Relationship between ENaC Regulators and SARS-CoV-2 Virus Receptor (ACE2) Expression in Cultured Adult Human Fungiform (HBO) Taste Cells.

In addition to the α, ß, and γ subunits of ENaC, human salt-sensing taste receptor cells (TRCs) also express the δ-subunit. At present, it is not clear if the expression and function of the ENaC δ-subunit in human salt-sensing TRCs is also modulated by the ENaC regulatory hormones and intracellular signaling effectors known to modulate salt responses in rodent TRCs. Here, we used molecular techniques to demonstrate that the G-protein-coupled estrogen receptor (GPER1), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and components of the renin-angiotensin-aldosterone system (RAAS) are expressed in δ-ENaC-positive cultured adult human fungiform (HBO) taste cells. Our results suggest that RAAS components function in a complex with ENaC and TRPV1 to modulate salt sensing and thus salt intake in humans. Early, but often prolonged, symptoms of COVID-19 infection are the loss of taste, smell, and chemesthesis. The SARS-CoV-2 spike protein contains two subunits, S1 and S2. S1 contains a receptor-binding domain, which is responsible for recognizing and binding to the ACE2 receptor, a component of RAAS. Our results show that the binding of a mutated S1 protein to ACE2 decreases ACE2 expression in HBO cells. We hypothesize that changes in ACE2 receptor expression can alter the balance between the two major RAAS pathways, ACE1/Ang II/AT1R and ACE2/Ang-(1-7)/MASR1, leading to changes in ENaC expression and responses to NaCl in salt-sensing human fungiform taste cells.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Kokumi Taste Active Peptides Modulate Salt and Umami Taste.

Kokumi taste substances exemplified by γ-glutamyl peptides and Maillard Peptides modulate salt and umami tastes. However, the underlying mechanism for their action has not been delineated. Here, we investigated the effects of a kokumi taste active and inactive peptide fraction (500-10,000 Da) isolated from mature (FIIm) and immature (FIIim) Ganjang, a typical Korean soy sauce, on salt and umami taste responses in humans and rodents. Only FIIm (0.1-1.0%) produced a biphasic effect in rat chorda tympani (CT) taste nerve responses to lingual stimulation with 100 mM NaCl + 5 µM benzamil, a specific epithelial Na+ channel blocker. Both elevated temperature (42 °C) and FIIm produced synergistic effects on the NaCl + benzamil CT response. At 0.5% FIIm produced the maximum increase in rat CT response to NaCl + benzamil, and enhanced salt taste intensity in human subjects. At 2.5% FIIm enhanced rat CT response to glutamate that was equivalent to the enhancement observed with 1 mM IMP. In human subjects, 0.3% FIIm produced enhancement of umami taste. These results suggest that FIIm modulates amiloride-insensitive salt taste and umami taste at different concentration ranges in rats and humans.

Nicotinic acetylcholine receptor (CHRN) expression and function in cultured human adult fungiform (HBO) taste cells.

In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca2+]i. Nicotine effects on [Ca2+]i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca2+]i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.

Nicotinic acetylcholine receptors (nAChRs) are expressed in Trpm5 positive taste receptor cells (TRCs).

Nicotine evokes chorda tympani (CT) taste nerve responses and an aversive behavior in Trpm5 knockout (KO) mice. The agonists and antagonists of nicotinic acetylcholine receptors (nAChRs) modulate neural and behavioral responses to nicotine in wildtype (WT) mice, Trpm5 KO mice and rats. This indicates that nicotine evokes bitter taste by activating a Trpm5-dependent pathway and a Trpm5-independent but nAChR-dependent pathway. Rat CT responses to ethanol are also partially inhibited by nAChR blockers, mecamylamine and dihydro-ß-erythroidine. This indicates that a component of the bitter taste of ethanol is also nAChR-dependent. However, at present the expression and localization of nAChR subunits has not been investigated in detail in taste receptor cells (TRCs). To this end, in situ hybridization, immunohistochemistry and q-RT-PCR techniques were utilized to localize nAChR subunits in fungiform and circumvallate TRCs in WT mice, Trpm5-GFP transgenic mice, nAChR KO mice, and rats. The expression of mRNAs for α7, ß2 and ß4 nAChR subunits was observed in a subset of rat and WT mouse circumvallate and fungiform TRCs. Specific α3, α4, α7, ß2, and ß4 antibodies localized to a subset of WT mouse circumvallate and fungiform TRCs. In Trpm5-GFP mice α3, α4, α7, and ß4 antibody binding was observed in a subset of Trpm5-positive circumvallate TRCs. Giving nicotine (100 µg/ml) in drinking water to WT mice for 3 weeks differentially increased the expression of α3, α4, α5, α6, α7, ß2 and ß4 mRNAs in circumvallate TRCs to varying degrees. Giving ethanol (5%) in drinking water to WT mice induced an increase in the expression of α5 and ß4 mRNAs in circumvallate TRCs with a significant decrease in the expression of α3, α6 and ß2 mRNAs. We conclude that nAChR subunits are expressed in Trpm5-positive TRCs and their expression levels are differentially altered by chronic oral exposure to nicotine and ethanol.

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.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and ß nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-ß-erythroidine, a α4ß2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and ß4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-ß-erythroidine, and CP-601932 (a partial agonist of the α3ß4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

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.