µM. Rat CT responses to NaCl+Bz and human salt sensory perception were not affected by the above four structurally related compounds. Above 10 µM, NGCC increased the CT response to MSG+Bz+SB-366791 and maximally enhanced the response between 40 and 60 µM. Increasing taste cell Ca(2+) inhibited the NGCC-induced increase but not the inosine monophosphate-induced increase in glutamate response. Addition of 45 µM NGCC to chicken broth containing 60 mM sodium enhanced the human umami taste intensity. Thus, depending upon its concentration, NGCC modulates salt taste by interacting with the putative TRPV1t-dependent salt taste receptor and umami taste by interacting with a Ca(2+)-dependent transduction pathway.

Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to bitter, sweet, and umami taste stimuli.

The relationship between taste receptor cell (TRC) intracellular Ca(2+) ([Ca(2+)](i)) and rat chorda tympani (CT) nerve responses to bitter (quinine and denatonium), sweet (sucrose, glycine, and erythritol), and umami [monosodium glutamate (MSG) and MSG + inosine 5'-monophosphate (IMP)] taste stimuli was investigated before and after lingual application of ionomycin (Ca(2+) ionophore) + Ca(2+), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Ca(2+) chelator), U73122 (phospholipase C blocker), thapsigargin (Ca(2+)-ATPase blocker), and diC8-PIP(2) (synthetic phosphatidylinositol 4,5-bisphosphate). The phasic CT response to quinine was indifferent to changes in [Ca(2+)](i). However, a decrease in [Ca(2+)](i) inhibited the tonic part of the CT response to quinine. The CT responses to sweet and umami stimuli were indifferent to changes in TRC [Ca(2+)](i). However, a decrease in [Ca(2+)](i) attenuated the synergistic effects of ethanol on the CT response to sweet stimuli and of IMP on the glutamate CT response. U73122 and thapsigargin inhibited the phasic and tonic CT responses to bitter, sweet, and umami stimuli. Although diC8-PIP(2) increased the CT response to bitter and sweet stimuli, it did not alter the CT response to glutamate but did inhibit the synergistic effect of IMP on the glutamate response. The results suggest that bitter, sweet, and umami taste qualities are transduced by [Ca(2+)](i)-dependent and [Ca(2+)](i)-independent mechanisms. Changes in TRC [Ca(2+)](i) in the BAPTA-sensitive cytosolic compartment regulate quality-specific taste receptors and ion channels that are involved in the neural adaptation and mixture interactions. Changes in TRC [Ca(2+)](i) in a separate subcompartment, sensitive to inositol trisphosphate and thapsigargin but inaccessible to BAPTA and ionomycin + Ca(2+), are associated with neurotransmitter release.

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.

Strain differences in the neural, behavioral, and molecular correlates of sweet and salty taste in naive, ethanol- and sucrose-exposed P and NP rats.

Strain differences between naive, sucrose- and ethanol-exposed alcohol-preferring (P) and alcohol-nonpreferring (NP) rats were investigated in their consumption of ethanol, sucrose, and NaCl; chorda tympani (CT) nerve responses to sweet and salty stimuli; and gene expression in the anterior tongue of T1R3 and TRPV1/TRPV1t. Preference for 5% ethanol and 10% sucrose, CT responses to sweet stimuli, and T1R3 expression were greater in naive P rats than NP rats. The enhancement of the CT response to 0.5 M sucrose in the presence of varying ethanol concentrations (0.5-40%) in naive P rats was higher and shifted to lower ethanol concentrations than NP rats. Chronic ingestion of 5% sucrose or 5% ethanol decreased T1R3 mRNA in NP and P rats. Naive P rats also demonstrated bigger CT responses to NaCl+benzamil and greater TRPV1/TRPV1t expression. TRPV1t agonists produced biphasic effects on NaCl+benzamil CT responses, enhancing the response at low concentrations and inhibiting it at high concentrations. The concentration of a TRPV1/TRPV1t agonist (Maillard reacted peptides conjugated with galacturonic acid) that produced a maximum enhancement in the NaCl+benzamil CT response induced a decrease in NaCl intake and preference in P rats. In naive P rats and NP rats exposed to 5% ethanol in a no-choice paradigm, the biphasic TRPV1t agonist vs. NaCl+benzamil CT response profiles were higher and shifted to lower agonist concentrations than in naive NP rats. TRPV1/TRPV1t mRNA expression increased in NP rats but not in P rats exposed to 5% ethanol in a no-choice paradigm. We conclude that P and NP rats differ in T1R3 and TRPV1/TRPV1t expression and neural and behavioral responses to sweet and salty stimuli and to chronic sucrose and ethanol exposure.

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.

The K+-H+ exchanger, nigericin, modulates taste cell pH and chorda tympani taste nerve responses to acidic stimuli.

The relationship between acidic pH, taste cell pH(i), and chorda tympani (CT) nerve responses was investigated before and after incorporating the K(+)-H(+) exchanger, nigericin, in the apical membrane of taste cells. CT responses were recorded in anesthetized rats in vivo, and changes in pH(i) were monitored in polarized fungiform taste cells in vitro. Under control conditions, stimulating the tongue with 0.15 M potassium phosphate (KP) or 0.15 M sodium phosphate (NaP) buffers of pHs between 8.0 and 4.6, KP or NaP buffers did not elicit a CT response. Post-nigericin (500 × 10(-6) M), KP buffers, but not NaP buffers, induced CT responses at pHs ≤ 6.6. The effect of nigericin was reversed by the topical lingual application of carbonyl cyanide 3-chloro-phenylhydrazone, a protonophore. Post-nigericin (150 × 10(-6) M), KP buffers induced a greater decrease in taste cell pH(i) relative to NaP buffers and to NaP and KP buffers under control conditions. A decrease in pH(i) to about 6.9 induced by KP buffers was sufficient to elicit a CT response. The results suggest that facilitating apical H(+) entry via nigericin decreases taste cell pH(i) and demonstrates directly a strong correlation between pH(i) and the magnitude of the CT response.

Regulation of the putative TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate.

Regulation of the putative amiloride and benzamil (Bz)-insensitive TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied by monitoring chorda tympani (CT) taste nerve responses to 0.1 M NaCl solutions containing Bz (5 x 10(-6) M; a specific ENaC blocker) and resiniferatoxin (RTX; 0-10 x 10(-6) M; a specific TRPV1 agonist) in Sprague-Dawley rats and in wildtype (WT) and TRPV1 knockout (KO) mice. In rats and WT mice, RTX elicited a biphasic effect on the NaCl + Bz CT response, increasing the CT response between 0.25 x 10(-6) and 1 x 10(-6) M. At concentrations >1 x 10(-6) M, RTX inhibited the CT response. An increase in PIP(2) by topical lingual application of U73122 (a phospholipase C blocker) or diC8-PIP(2) (a short chain synthetic PIP(2)) inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. A decrease in PIP(2) by topical lingual application of phenylarsine oxide (a phosphoinositide 4 kinase blocker) enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 x 10(-6) M. The ENaC-dependent NaCl CT responses were not altered by changes in PIP(2). An increase in PIP(2) enhanced CT responses to sweet (0.3 M sucrose) and bitter (0.01 M quinine) stimuli. RTX produced the same increase in the Bz-insensitive Na(+) response when present in salt solutions containing 0.1 M NaCl + Bz, 0.1 M monosodium glutamate + Bz, 0.1 M NaCl + Bz + 0.005 M SC45647, or 0.1 M NaCl + Bz + 0.01 M quinine. No effect of RTX was observed on CT responses in WT mice and rats in the presence of the TRPV1 blocker N-(3-methoxyphenyl)-4-chlorocinnamide (1 x 10(-6) M) or in TRPV1 KO mice. We conclude that PIP(2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste, although in the last case, PIP(2) seems to directly regulate the taste receptor protein itself, i.e., the TRPV1 ion channel or its taste receptor variant, TRPV1t.

Regulation of the benzamil-insensitive salt taste receptor by intracellular Ca2+, protein kinase C, and calcineurin.

The regulation of the benzamil (Bz)-insensitive salt taste receptor was investigated by intracellular Ca2+ ([Ca2+]i), protein kinase C (PKC), and the Ca2+-dependent serine-threonine phosphatase, calcineurin (PP2B), by monitoring chorda tympani taste nerve responses to 0.1 M NaCl solutions containing Bz (5x10(-6) M) and resiniferatoxin (RTX; 0-10x10(-6) M) in Sprague-Dawley rats and in wild-type (WT) and transient receptor potential vanilloid-1 knockout (TRPV1 KO) mice. In rats and WT mice, RTX increased the NaCl+Bz chorda tympani responses between 0.25x10(-6) and 1x10(-6) M and inhibited the responses above 1x10(-6) M. Decreasing taste receptor cell (TRC) [Ca2+]i with BAPTA loading, activation of PKC with 4alpha-phorbol-12,13-didecanoate (PMA), or inhibition of PP2B by cyclosporin A or FK-506, enhanced the magnitude of the Bz-insensitive NaCl chorda tympani responses in the presence of RTX and either minimized or completely eliminated the decrease in the chorda tympani response>1x10(-6) M RTX. In contrast, increasing TRC [Ca2+]i with ionomycin inhibited Bz-insensitive NaCl chorda tympani responses in the presence of RTX. No effect of the cited modulators was observed on the chorda tympani responses in WT mice and rats in the presence of TRPV1 blocker SB-366791 (1x10(-6) M) or in TRPV1 KO mice. 32P-labeling demonstrated direct phosphorylation of TRPV1 or TRPV1t in anterior lingual epithelium by PMA, cyclosporin A, or FK-506. PMA also enhanced the RTX-sensitive unilateral apical Na+ flux in polarized fungiform TRC in vitro. We conclude that TRPV1 or its variant TRPV1t is phosphorylated and dephosphorylated by PKC and PP2B, respectively, and either sensitizes or desensitizes the Bz-insensitive NaCl chorda tympani responses to RTX stimulation.

Nicotine activates TRPM5-dependent and independent taste pathways.

The orosensory responses elicited by nicotine are relevant for the development and maintenance of addiction to tobacco products. However, although nicotine is described as bitter tasting, the molecular and neural substrates encoding the taste of nicotine are unclear. Here, rats and mice were used to determine whether nicotine activates peripheral and central taste pathways via TRPM5-dependent mechanisms, which are essential for responses to other bitter tastants such as quinine, and/or via nicotinic acetylcholine receptors (nAChRs). When compared with wild-type mice, Trpm5(-/-) mice had reduced, but not abolished, chorda tympani (CT) responses to nicotine. In both genotypes, lingual application of mecamylamine, a nAChR-antagonist, inhibited CT nerve responses to nicotine and reduced behavioral responses of aversion to this stimulus. In accordance with these findings, rats were shown to discriminate between nicotine and quinine presented at intensity-paired concentrations. Moreover, rat gustatory cortex (GC) neural ensemble activity could also discriminate between these two bitter tastants. Mecamylamine reduced both behavioral and GC neural discrimination between nicotine and quinine. In summary, nicotine elicits taste responses through peripheral TRPM5-dependent pathways, common to other bitter tastants, and nAChR-dependent and TRPM5-independent pathways, thus creating a unique sensory representation that contributes to the sensory experience of tobacco products.

Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t).

Maillard reacted peptides (MRPs) were synthesized by conjugating a peptide fraction (1000-5000 Da) purified from soy protein hydrolyzate with galacturonic acid, glucosamine, xylose, fructose, or glucose. The effect of MRPs was investigated on human salt taste and on the chorda tympani (CT) taste nerve responses to NaCl in Sprague-Dawley rats, wild-type, and transient receptor potential vanilloid 1 (TRPV1) knockout mice. MRPs produced a biphasic effect on human salt taste perception and on the CT responses in rats and wild-type mice in the presence of NaCl + benzamil (Bz, a blocker of epithelial Na+ channels), enhancing the NaCl response at low concentrations and suppressing it at high concentrations. The effectiveness of MRPs as salt taste enhancers varied with the conjugated sugar moiety: galacturonic acid = glucosamine > xylose > fructose > glucose. The concentrations at which MRPs enhanced human salt taste were significantly lower than the concentrations of MRPs that produced increase in the NaCl CT response. Elevated temperature, resiniferatoxin, capsaicin, and ethanol produced additive effects on the NaCl CT responses in the presence of MRPs. Elevated temperature and ethanol also enhanced human salt taste perception. N-(3-methoxyphenyl)-4-chlorocinnamid (a blocker of TRPV1t) inhibited the Bz-insensitive NaCl CT responses in the absence and presence of MRPs. TRPV1 knockout mice demonstrated no Bz-insensitive NaCl CT response in the absence or presence of MRPs. The results suggest that MRPs modulate human salt taste and the NaCl + Bz CT responses by interacting with TRPV1t.

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.

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.