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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.

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-β-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, β2 and β4 nAChR subunits was observed in a subset of rat and WT mouse circumvallate and fungiform TRCs. Specific α3, α4, α7, β2, and β4 antibodies localized to a subset of WT mouse circumvallate and fungiform TRCs. In Trpm5-GFP mice α3, α4, α7, and β4 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, β2 and β4 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 β4 mRNAs in circumvallate TRCs with a significant decrease in the expression of α3, α6 and β2 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.

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.

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 β 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-β-erythroidine, a α4β2 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 β4 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.

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.