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

Abstract

The relationship between taste receptor cell (TRC) Ca²⁺ concentration ([Ca²⁺]_i) and rat chorda tympani (CT) nerve responses to salty [NaCl and NaCl+benzamil (Bz)] and sour (HCl, CO₂, and acetic acid) taste stimuli was investigated before and after lingual application of ionomycin+Ca²⁺, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), U73122 (phospholipase C blocker), and thapsigargin (Ca²⁺-ATPase inhibitor) under open-circuit or lingual voltage-clamp conditions. An increase in TRC [Ca²⁺]_i attenuated the tonic Bz-sensitive NaCl CT response and the apical membrane Na⁺ conductance. A decrease in TRC [Ca²⁺]_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 NH₄Cl. An increase in TRC [Ca²⁺]_i did not alter the phasic response but attenuated the tonic CT response to acidic stimuli. A decrease in [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_i-dependent and [Ca²⁺]_i-independent mechanisms. Changes in TRC [Ca²⁺]_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 [Ca²⁺]_i in a separate subcompartment, sensitive to inositol trisphosphate and thapsigargin but inaccessible to BAPTA, are associated with neurotransmitter release.

several studies suggest that taste receptor cells (TRCs) respond with an increase in Ca²⁺ concentration ([Ca²⁺]_i) when stimulated with sour (Lyall et al. 2003; Richter et al. 2003), sweet (Rebello and Medler 2010), bitter (Akabas et al. 1988; Medler 2010; Ogura et al. 2002; Rebello and Medler 2010), umami (Narukawa et al. 2006; Rebello and Medler 2010), and a mixture containing sweet and bitter taste stimuli (Huang and Roper 2010). Type III cells, termed presynaptic cells that express the polycystic kidney disease-like proteins PKD1L3 and PKD2L1, function as sour-sensing cells (Huang et al. 2006; Ishimaru et al. 2006; LopezJimenez et al. 2006) and possess specialized chemical synapses. Whereas PKD2L1 and PKD1L3 are reliable markers of sour-sensitive taste cells, PKD2L1 may have some role in sour transduction in fungiform taste cells, but neither PKD2L1 nor PKD1L3 plays a role in sour transduction in circumvallate taste cells (Horio et al. 2011). Weak organic acids enter across the apical membranes of TRCs as neutral molecules and decrease intracellular pH (pH_i) (DeSimone and Lyall 2006). For strong acids, H⁺ entry is dependent on at least two proton-conductive pathways in the apical membranes of sour-sensing TRCs (Chang et al. 2010; DeSimone et al. 2011; Lyall et al. 2002a) that are amiloride and Ca²⁺ insensitive. One conductive pathway is activated by cAMP, and the second pathway depends on gp91^phox, a component of the NADPH oxidase enzyme (DeSimone et al. 2011; Lyall et al. 2002a). Acidic stimuli depolarize type III cells and increase Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) that are involved in the release of the neurotransmitters serotonin and norepinephrine from intracellular vesicles (Huang et al. 2008).

In fungiform TRCs, Na⁺ ions enter across the apical membrane by at least two pathways: one involving the apical amiloride- and Bz-sensitive epithelial Na⁺ channels (ENaCs) (Bosak et al. 2010; Chandrashekar et al. 2010; DeSimone et al. 1981) and the second involving putative transient receptor potential vanilloid (TRPV1t) nonspecific cation channels (Lyall et al. 2004b). However, at present, in type I TRCs involved in Na⁺ sensing through ENaC (Dvoryanchikov et al. 2009; Vandenbeuch et al. 2008)], the relationship between the increase in [Ca²⁺]_i and neurotransmitter release is not clear. In addition to its role in the neurotransmitter release, alterations in TRC [Ca²⁺]_i have been shown to modulate the chorda tympani (CT) taste nerve responses to salty (DeSimone and Lyall 2008; Lyall et al. 2009) and sour stimuli (Lyall et al. 2002a, 2004a, 2006). Although an increase in cytosolic [Ca²⁺]_i seems to be critical for normal responses in TRCs (Medler 2010), at present, a detailed understanding of the relationship between TRC [Ca²⁺]_i and neural responses to salty and sour taste stimuli is lacking.

The objective of this study was to investigate the effects of changes in TRC [Ca²⁺]_i on rat CT responses to representative salty and sour taste stimuli. TRC [Ca²⁺]_i was either increased or decreased by topical lingual application of a Ca²⁺ ionophore, ionomycin, plus Ca²⁺ or a membrane-permeable Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), respectively. The effects of BAPTA loading in TRCs were reversed by saturating the Ca²⁺-binding sites on intracellular BAPTA by treating rat lingual TRCs in vivo with ionomycin+Ca²⁺. We also monitored changes in [Na⁺]_i, [Ca²⁺]_i, and [H⁺]_i in TRCs using imaging techniques in vitro. The data presented in this article suggest that changes in TRC [Ca²⁺]_i in two subcompartments, namely, an ionomycin- and BAPTA-sensitive cytosolic compartment and an intracellular inositol trisphosphate (IP₃)- and thapsigargin-sensitive subcompartment, play an important role in regulating the neural responses to salty and sour taste stimuli. Changes in [Ca²⁺]_i in the cytosolic compartment regulate taste receptors/ion channels and cotransporters involved in the taste transduction mechanism and are involved in the neural adaptation to different taste qualities. Changes in TRC [Ca²⁺]_i in a separate subcompartment, inaccessible to Ca²⁺ buffering by BAPTA, are associated with neurotransmitter release and subsequent neural response.

MATERIALS AND METHODS

In Vivo Studies

CT taste nerve recordings.

Animals were housed in the Virginia Commonwealth University (VCU) animal facility in accordance with institutional guidelines. All in vivo and in vitro animal protocols were approved by the Institutional Animal Care and Use Committee of VCU. Sixty female Sprague-Dawley rats (150–200 g) were used in this study. For CT recordings, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg), and supplemental pentobarbital sodium (20 mg/kg) was administered as necessary to maintain surgical anesthesia. The animal's corneal reflex and toe-pinch reflex were used to monitor the depth of surgical anesthesia. Body temperatures were maintained at 37° with a Deltaphase isothermal pad (model 39 DP; Braintree Scientific, Braintree, MA). The left CT nerve was exposed laterally as it exited the tympanic bulla and was placed onto a 32-gauge platinum-iridium wire electrode. An indifferent electrode was placed in nearby tissue. Neural responses were differentially amplified with an optically coupled isolated bio-amplifier (ISO-80; World Precision Instruments, Sarasota, FL). Stimulus solutions were injected into a Lucite chamber (3 ml; 1 ml/s) affixed by vacuum to a 28-mm² patch of anterior dorsal lingual surface. The chamber was fitted with separate Ag-AgCl electrodes for passing of current and measuring potential. These electrodes served as inputs to a voltage- and current-clamp amplifier that permitted the recording of neural responses with the chemically stimulated receptive field under zero current clamp or voltage clamp. The clamp voltages were referenced to the mucosal side of the tongue (Ye et al. 1993, 1994). Integrated neural responses and lingual current and voltage changes were captured on disk using LabView software (National Instruments, Austin, TX) and analyzed off-line as described previously (Lyall et al. 2002a, 2004b, 2010). Student's t-test was employed to analyze the differences between sets of data.

Solutions.

The composition of the rinse and stimulating solutions is shown in Table 1. For the majority of the CT recordings, the rinse (R) and stimulating solutions were used without added CaCl₂. However, in those experiments in which we used ionomycin to load the cells with Ca²⁺, 1 or 10 mM CaCl₂ was added to the rinse and stimulating solutions.

Table 1.

Composition of solutions used in the CT experiments

Rinse	Stimulus
R (10 mM KCl)	NaCl (10 mM KCl + 100 mM NaCl)
R (10 mM KCl)	NaCl+Bz (10 mM KCl + 100 mM NaCl + 0.005 mM Bz)
*R+Ca2+ (10 mMKCl + 10 mM CaCl2)	*NaCl+Ca2+ (10 mM KCl +10 mM CaCl2 + 100 mM NaCl)
*R+Ca2+ (10 mM KCl + 10 mM CaCl2)	*NaCl+Bz+Ca2+ (10 mM KCl + 10 mM CaCl2 + 100 mM NaCl + 0.005 mM Bz)
*R+Ca2+ (10 mM KCl + 10 mM CaCl2)	*NaCl+Bz+Ca2++CPC (10 mM KCl + 10 mM CaCl2 + 100 mM NaCl + 0.005 mM Bz + 2 mM CPC)
R (10 mM KCl)	HCl (10 mM KCl + 20 mM HCl)
*R+Ca2+ (10 mM KCl + 10 mM CaCl2)	*HCl+Ca2+ (10 mM KCl + 10 mM CaCl2 + 20 mM HCl)
RAA (175 mM KCl + 10 mM HEPES, pH 6.1)	Acetic acid (10 mM CH3COOH + 175 mM CH3COOK, pH 6.1)
*RAA+Ca2+ (175 mM KCl + 1 mM CaCl2 + 10 mM HEPES, pH 6.1)	*Acetic acid+Ca2+ (10 mM CH3COOH + 175 mM CH3COOK + 10 mM CaCl2, pH 6.1)
RCO2 (72 mM KCl +10 mM HEPES, pH 7.4)	CO2 (72 mM KHCO3 + 10% CO2-90% O2, pH 7.4)
*RCO2+Ca2+ (72 mM KCl + 1 mM CaCl2 +10 mM HEPES, pH 7.4)	*CO2+Ca2+ (72 mM KHCO3 + 10% CO2-90% O2 + 10 mM CaCl2, pH 7.4)
R (10 mM KCl)	Control-1 (300 mM NH4Cl)
R (10 mM KCl)	Control-2 (300 mM NaCl)

AA, acetic acid; Bz, benzamil; CPC, cetylpyridinium chloride. Rinse and stimulating solutions indicated with asterisks were used only in experiments with ionomycin. In some experiments with ionomycin, the rinse and stimulating solutions contained 1 mM CaCl₂.

TRCs were loaded in vivo with Ca²⁺ using 150 μM ionomycin (Sigma) in dimethyl sulfoxide (DMSO) for 45 min. In addition, rinse and stimulating solutions (Table 1) used for CT recordings following ionomycin treatment contained either 1 or 10 mM CaCl₂ (Lyall et al. 2002a, 2004a, 2009). TRCs were loaded in vivo with the Ca²⁺ chelator BAPTA-AM (Sigma) by dissolving BAPTA-AM directly in DMSO and applying it topically to the tongue for 45 min at a concentration of 13 or 33 mM as described previously (Lyall et al. 2009). The effects of BAPTA loading in TRCs were reversed by saturating the Ca²⁺-binding sites on the intracellular BAPTA by treating the cells with ionomycin+Ca²⁺. To rat tongues already treated with BAPTA-AM, we topically applied 150 μM ionomycin to the lingual surface for 30 min. Following ionomycin treatment, the lingual surface was perfused for 10 min with the rinse solution containing 10 mM KCl + 10 mM CaCl₂ and then perfused with 10 mM KCl rinse solution without CaCl₂ for another 10 min. This was done to remove external ionomycin and CaCl₂. For the CT recordings under control conditions, post-BAPTA-AM treatment and post-BAPTA-AM–post-ionomycin+Ca²⁺ treatment, the rinse and stimulating solutions used did not contain CaCl₂ (Table 1). CT responses were also recorded after topical lingual application of 250 μM U73122, a nonspecific blocker of phospholipase Cs (PLCs); its inactive analog, U73343 (Coleman et al. 2011); or 250 μM thapsigargin, a noncompetitive inhibitor of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) (Rogers et al. 1995). All drugs were obtained from Sigma. Benzamil (Bz; 5 μM) and cetylpyridinium chloride (CPC; 2 mM) were added to the stimulating solutions to block Na⁺ entry into TRCs through ENaC and the putative TRPV1t, respectively (DeSimone et al. 2001; Lyall et al. 2004a). DMSO (Lyall et al. 1999), CaCl₂ (1 or 10 mM) (Lyall et al. 2002a, 2004a), or topical lingual application of 150 μM ionomycin by themselves (data not shown) did not alter the CT responses to taste stimuli.

In Vitro Studies

Studies with polarized fungiform taste bud preparations: Ca²⁺, Na⁺, and pH imaging.

Rat lingual epithelium was isolated by collagenase treatment (Vinnikova et al. 2004). A small piece of the anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber as described previously (Lyall et al. 2001, 2002a). Relative changes in [Ca²⁺]_i were monitored in polarized TRCs by loading the tissue with the Ca²⁺-sensitive fluoroprobe Fura-2 AM (10 μM; Molecular Probes) in the presence of 0.15% Pluronic at room temperature for 4 h. Before the experiment was started, the tissues were perfused on both sides with control solution for 15 min at the rate of 1 ml/min. TRCs were alternately excited at 340 and 380 nm and imaged at 15-s intervals. The emitted light was imaged at 510 nm. All experiments were done at room temperature (∼22°C).

Relative changes in [Na⁺]_i were monitored in polarized TRCs by loading the tissue with a Na⁺-sensitive fluoroprobe, Na-green. Before the experiment was started, the tissues were perfused on both sides with control Ringer solution for 15 min at the rate of 1 ml/min. TRCs were excited at 490 nm and imaged at 15-s intervals. The emitted light was imaged at 530 nm. All experiments were done at room temperature (∼22°C). Small regions of interest (ROIs) in the taste bud (diameter 2–3 μm) were chosen in which the changes in the fluorescence intensity at 490 nm (F₄₉₀) were analyzed using imaging software (TILLvisIon version 4.0.7.2; TILL Photonics, Martinsried, Germany). The background and autofluorescence at 490 nm were corrected from images of a taste bud without the dye. The F₄₉₀ value under control conditions for each ROI was taken as 100%. Student's t-test was employed to analyze the differences between sets of data (Lyall et al. 2002b).

In some experiments, taste bud cells were loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and changes in TRC pH_i were measured as described previously (Lyall et al. 2001, 2004a, 2006; Vinnikova et al. 2004).

Studies with isolated taste bud cells using confocal microscopy.

Changes in [Ca²⁺]_i were also monitored in isolated fungiform taste bud cells (Vinnikova et al. 2004) using confocal microscopy. The isolated taste buds were placed on a CellTak (Sigma)-coated coverslip that formed the bottom of a perfusion chamber and were perfused with Ringer solution (pH 7.4; Table 2). TRCs were loaded with fluo 3-AM (Molecular Probes). The taste buds were incubated at room temperature with Ringer solution containing 30 μM of the dye for 1 h. After this, the cells were washed with control solution. The measurements were made using a Zeiss LSM 410 confocal microscope. The cells were excited by the 488-nm spectral line of the argon-ion laser, and the images were taken at the emission wavelength of 530 ± 10 nm (E₅₃₀). In TRCs loaded with fluo-3, the changes in E₅₃₀ were expressed relative to the fluorescence emission intensity of different ROIs under control conditions. The E₅₃₀ value under control conditions for each ROI was taken as 100% and was compared between different ROIs under different experimental conditions. Student's t-test was employed to analyze the differences between sets of data.

Table 2.

Composition of Ringer solutions used in imaging experiments

	Concentration, mM
	Control	0 Na+	0Ca2+	0Na+-0 Ca2+	Na-acetate	NH4Cl
NaCl	140	0	140	0	110	125
KCl	5	5	5	5	5	5
CaCl2	1	1	0	0	1	1
MgCl2	1	1	1	1	1	1
Na-pyruvate	10	0	10	0	10	10
Glucose	10	10	10	10	10	10
NMDG-Cl	0	140	0	140	0	0
Pyruvic acid	0	10	0	10	0	0
EGTA	0	0	2	2	0	0
Na-acetate	0	0	0	0	30	0
NH4Cl	0	0	0	0	0	15
HEPES	10	10	10	10	10	10
pH	7.4	7.4	7.4	7.4	7.4	7.4

NMDG, N-methy-d-glucamine.

Solutions.

The composition of the Ringer solutions used in the imaging experiments is shown in Table 2. At the physiological pH and osmolarity, the relationship between changes in pH_i and [Ca²⁺]_i was investigated by briefly exposing the isolated fungiform TRCs or the basolateral membrane of polarized fungiform TRCs to Ringer solutions containing 30 mM Na-acetate, 15 mM NH₄Cl or unilaterally decreasing the basolateral Na⁺ concentration from 150 mM to 0 as described previously (Lyall et al. 2004a, 2006; Vinnikova et al. 2004).

RESULTS

Effect of Changes in TRC [Ca²⁺]_i on the CT Responses to Mineral Salts

Studies with ionomycin+Ca²⁺.

The CT response to 100 mM NaCl + 10 mM CaCl₂ (Table 1, NaCl+Ca²⁺) exhibited a Bz-sensitive and a Bz-insensitive component (Fig. 1A, control). The Bz-insensitive component (NaCl+Bz+Ca²⁺) was inhibited to the rinse baseline level by perfusing the tongue with NaCl+Bz+Ca²⁺ + 2 mM CPC (DeSimone et al. 2001; Lyall et al. 2004b). Following ionomycin treatment, both the phasic (transient) and tonic (steady state) CT responses to NaCl+Ca²⁺ were smaller relative to control. However, the tonic CT responses to NaCl+Bz+Ca²⁺ and NaCl+Bz+Ca²⁺+CPC were not different from control (Fig. 1A, post-ionomycin). Following treatment with ionomycin + 10 mM CaCl₂, the decrease in tonic NaCl CT response was due to a specific decrease in the Bz-sensitive component [(NaCl+Ca²⁺) − (NaCl+Bz+Ca²⁺)] of the NaCl CT response (Fig. 1B; P = 0.0001, unpaired; n = 3). The Bz-insensitive but CPC-sensitive tonic NaCl CT response was not affected. In contrast, when the rinse and NaCl solutions contained 1 mM CaCl₂, ionomycin treatment had no effect on the CT response to NaCl+Ca²⁺ or NaCl+Bz+Ca²⁺ (Table 1) (data not shown). Presumably, with only 1 mM extracellular Ca²⁺ in the rinse and stimulating solutions, the increase in TRC [Ca²⁺] in the presence of ionomycin is insufficient to inhibit the Bz-sensitive NaCl CT response.

Fig. 1.

Effect of ionomycin+Ca²⁺ on the chorda tympani (CT) response to NaCl. A: a representative CT response recorded while the tongue was superfused with 10 mM KCl + 10 mM CaCl₂ (rinse solution, *R+Ca²⁺, see Table 1 for composition of rinse and stimulus solutions), 10 mM KCl + 10 mM CaCl₂ + 100 mM NaCl (NaCl+Ca²⁺), 10 mM KCl + 10 mM CaCl₂ + 100 mM NaCl + 5 μM benzamil (Bz) (NaCl+Bz+Ca²⁺), and 10 mM KCl + 10 mM CaCl₂ + 100 mM NaCl + 5 μM Bz + 2 mM cetylpyridinium chloride (CPC) (NaCl+Bz+Ca²⁺+CPC). The CT response was recorded before (control) and after the topical lingual application of 150 μM ionomycin (post-ionomycin). B: means ± SE of the normalized changes in the tonic CT response to NaCl+Ca²⁺, NaCl+Bz+Ca²⁺, and the Bz-sensitive component in 3 rats under control conditions and post-ionomycin. In each case, the NaCl CT responses were normalized to the corresponding CT responses obtained with 300 mM NH₄Cl. *P = 0.0001 (unpaired; n = 3). C: effect of ionomycin+Ca²⁺ on the voltage sensitivity of the tonic NaCl CT response. The NaCl CT responses were recorded at zero-current clamp and at −60 and +60 mV applied transepithelial voltage across the receptive field before (control) and after ionomycin + CaCl₂ treatment (post-ionomycin+Ca²⁺). In each case, the NaCl CT responses were normalized to the corresponding CT responses obtained with 300 mM NH₄Cl. Each point represents the mean ± SE of 3 animals. M ± SEM, means ± SE.

Relative to the response at zero-current clamp (0 mV), the magnitude of the tonic NaCl+Ca²⁺ CT response increased at −60 mV and decreased at +60 mV (Fig. 1C; control). Treatment with ionomycin + 10 mM CaCl₂ significantly attenuated both the magnitude of the tonic NaCl+Ca²⁺ CT response at 0 mV and the voltage sensitivity of the response (slope of the CT response with change in voltage) (Fig. 1C, post-ionomycin+Ca²⁺). Following ionomycin treatment, the slope of the response (2.7 ± 0.2 ×10⁻³ response units/mV) was smaller relative to its value (4.8 ± 0.3 × 10⁻³ response units/mV) under control conditions (P = 0.009, unpaired; n = 3). The observation that the size of the response and the slope of the response (i.e., the voltage sensitivity or response conductance) are proportional suggests that a decrease in the Bz-sensitive NaCl CT response is due to a decrease in the apical membrane Na⁺ conductance. No significant effect was observed on the CT responses to KCl after ionomycin + 10 mM CaCl₂ treatment relative to control (data not shown). These results suggest that an increase in TRC [Ca²⁺]_i specifically inhibits the Bz-sensitive NaCl CT response.

Studies with BAPTA-AM.

Chelating TRC [Ca²⁺]_i with 33 mM BAPTA-AM enhanced both the phasic and tonic NaCl CT responses relative to control (Fig. 2A, NaCl). Most of the increase in the tonic NaCl CT response in the presence of BAPTA was due to an increase in the Bz-sensitive [NaCl − (NaCl+Bz)] component of the NaCl CT response. Treating the tongue with 33 mM BAPTA-AM enhanced the tonic Bz-sensitive part of the NaCl CT response by 44 ± 2% relative to control (Fig. 2B; P = 0.005, unpaired; n = 3). A small but significant increase in the tonic Bz-insensitive component of the NaCl CT response was also observed after BAPTA treatment (P = 0.024).

Fig. 2.

Effect of BAPTA acetoxymethyl ester (BAPTA-AM) on the CT response to NaCl. A: a representative CT response recorded while the tongue was superfused with 10 mM KCl (rinse solution; R), 10 mM KCl + 100 mM NaCl (NaCl), and 10 mM KCl + 100 mM NaCl + 5 μM Bz (NaCl+Bz). The CT response was recorded before (control) and after the topical application of 33 mM BAPTA-AM (post-BAPTA). B: means ± SE of the normalized changes in the tonic CT response to NaCl, NaCl+Bz, and the Bz-sensitive component in 3 rats under control conditions and post-BAPTA. In each case, the NaCl CT responses were normalized to the corresponding CT responses obtained with 300 mM NH₄Cl. *P = 0.024; **P = 0.005 (unpaired; n = 3). C: effect of BAPTA-AM on the voltage sensitivity of the tonic NaCl CT response. The NaCl CT responses were recorded at zero-current clamp and at −60 and +60 mV applied transepithelial voltage across the receptive field before and after BAPTA-AM treatment. In each case, the NaCl CT responses were normalized to the corresponding CT responses obtained with 300 mM NH₄Cl. Each point represents the mean ± SE of 3 animals.

BAPTA-AM treatment significantly enhanced the magnitude of the tonic NaCl CT response at 0 mV and the voltage sensitivity of the CT response (Fig. 2C, post-BAPTA) relative to control (Fig. 2C, control). Following BAPTA treatment, the slope of the response (4.7 ± 0.04 × 10⁻³ response units/mV) was greater relative to its value (3.4 ± 0.07 × 10⁻³ response units/mV) under control conditions (P = 0.0001, unpaired; n = 3). These results suggest that the observed increase in the Bz-sensitive and Bz-insensitive NaCl CT responses is due to an increase in the TRC apical membrane Na⁺ conductance.

Studies with post-BAPTA–post-ionomycin+Ca²⁺ treatment.

BAPTA-induced enhancement of the phasic and tonic components of the NaCl CT response was reversed by saturating the Ca²⁺-binding sites on the intracellularly trapped BAPTA with ionomycin + 10 mM CaCl₂ (Fig. 3, A–C). It is important to note that among several salty and sour taste stimuli tested, BAPTA (13 mM) specifically enhanced the CT response to NaCl. BAPTA treatment produced no significant effect on the phasic and tonic components of the CT response to 100 mM KCl or 300 mM NH₄Cl (Fig. 3, D and E). These results suggest that K⁺ and NH₄⁺ conductive pathways in the apical membrane of TRCs are not affected by a decrease in TRC [Ca²⁺]_i.

Fig. 3.

A–C: effect of BAPTA and post-BAPTA–post-ionomycin+Ca²⁺ on the CT responses to NaCl, HCl, CO₂, and acetic acid (AA). A representative CT trace is shown in which the rat tongue was first stimulated with R and then with 100 mM NaCl, 20 mM HCl, R_CO2, 10% CO₂, R_AA and AA, (see Table 1 and Fig. 6 legend for definitions of R_CO2 and R_AA) under control conditions (A) and after topical lingual application of 13 mM BAPTA-AM for 30 min (B; post-BAPTA 13 mM). Following BAPTA treatment, the tongue was treated with 150 μM ionomycin + 10 mM CaCl₂ (C; post-BAPTA–post-ionomycin+Ca²⁺). Arrows represent the time periods when the rat tongue was superfused with the rinse solutions R_CO2, R_AA, and R. D and E: effect of BAPTA on the CT responses to KCl and NH₄Cl. Representative CT traces are shown in which the rat tongue was first stimulated with R and then with 100 mM KCl (D) or 300 mM NH₄Cl (E) under control conditions and after topical lingual application of 33 mM BAPTA-AM for 30 min (post-BAPTA 33 mM). Arrows represent the time periods when the rat tongue was superfused with the rinse solution and the stimulating solutions.

Effect of Changes in [Ca²⁺]_i on the Unilateral Apical Na⁺ Flux in Polarized Fungiform TRCs

In polarized fungiform taste buds loaded with Na-green (Fig. 4, A and B), the unilateral Na⁺ flux was measured as a temporal increase in F₄₉₀ in response to an increase in apical Na⁺ concentration from 0 to 150 mM (Fig. 4C) (Lyall et al. 2002b). The unilateral apical Na⁺ flux was measured when the polarized fungiform taste bud preparations were perfused bilaterally with Ringer solutions containing 1 mM Ca²⁺ or 0 Ca²⁺ (pH 7.4; Table 2) (Fig. 4C). The mean unilateral apical Na⁺ flux was enhanced significantly (P = 0.0073, unpaired; n = 3; 23 ROIs) in Ca²⁺-free Ringer solution (Fig. 4D). When the polarized taste buds were reperfused with control Ringer solution (pH 7.4; Table 2), the unilateral apical Na⁺ flux decreased to its control level (Fig. 4D). The changes in unilateral apical Na⁺ influx observed in our in vitro experiments are most likely related to the ionomycin- and BAPTA-induced effects on the NaCl CT responses in vivo shown in Figs. 1, 2, and 3, A–C.

Fig. 4.

Regulation of unilateral apical Na⁺ influx by taste receptor cell (TRC) Ca²⁺ concentration ([Ca²⁺]_i). A: the transmitted image of a polarized fungiform taste bud mounted in our custom perfusion chamber and viewed with a ×40 objective from the basolateral side. B: the fluorescence image excited at 490 nm after the cells were loaded with Na-green. The image in A shows that the dye is specifically loaded in the TRCs within the taste bud (arrow). Bar, 50 μm. C: a polarized fungiform taste bud preparation loaded with Na-green was initially perfused on both sides with Na⁺-free solution containing 150 mM N-methyl-d-glucamine-Cl (pH 7.4; 1 mM CaCl₂). Temporal changes in fluorescence intensity at 490 nm (F₄₉₀) were monitored while the apical compartment was perfused with the control Ringer solution containing 150 mM NaCl (pH 7.4; 1 mM CaCl₂). In the second part of the experiment, the apical and basolateral membranes were perfused with 0 Na⁺-0 Ca²⁺ solution for 15 min. Following this, the temporal changes in F₄₉₀ were monitored while the apical membrane was perfused with 150 mM Na⁺-0 Ca²⁺ Ringer solution (pH 7.4; 0 Ca²⁺). In each region of interest (ROI), the F₄₉₀ value in 0-Na⁺ Ringer solution was normalized to 100%. The changes in F₄₉₀ are presented as %change in fluorescence relative to 0 Na⁺, and values are means ± SE of n, where n = no. of ROIs within the taste bud. D: summary of the data from 3 individual polarized fungiform taste bud preparations. *P = 0.0073 (unpaired).

Effect of Changes in TRC [Ca²⁺]_i on the CT Responses to Sour Stimuli

Studies with ionomycin+Ca²⁺.

We have previously shown that tonic CT responses to HCl, CO₂, or acetic acid solutions containing 10 mM CaCl₂ are decreased after the tongue is treated with 150 μM ionomycin (Lyall et al. 2003, 2004a).

Studies with BAPTA-AM.

Loading TRCs in vivo with 13 mM BAPTA-AM inhibited the tonic CT response to 10, 20, and 30 mM HCl without affecting the phasic component of the HCl CT response (Fig. 5A). The mean phasic CT response to 20 mM HCl was not affected by BAPTA-AM treatment relative to control (Fig. 5B) (Lyall et al. 2003, 2004a). BAPTA-AM at 13 mM (Fig. 6, A and B) and 33 mM (Fig. 6, C and D) induced a concentration-dependent decrease in the tonic CT response to CO₂, acetic acid, and HCl (raw data not shown) without affecting the phasic component of the CT response. The mean phasic CT responses to CO₂ and acetic acid were not affected by BAPTA-AM treatment relative to control (data not shown). BAPTA induced a dose-dependent decrease in the tonic CT response to acidic stimuli (Figs. 7, A–C). In each case, titrating the Ca²⁺-binding sites on the intracellular BAPTA with ionomycin+Ca²⁺ restored the CT response to acidic stimuli close to their control values. Taken together, the results with ionomycin+Ca²⁺ and BAPTA-AM treatment indicate that the transduction mechanism for both strong and weak organic acids involves an increase in TRC [Ca²⁺]_i.

Fig. 5.

Effect of BAPTA on the CT responses to HCl. A: a representative CT trace in which the rat tongue was first stimulated with R and then with 10, 20, and 30 mM HCl under control conditions and after topical lingual application of 13 mM BAPTA-AM for 30 min. Arrows represent the time periods when the rat tongue was superfused with the rinse solution and the HCl solution. B: in each animal the phasic CT responses to 20 mM HCl were normalized to the tonic CT responses to 300 mM NH₄Cl, and values are means ± SE of 3 rats.

Fig. 6.

Effect of BAPTA and post-BAPTA–post-ionomycin+Ca²⁺ on the CT responses to HCl, CO₂, and AA. The CT responses to dissolved 10% CO₂ were recorded with 72 mM KHCO₃ + 10% CO₂-90% O₂ (pH 7.4) relative to the rinse solution (R_CO2 = 72 mM KCl + 10 mM HEPES, pH 7.4). The CT responses to 10 mM AA were recorded with 10 mM AA + 175 mM K-acetate (pH 6.1) relative to the rinse solution (R_AA = 175 mM KCl + 10 mM HEPES, pH 6.1) (Table 1). A and B: the topical lingual application of 13 mM BAPTA-AM inhibited the tonic part of the CT responses to dissolved CO₂ and AA by 42.5 and 41.7%, respectively. However, post-BAPTA treatment, topical lingual application of 150 μM ionomycin + 10 mM CaCl₂ (post-BAPTA–post-ionomycin+Ca²⁺) reversed the BAPTA-induced inhibition of the tonic part of the CT responses to CO₂ and AA stimulation by 99.0 and 112.2%, respectively. C and D: the topical lingual application of 33 mM BAPTA-AM completely inhibited the tonic part of the CT responses to dissolved CO₂ and AA, without affecting the transient phasic part of the neural response. However, post-BAPTA–post-ionomycin+Ca²⁺ treatment completely reversed the BAPTA-induced inhibition of the tonic part of the CT responses to CO₂ and AA stimulation. Arrows represent the time period for which the tongue was superfused with the acidic stimuli.

Fig. 7.

Summary of the effects of BAPTA and post-BAPTA–post-ionomycin+Ca²⁺ on the CT responses to sour taste stimuli. Values are means ± SE of the normalized CT responses to n, where n represents the number of animals in each group and is given in parentheses above each bar. In each animal, the CT responses under different experimental conditions were normalized to the CT response to 300 mM NH₄Cl. Effects of 0.1, 13, and 33 mM BAPTA treatment (post-BAPTA) and ionomycin + 10 mM CaCl₂ (post-BAPTA–post-ionomycin+Ca²⁺) are shown for 20 mM HCl (A), 10% CO₂ (pH 7.4) (B), and 10 mM AA (pH 6.1) (C). In each case, the topical lingual application of BAPTA-AM inhibited CT responses to the above stimuli in a dose-dependent manner. However, post-BAPTA–post-ionomycin+Ca²⁺ treatment completely reversed the BAPTA-induced inhibition of the CT responses to the above stimuli. *P < 0.01 (unpaired of n).

Relationship Between pH_i and [Ca²⁺]_i in Isolated Fungiform Taste Buds

Figure 8A shows a pseudocolor confocal fluorescence emission image of an isolated taste bud loaded with fluo 3 and perfused with control Ringer solution (Table 2; pH 7.4). Figure 8A shows that the fluorescent dye is taken up by all TRCs within the plane of the imaged taste bud. Figure 8B shows the same plane after 1 min following the replacement of the control Ringer solution with a Ringer solution containing 30 mM Na-acetate in the perfusion chamber (Table 2; pH 7.4). The changes in fluo 3 fluorescence are related to the pH_i changes observed in a separate set of isolated fungiform taste bud cells (Fig. 8E) loaded with BCECF and exposed to a basolateral Na-acetate pulse as in Fig. 8D. Figure 8C shows that individual TRCs and specific regions within a single TRC respond with changes in fluorescence intensity differently. In 16 ROIs investigated within the taste bud, 8 ROIs demonstrated an increase in fluorescence intensity (i.e., an increase in [Ca²⁺]_i) during the acidification phase of the Na-acetate pulse (Fig. 8C, a–b). Although not shown, 5 ROIs responded with a decrease in fluorescence (i.e., a decrease in [Ca²⁺]_i), and 3 ROIs demonstrated no significant change in fluorescence. In another isolated taste bud, 12 of 18 ROIs demonstrated an increase in fluo 3 emission fluorescence (i.e., an increase in [Ca²⁺]_i) during the acidification phase of the basolateral Na-acetate pulse (Fig. 8D, a–b) and a partial decrease in fluorescence during the spontaneous recovery from acidic pH (Fig. 8D, b–c). Upon Na-acetate washout, the alkalinization phase of the Na-acetate pulse was accompanied by a decrease in fluo 3 emission fluorescence (i.e., a decrease in [Ca²⁺]_i) (Fig. 8D, c–d), and the spontaneous recovery phase from the alkaline pH_i (Fig. 8D, d–e) was accompanied by an increase in fluo 3 emission fluorescence close to its baseline value.

Fig. 8.

Changes in TRC [Ca²⁺]_i and intracellular pH (pH_i) induced by a basolateral Na-acetate pulse. An isolated fungiform taste bud was loaded with fluo 3 and perfused with control Ringer solution. The temporal changes in the emission fluorescence intensity (E₅₃₀) were monitored while the taste bud was perfused with control Ringer solution and with Ringer solution containing 30 mM Na-acetate. A and B: a pseudocolor fluorescence emission image of an isolated unstimulated taste bud loaded with fluo 3 (A) and the same plane after 1 min following a Na-acetate pulse (B). In the unstimulated state for each ROI within the TRCs, the fluorescence intensity was normalized to 100%. In 16 ROIs investigated within the taste bud, 8 ROIs demonstrated an increase in fluorescence intensity relative to control (C), 5 ROIs responded with a decrease in fluorescence (data not shown), and 3 ROIs demonstrated no significant change in fluorescence (data not shown). Values are means ± SE of n, where n = no. of ROIs investigated in different TRCs. D: a separate set of isolated taste bud cells loaded with fluo 3 were initially bathed in control Ringer solution (Table 2; pH 7.4). During the time period shown by the top horizontal bar, the cells were perfused with Ringer solution containing 30 mM Na-acetate (Table 2; pH 7.4). Following this, the TRCs were perfused again with the control Ringer solution. Twelve of 18 ROIs responded with changes in fluo 3 emission intensity monitored with confocal microscopy during the basolateral Na-acetate pulse and return to control Ringer solution. Values are means ± SE of n, where n = no. of ROIs investigated. E: a separate set of isolated taste bud cells loaded with BCECF were initially bathed in control Ringer solution (Table 2; pH 7.4). During the time period shown by the top horizontal bar, the cells were perfused with Ringer solution containing 30 mM Na-acetate (Table 2; pH 7.4). Following this, the TRCs were perfused again with the control Ringer solution. Five of 9 ROIs responded with changes in pH_i as shown by conventional imaging during the basolateral Na-acetate pulse and return to control Ringer solution. The pH_i values are means ± SE of n, where n = no. of ROIs investigated.

The relationship between pH_i and [Ca²⁺]_i was also investigated in intact polarized fungiform taste bud preparations by briefly exposing the basolateral membrane of TRCs to Ringer solution containing 15 mM NH₄Cl or 0 Na⁺ (Vinnikova et al. 2004). The results presented in Fig. 9A show that during the NH₄Cl pulse, the initial increase in TRC pH_i (Fig. 9B, a–b) is accompanied by a decrease in [Ca²⁺]_i [a decrease in fura 2 fluorescence intensity ratio (FIR)], and upon NH₄Cl washout, a decrease in pH_i (Fig. 9B, c–d) is accompanied by an increase in [Ca²⁺]_i (increase in fura 2 FIR). A similar relationship between pH_i and [Ca²⁺]_i was observed in four individual polarized fungiform taste bud preparations exposed to brief basolateral NH₄Cl pulses (a total of 26 ROIs within the 4 taste buds). In a separate polarized fungiform taste bud preparation, [Ca²⁺]_i increased (Fig. 9C, a–b) as TRC pH_i acidified during the basolateral Na⁺ removal (Fig. 9D, a–b). Upon reperfusion of the basolateral membrane with 150 mM NaCl, the pH_i recovered to its resting value (Fig. 9D, c–d), accompanied by a decrease in [Ca²⁺]_i. A similar relationship between pH_i and [Ca²⁺]_i was observed in three individual polarized fungiform taste bud preparations exposed to basolateral 0 Na⁺ Ringer solution (a total of 18 ROIs within the 3 taste buds).These results suggest that within a taste bud TRCs are heterogeneous with respect to their responses to acid stimulation. Only those cells which demonstrate a strict inverse relationship between pH_i and [Ca²⁺]_i are likely to be involved in sour taste transduction.

Fig. 9.

Changes in TRC [Ca²⁺]_i and pH_i induced by a basolateral NH₄Cl pulse and Na⁺ removal. Separate polarized fungiform taste bud preparations loaded with fura 2 (A) or BCECF (B) were initially perfused on both sides with control Ringer solution (pH 7.4; Table 2). Temporal changes in fluorescence intensity ratio (FIR: F₃₄₀/F₃₈₀) of fura 2-loaded taste bud cells or pH_i in BCECF-loaded taste bud cells were monitored in different ROIs within the taste buds while the basolateral compartment was unilaterally perfused for 100 s with the Ringer solution containing 15 mM NH₄Cl (pH 7.4; Table 2) and then switched to control Ringer solution. In separate experiments, polarized fungiform taste bud preparations loaded with fura 2 (C) or BCECF (D) were initially perfused on both sides with control Ringer solution (pH 7.4; Table 2). Temporal changes in FIR of fura 2-loaded taste bud cells or pH_i in BCECF-loaded taste bud cells were monitored in different ROIs within the taste buds while the basolateral compartment was unilaterally perfused with the 0-Na⁺ Ringer solution (pH 7.4; Table 2) and then switched to control Ringer solution. In each ROI, the FIR of fura 2-loaded taste bud cells in control Ringer solution was normalized to 1. The FIR or pH_i values are means ± SE of n, where n = no. of ROIs investigated.

Effect of U73122 on the CT Responses to Salty Stimuli

U73122 inhibited the tonic CT responses to 100 mM NaCl (Fig. 10A). In the presence of U73122, the decrease in NaCl CT response was due to a decrease in both the Bz-sensitive and Bz-insensitive components of the NaCl CT response (Fig. 10, A and B). U73122 did not inhibit the control CT response to 0.3 M NH₄Cl (Fig. 10B). U73343, its inactive analog, had no effect on CT responses to any taste stimuli tested (Coleman et al. 2011; Lyall et al. 2010). These data suggest that CT responses to salty stimuli are also regulated by one or more PLCs in salt-sensing TRCs.

Fig. 10.

Effect of U73122 on the CT responses to taste stimuli. A: CT responses were recorded while the tongue was first superfused with R and then with 100 mM NaCl (NaCl) and 100 mM NaCl + 5 μM Bz (NaCl+Bz) under control conditions and after the topical application of 250 μM U73122 (post-U73122). Arrows represent the time period for which the tongue was superfused with different taste stimuli. B: summary of the effects on U73122 on the tonic CT response to 300 mM NH₄Cl, 300 mM NaCl, 100 mM NaCl, 100 mM NaCl + 5 μM Bz, and the Bz-sensitive component [NaCl − (NaCl+Bz)] in 4 rats. In each animal, the NaCl CT responses were normalized to the corresponding CT responses obtained with 300 mM NH₄Cl, and values are means ± SE of 4 rats. *P = 0.011; **P = 0.0044; ***P = 0.0028 (unpaired).

Effect of Thapsigargin on the CT Responses to Salty and Sour Stimuli

Topical lingual application of thapsigargin inhibited the tonic CT responses to NH₄Cl, NaCl, and HCl relative to control (Fig. 11). These data suggest that CT responses to salty and sour stimuli are also partially dependent on Ca²⁺ release from internal stores.

Fig. 11.

Effect of thapsigargin on the CT responses to taste stimuli. CT responses were recorded while the tongue was first superfused with R and then with 300 mM NH₄Cl, 300 mM NaCl, 100 mM NaCl, and 20 mM HCl before (control) and after topical lingual application of 250 μM thapsigargin for 40 min (post-thapsigargin). In each animal, the CT responses under different experimental conditions were normalized to the CT response to 300 mM NH₄Cl at the beginning of the experiment, and values are means ± SE of 3 rats. *P = 0.0087; **P = 0.0001 (unpaired).

DISCUSSION

The main effects of changes in TRC cytosolic [Ca²⁺]_i on the tonic CT responses to salty and sour taste stimuli along with the proposed taste receptor/ion channels, transporters, and intracellular signaling intermediates regulated by [Ca²⁺]_i are summarized in Table 3. Overall, the data suggest that, similar to the sweet, bitter, and umami taste qualities (DeSimone et al. 2012), salty and sour taste qualities are also transduced by [Ca²⁺]_i-dependent and [Ca²⁺]_i-independent mechanisms.

Table 3.

Summary of effects of BAPTA and ionomycin+Ca²⁺ on tonic CT responses to salty and sour taste stimuli, apical Na⁺ conductance, and unilateral apical Na⁺ influx

Taste Stimuli	Response Measured	Ionomycin+Ca2+ (↑TRC [Ca2+]i)	BAPTA-AM (↓TRC [Ca2+]i)	Target
NaCl, NaCl+Bz	Bz-sensitive NaCl CT response [NaCl − (NaCl+Bz)]	↓	↑	ENaC
NaCl, NaCl+Bz	Bz-insensitive NaCl CT response (NaCl+Bz)	↔	↑	TRPV1t
NaCl+Bz, NaCl+Bz+RTX*	RTX concentration vs. magnitude of Bz-insensitive NaCl CT response	↓	↑	TRPV1t
NaCl	Apical membrane Na+ conductance	↓	↑	ENaC
KCl	KCl CT response	↔	↔	‡TRPV1t
NH4Cl	NH4Cl CT response	↔	↔	‡TRPV1t
HCl, CO2, acetic acid	HCl, CO2, acetic acid CT response	↓	↓	†NHE-1
NaCl	Unilateral apical Na+ influx	↓	↑	ENaC

Arrows indicate effects (↓, decrease; ↑, increase; or ↔, no change) of ionomycin+Ca²⁺ (increased taste receptor cell [Ca²⁺]_i) and BAPTA-AM (decreased taste receptor cell [Ca²⁺]_i) in chorda tympani (CT), Na⁺ conductance, or Na⁺ influx response to various taste stimuli. ENaC, epithelial Na⁺ channels; RTX, resiniferatoxin.

^*Data are from Lyall et al. (2010). The phasic CT responses to acidic stimuli are Ca²⁺ independent.

^†The increase in adaptation occurs due to [Ca²⁺]_i-induced activation of the basolateral Na+-H+ exchanger 1 (NHE-1) (Lyall et al. 2002a, 2004a).

^‡About 30–40% of the CT response to KCl and NH₄Cl can be accounted for by the flux of K⁺ and NH₄⁺ through the putative TRPV1t (variant of transient receptor potential vanilloid-1) cation channels (DeSimone et al. 2001).

Changes in Cytosolic [Ca²⁺]_i Regulate ENaC and the Bz-Sensitive NaCl CT Response

In inside-out patch recordings of M1 cells (mouse kidney cortical collecting duct cells) elevating [Ca²⁺]_i inhibited ENaC open probability without altering the channel conductance. The inhibitory effect was due to a direct interaction between Ca²⁺ and ENaC, and was dependent on [Ca²⁺]_i (Gu 2008). Here, we show that changes in TRC [Ca²⁺]_i regulate 1) unilateral apical Na⁺ influx (Fig. 4); 2) apical membrane Na⁺ conductance (Figs. 1C and 2C); and 3) the magnitude of the Bz-sensitive NaCl CT response (Fig. 1, A and B; Fig. 2, A and B; and Fig. 3, A–C). Decreasing TRC [Ca²⁺]_i with BAPTA specifically enhanced the CT response to NaCl (Fig. 3, A–C). Taken together, these results suggest that salt sensing involves changes in TRC Ca²⁺ in at least two separate subcompartments. TRCs have a cytosolic [Ca²⁺]_i compartment in which [Ca²⁺]_i involved in taste transduction may be buffered with BAPTA and is associated with regulating ENaC activity. In a separate subcompartment that does not seem to be accessible to BAPTA, [Ca²⁺]_i is mobilized during neurotransmitter release.

In a subset of TRCs, a decrease in pH_i is accompanied by an increase in [Ca²⁺]_i and an increase in pH_i with a decrease in [Ca²⁺]_i (Figs. 8 and 9) (Lyall et al. 2003). We have previously shown that a decrease in TRC pH_i decreases ENaC activity and inhibits the magnitude of the Bz-insensitive NaCl CT response, and an increase in pH_i enhances ENaC activity and increases the Bz-insensitive NaCl CT response (Lyall et al. 2002b). This suggests that changes in TRC pH_i can directly and/or indirectly regulate ENaC activity via secondary changes in [Ca²⁺]_i (Gu 2008). It is expected that an increase in both [H⁺]_i (i.e., a decrease in pH_i) and [Ca²⁺]_i will have an additive effect in inhibiting ENaC activity and thus in decreasing the Bz-sensitive NaCl CT response. Similarly, an increase in pH_i and the accompanying decrease in [Ca²⁺]_i could have an additive effect in enhancing the Bz-sensitive NaCl CT response. Thus [Ca²⁺]_i and [H⁺]_i are important regulators of ENaC in salt-sensing TRCs and of the Bz-sensitive NaCl CT response.

The regulation of ENaC activity by pH varies in different tissues and in different species. In Xenopus oocytes expressing human α-, β-, and γ-ENaC subunits, amiloride-sensitive current was enhanced at pH 6.0 and decreased at pH 8.5 relative to pH 7.4. In contrast, the pH-induced changes were not observed with rat α-, β-, and γ-ENaC subunits expressed in oocytes (Collier and Snyder 2009a). This difference in pH sensitivity was attributed to species differences in the γ-ENaC subunit. An additional ENaC subunit, the δ-subunit, is activated by acidic pH (Ji and Benos 2004). However, the δ-ENaC subunit is not expressed in the rat or in human renal epithelia (Collier and Snyder 2009a). Since this subunit is expressed in a subset of human TRCs (Huque et al. 2009), it raises the possibility that the regulation of ENaC in human TRCs by pH may differ from the rat responses reported in this study.

It is suggested that changes in pH alter human ENaC activity by modulating Na⁺ self-inhibition (Collier and Snyder 2009b). In rats, TRC ENaC activity and the Bz-sensitive NaCl CT response are also regulated by Na⁺ self-inhibition (DeSimone and Lyall 2008; Gilbertson and Zhang 1998). However, the modulation of NaCl CT responses by pH_i and [Ca²⁺]_i under the experimental conditions described in this study cannot be explained by alterations in Na⁺ self-inhibition of the ENaC activity in TRCs. Changes in H⁺ and [Ca²⁺]_i most likely inhibit the Bz-insensitive NaCl CT responses in rats by directly inhibiting the ENaC activity in TRCs (Fig. 4) (Gu 2008; Lyall et al. 2002b).

Changes in Cytosolic [Ca²⁺]_i Regulate TRPV1t and the Bz-Insensitive NaCl CT Responses

The Bz-insensitive NaCl CT response is enhanced by decreasing TRC [Ca²⁺]_i (Fig. 2B). We have previously shown that the TRPV1t-dependent Bz-insensitive NaCl CT response is 1) modulated by vanilloids, CPC, ethanol, nicotine, Maillard reacted peptides, and elevated temperature; 2) inhibited by capsazepine and SB-366791; and 3) absent in TRPV1 knockout mice (Lyall et al. 2004b). Changes in TRC [Ca²⁺]_i modulate the agonist concentration versus the magnitude of the Bz-insensitive NaCl CT response relationships by altering channel activity indirectly through protein kinase C- and calcineurin-dependent changes in the phosphorylation-dephosphorylation state of the channel protein (Lyall et al. 2009).

About 40% of the CT response to KCl and NH₄Cl can be accounted for by the influx of K⁺ and NH₄⁺ via the putative apical TRPV1t cation channel (Lyall et al. 2004b). Since BAPTA treatment only produced a small change in the Bz-insensitive NaCl CT response (Fig. 2B), it is expected that changes in TRC [Ca²⁺]_i would also have only minimal effect on the K⁺ and NH₄⁺ flux through the putative TRPV1t cation channel (Fig. 3, D and E). Thus an increase in TRC [Ca²⁺]_i is not a prerequisite requirement for eliciting a CT response to KCl or NH₄Cl. The CT responses to KCl are also insensitive to changes in [Ca²⁺]_i and pH (Lyall et al. 2002b).

In TRCs loaded with 33 mM BAPTA-AM, it is expected that the cytosolic [Ca²⁺]_i is buffered at a value much below normal. However, under the conditions where the cytosolic [Ca²⁺]_i is close to zero, we observed a bigger NaCl CT response relative to control (Fig. 2, A and B, and Fig. 3, A–C). If an increase in the cytosolic [Ca²⁺]_i is a prerequisite for the release of the neurotransmitter (Finger et al. 2005; Huang and Roper 2010), one would predict that in the presence of intracellular BAPTA, no CT response would have been observed. This suggests that changes in cytosolic [Ca²⁺]_i are mostly involved in the regulation of ENaC and TRPV1t activity and are not associated with the neurotransmitter release from TRCs onto CT nerve fibers. The latter compartment associated with the neurotransmitter release does not seem to be affected by ionomycin or BAPTA treatment under the in vivo and in vitro experimental conditions used in this study.

Changes in Cytosolic [Ca²⁺]_i Regulate CT Responses to Acidic Stimuli

In polarized TRCs, stimulating the apical membrane with acidic stimuli elicited a decrease in TRC pH_i (Lyall et al. 2001). At constant external pH (pH_o), inducing a rapid decrease in pH_i with the use of short basolateral pulses of Na-acetate or NH₄Cl induced an increase in [Ca²⁺]_i in a subset of TRCs (Figs. 8 and 9). Conversely, intracellular alkalinization was accompanied by a decrease in [Ca²⁺]_i (Lyall et al. 2003). This positive relationship between [H⁺]_i and [Ca²⁺]_i is important for acid taste transduction for both strong and weak organic acids (Richter et al. 2003). In many cases, weak organic acids are good stimuli even when presented at near-neutral pH. In this case, weak organic acids can permeate the apical membrane and enter TRCs as neutral undissociated molecules. Once inside the cell, the undissociated molecules dissociate to yield H⁺ ions (Lyall et al. 2001). Facilitating apical H⁺ entry by incorporating the K⁺/H⁺ exchanger nigericin in the apical membrane of TRCs in in vivo elicits a CT response at pHs close to the neutral pH (Sturz et al. 2011). Thus the proximate signal for both strong and weak acid transduction is a decrease in TRC pH_i, and a downstream signaling event is the pH_i-induced increase in [Ca²⁺]_i (DeSimone and Lyall 2006).

At present, the exact mechanism as to how a change in pH_i induces an increase in [Ca²⁺]_i is not clear. Since in mouse TRCs removing extracellular Ca²⁺ reduced acid-evoked Ca²⁺ responses but depleting intracellular Ca²⁺ stores with thapsigargin had no effect, it was suggested that acid taste responses are generated by an influx of extracellular Ca²⁺. Based on the observations that acid-evoked Ca²⁺ responses could be blocked by Ba²⁺ and Cd²⁺, the data further suggested that Ca²⁺ influx occurred through the VGCCs (Richter et al. 2003).

BAPTA treatment produced a differential effect on the phasic and tonic components of the CT responses to acidic stimuli (Figs. 5 and 6). This suggests that the initial transient phasic CT response is independent of changes in [Ca²⁺]_i in the cytosolic compartment but depends on Ca²⁺ increase in a subcompartment in TRCs that is not accessible by BAPTA. The transduction mechanisms for phasic and tonic components of the CT responses to acidic stimuli are different. The phasic component of the CT response depends on the pH_i-induced cell shrinkage and the activation of a basolateral flufenamic acid-sensitive shrinkage-activated nonselective cation channel (Lyall et al. 2006).

The increase in cytosolic [Ca²⁺]_i is not only essential for the generation of the tonic part of the CT response to acids, but it also regulates the tonic part of the response. Topical lingual application of zoniporide (a specific blocker of the basolateral Na⁺/H⁺ exchanger-1, NHE-1) reversed the effect of ionomycin+Ca²⁺ on the CT responses to acidic stimuli (Lyall et al. 2003, 2004a). These results suggest that an increase in [Ca²⁺]_i activates the basolateral NHE-1 in TRCs, which in turn increases the rate of intracellular pH and cell volume recovery and increases neural adaptation to acidic stimuli (Lyall et al. 2002a, 2004a).

IP₃- and Thapsigargin-Dependent [Ca²⁺]_i Subcompartment

An increase in [Ca²⁺]_i is required for the release of neurotransmitter from the presynaptic terminals in all neurons (Llinas and Moreno 1998). An increase in [Ca²⁺]_i in the microdomains against the cytoplasmic surface of the plasmalemma during transmitter release suggests that the synaptic vesicle fusion responsible for transmitter release is triggered by the activation of a low-affinity Ca²⁺-binding site at the active zone (Llinas and Moreno 1998). However, the relationship between changes in [Ca²⁺]_i in the cytosolic compartment and the synaptic regions of the TRCs is not known at present. In our studies, depleting intracellular Ca²⁺ stores with thapsigargin reduced CT responses to salty and sour taste stimuli (Fig. 11). Ryanodine receptors contribute to some taste-evoked signals that are dependent on Ca²⁺ release from internal stores. In a subset of type III cells, blocking ryanodine receptors reduced an increase in Ca²⁺ induced by depolarizing the membrane potential with high K⁺ (Rebello and Medler 2010). This suggests that ryanodine receptors may be functionally coupled to VGCCs in a subset of type III cells and may contribute to the depolarization-induced Ca²⁺ signal.

The decrease in the Bz-insensitive NaCl CT response in the presence of U73122 is most likely due to changes in PIP₂ and its interactions with TRPV1t (Lyall et al. 2010). Besides PLCβ₂, several other PLC isoforms (PLCβ₁, PLCβ₃, PLCβ₄, and PLCγ₁) have been shown to be present in fungiform and circumvallate TRCs (Chandrashekar et al. 2006; Hacker et al. 2008; Lyall et al. 2010; Toyono et al. 2005). U73122 affects the Bz-insensitive NaCl CT response by inhibiting one or more PLC isoforms other than the PLCβ₂ isoform and increasing membrane PIP₂ levels. The Bz-sensitive NaCl CT response decreased in the presence of U73122 (Fig. 10, A and B). In A6 cells, an increase in PIP₂ increased ENaC activity by direct interaction with β- or γ-ENaC subunits (Yue et al. 2002), and inhibiting PLC with U73122 enhanced ENaC activity by increasing the mean open time of the channel (Pochynyuk et al. 2008a). These results suggest that PIP₂ regulation of ENaC may be tissue and cell specific. In addition to PIP₂, a physiological role for phosphatidylinositide 3-OH kinase (PI3-K) and its product, phosphatidylinositiol 3,4,5-trisphosphate (PIP₃), in modulating ENaC activity has been documented (Pochynyuk et al. 2008b). It is likely that U73122-induced changes in membrane PIP₂ levels affect ENaC activity indirectly via changes in PIP₃.

Thus, although a decrease in BAPTA-sensitive cytosolic [Ca²⁺]_i enhances the Bz-sensitive NaCl CT response, inhibiting IP₃ generation (Fig. 10, A and B) or depleting intracellular Ca²⁺ stores with thapsigargin (Fig. 11) attenuated the CT response. Thapsigargin also attenuated CT responses to HCl relative to control (Fig. 11). These results tend to suggest that even in salt-sensing and sour-sensing TRCs, neurotransmitter release may also be partially dependent on a IP₃- and thapsigargin-sensitive [Ca²⁺]_i subcompartment.

In summary, changes in pH_i and cytosolic [Ca²⁺]_i modulate Na⁺ flux through apical ENaCs and hence modulate the Bz-sensitive NaCl CT response. In contrast, Na⁺ transport through the putative TRPV1t nonspecific cation channel is much less sensitive to changes in cytosolic [Ca²⁺]_i and [H⁺]_i (Lyall et al. 2002b). For acidic stimuli, a decrease in TRC pH_i (Lyall et al. 2004a) elicits an increase in cytosolic Ca²⁺ and activation of basolateral NHE-1, which leads to a decrease in the tonic CT response to acid stimulation, and hence causes adaptation of the neural response. The phasic part of the CT response to acidic stimuli is independent of cytosolic Ca²⁺ but must involve an increase in [Ca²⁺]_i in a subcompartment. Since HCl CT responses are enhanced by cAMP (DeSimone et al. 2011; Lyall et al. 2002a), there may be cross talk between cAMP and Ca²⁺ signaling to integrate tastant-evoked signals in a subset of type III cells (Roberts et al. 2009). Sour-sensing type III TRCs (presynaptic cells) possess specialized chemical synapses, synthetic enzymes for aminergic neurotransmitters and proteins involved in vesicular release (Roper 2007). In type I taste cells involved in Na⁺ sensing, depolarization caused by the entry of Na⁺ could be sufficient to cause the release of a neurotransmitter.

GRANTS

This work was supported by National Institute of Deafness and other Communication Disorders Grants DC-000122, DC-005981, and DC-011569 (to V. Lyall). Imaging cytometry was supported in part by National Cancer Institute Grant P30 CA16059.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.A.D. and V.L. conception and design of research; J.A.D., Z.R., T.-H.T.P., G.L.H., S.M., and V.L. analyzed data; J.A.D. and V.L. interpreted results of experiments; J.A.D., Z.R., S.M., and V.L. prepared figures; J.A.D. and V.L. drafted manuscript; J.A.D. and V.L. edited and revised manuscript; J.A.D. and V.L. approved final version of manuscript; Z.R., T.-H.T.P., G.L.H., S.M., and V.L. performed experiments.