Although part of this study was funded by the financial support from Senomyx, this does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: VL JAD GLH MR. Performed the experiments: SM THTP JQ. Analyzed the data: VL JAD SM JQ. Contributed reagents/materials/analysis tools: SM THTP JQ. Wrote the paper: VL JAD.
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 (
In rats, about 70% of the chorda tympani (CT) taste nerve response to NaCl is due to Na+ influx through the amiloride- and benzamil (Bz)-sensitive epithelial Na+ channel (ENaC) expressed in the apical membrane of a subset of fungiform taste bud cells, and is associated with appetitive behavioral responses to low NaCl concentrations
Recently, a small molecule activator, N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide of hENaC has been described. It demonstrated a threshold for activating hENaC expressed in frog oocytes at around 30 nM and produced a half-maximal response at 1.2 µM. It produced around 700% increase in the amiloride-sensitive Na+ current above baseline with a Hill coefficient of 1.0
Our results show that Compound 1 and Compound 2 increased the open-circuit Bz-sensitive NaCl CT response (
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 obtained from Senomyx Inc., San Diego, CA. 8-(4-Chlorophenylthio) adenosine 3′,5′-cyclic monophosphate (8-CPT-cAMP), 8-(4- Chlorophenylthio)adenosine- 3′,5′- cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS), 8-(4- Chlorophenylthio) guanosine- 3′,5′- cyclic monophosphorothioate (8-CPT-cGMP); 3-Isobutyl-1-methylxanthine (IBMX), forskolin, benzamil (Bz), 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS) and N-methyl -D-glucamine hydrochloride (NMGD-Cl) were obtained from Sigma-Aldrich.
The animals were housed in the Virginia Commonwealth University (VCU) animal facility in accordance with institutional guidelines. All
The dependence of the Bz-sensitive part of the NaCl CT response on the concentration of agonists was fitted to a modified Hill Equation of the form:
Here,
Stimulating solutions (mM) | Rinse (mM) | |
100 NaCl | 10 KCl | |
100 NaCl +0.005 Bz | 10 KCl | |
100 NaCl +0.25 to 1.0 Compound 1 | 10 KCl | |
100 NaCl +0.005 Bz +0.25–1.0 Compound 1 | 10 KCl | |
100 NaCl +0.25 to 1.0 Compound 2 | 10 KCl | |
100 NaCl +0.005 Bz +0.25–1.0 Compound 2 | 10 KCl | |
100 NaCl +10 HEPES (pH 7.0) | 10 KCl +10 HEPES (pH 7.0) | |
100 NaCl +10 HEPES +0.005 Bz (pH 7.0) | 10 KCl +10 HEPES (pH 7.0) | |
100 NaCl +10 TRIS (pH 10.3) | 10 KCl +10 TRIS (pH 10.3) | |
100 NaCl +10 TRIS +0.005 Bz (pH 10.3) | 10 KCl +10 TRIS (pH 10.3) | |
50–1000 NaCl | 10 KCl | |
50–1000 NaCl +0.005 Bz | 10 KCl | |
300 NH4Cl | 10 KCl | |
↓ [Ca2+]i | 33 BAPTA-AM | 30 |
↑ [Ca2+]i | 0.15 ionomycin +10 CaCl2 | 30 |
↑ cAMP | 20 8-CPT-cAMP | 30 |
↑ cAMP | 0.1 IBMX +0.1 forskolin | 20 |
↑ cGMP | 20 8-CPT-cGMP | 30 |
↓ PKA | 4 Rp-8-CPT-cAMP | 20 |
↑ pHi | 10 HEPES (pH 7.0) to 10 TRIS (pH 10.3) |
CT responses were monitored under open-circuit conditions and under ±60 mV lingual voltage clamp.
pH of the solutions containing HEPES or TRIS was adjusted with HCl or NaOH.
In preliminary studies, 8-CPT-cAMP was applied topically to the anterior lingual surface of rat tongues at varying concentrations ranging between 5 and 20 mM and for different time intervals between 0 and 30 min. Application of 8-CPT-cAMP produced a dose-dependent increase in the NaCl CT response producing the maximum increase in the response between 15 and 20 mM (data not shown). The effects of 8-CPT-cAMP could only be observed after 10–15 min of its lingual application (data not shown). Accordingly, here we monitored the NaCl CT response before and after topical lingual application of 20 mM 8-CPT-cAMP for 30 min. In some studies rat tongue was pretreated with Rp-8-CPT-cAMP before applying 8-CPT-cAMP. Rp-8-CPT-cAMP is an inhibitor of the activation by cAMP of cAMP-dependent protein kinase I and II
Four rats were anesthetized by exposing them to an inhalation anesthetic, isoflurane (1.5 ml) in a desiccator. When rats were fully unconscious, a midline incision was made in the chest wall and the aorta severed. The tongues were then rapidly removed and stored in ice-cold control Ringer’s solution (
Salt | Control Ringer’s | 150 Na+- Ringer’s | |
NaCl | 140 | 150 | 0 |
KCl | 5 | 5 | 5 |
CaCl2 | 1 | 1 | 1 |
MgCl2 | 1 | 1 | 1 |
Na-pyruvate | 10 | 0 | 0 |
Glucose | 10 | 10 | 10 |
HEPES | 10 | 10 | 10 |
NMDG-Cl | 0 | 0 | 150 |
pH | 7.4 | 7.4 | 7.4 |
In some experiments the 0 Na+ Ringer’s solution contained in addition, 150 µM 8-CPT-cAMP or 3 µM ionomycin.
The pH of the solutions was adjusted with HCl or NaOH.
For the measurement of unilateral apical Na+ influx, taste cells within the taste bud were loaded with sodium green (10 µM) dissolved in control Ringer’s solution (
In taste bud cells loaded with Na-green the changes in Na+ were expressed relative to the fluorescence intensity (F490) under control conditions. The F490 under control conditions for each ROI was taken as 100%. Student’s t-test was employed to analyze the differences between sets of data
(
(
Since Compound 1 modulates the Bz-sensitive part of the response exclusively (
ENaC Modulator | ||
0.0019±0.0004 | 0.388±0.020 | |
0.0038±0.0009 | 0.659±0.046 | |
0.0019±0.0005 | 0.390±0.020 | |
0.0032±0.0004 | 0.527±0.019 | |
0.0016±0.0004 | 0.280±0.018 | |
0.0037±0.0008 | 0.629±0.038 | |
0.0021±0.0001 | 0.474±0.005 | |
0.0036±0.0002 | 0.699±0.008 | |
0.0018±0.0003 | 0.373±0.017 | |
0.0040±0.0005 | 0.611±0.027 | |
0.0022±0.0004 | 0.318±0.002 | |
−0.0001±0.0001 | −0.009±0.004 | |
0.0019±0.0001 | 0.370±0.027 |
Values are mean ± SEM of 3 animals in each group.
The Mean control value represents the mean of all control parameters under the 6 different experimental conditions.
(
(
Open-circuit normalized CT responses to 50, 100, 200, 300 and 500 mM NaCl in the absence and presence of 5 µM Bz. The Bz-sensitive component was obtained as the difference between the CT response in the absence and presence of Bz. The values are mean ± SEM of 3 rats. The curve for the CT response to NaCl+Bz (Bz-insensitive curve) as a function of the NaCl concentration was linear up to 500 mM NaCl and was accordingly fitted to a regression line (R = 0.998) with intercept = −0.011±0.006 and slope = 0.772±0.023. The curve for the Bz-sensitive response was fitted to eq. 3 which describes the saturation of the open-circuit response,
NaCl (mM) | 100 NaCl+Compound 1 (mM) | BAPTA-AM | pH | 8-CPT-cAMP | |||||
100 | 0.33±0.05 | 0.0 | 0.37±0.03 | Control | 0.37±0.03 | 7.0 | 0.37±0.03 | Control | 0.37±0.03 |
200 | 0.51±0.03 | 0.5 | 0.49±0.02 | ||||||
300 | 0.63±0.05 | 1.0 | 0.66±0.05 | Post- | 0.70±0.01 | 10.3 | 0.61±0.03 | Post- | 0.63±0.04 |
Control = Mean control value from
Arginine vasopressin and cAMP have been shown to increase the amiloride-sensitive Na+ current in isolated taste bud cells
(
The enhanced response due to 8-CPT-cAMP also varied linearly with voltage (
Topical application of an equivalent concentration of 8-CPT-cGMP did not produce changes in the NaCl CT response relative to control (data not shown). This suggests that these effects are specific for 8-CPT-cAMP. The 8-CPT-cAMP induced increase in the Bz-sensitive NaCl CT response was not observed when the rat tongue was treated with Rp-8-CPT-cAMP (
Similar results for BAPTA-AM, pHi and ionomycin+Ca2+ are shown respectively in
Treating the basolateral membrane of polarized fungiform taste bud cells with 150 µM 8-CPT-cAMP increased the F490 fluorescence, and thus the unilateral apical Na+ influx into taste bud cells (
Each ENaC agonist investigated caused
(
The fact that
Since
Here,
Investigating the effect of ENaC enhancers on neural and behavioral responses to NaCl is important in identifying salt taste enhancers that may be useful in lowering salt intake. Compound 2 has been identified as a potent small molecule activator of ENaC. At a concentration of 1 µM and 30 µM Compound 2 enhanced the αβγ and δβγ hENaC expressed in oocytes by 300% and 700%, respectively. In contrast, only weak αβγ mENaC activation was observed at 100–300 µM concentrations of Compound 2
In rats, typically about 70% of the NaCl CT response is Bz-sensitive (
Both Compound 1 and Compound 2 increased the slope of the line representing the response as a function of voltage as well as the open-circuit response (
Compound 2 seems to exert its agonist action by binding directly to the β subunit of hENaC
Both an exogenous (
The Bz-sensitive component of the NaCl CT response is enhanced by systemic administration of aldosterone
While these sites are likely to be different for each agonist tested, the effect in each case of ENaC conductance on the NaCl CT response seems to be the same, i.e. an increase in
BAPTA-AM and ionomycin+Ca2+ alter [Ca2+]i in all fungiform taste bud cells. In our previous studies
No CT response is observed by changing the rinse solution pH from 7.0 to 10.3
Our results further tend to suggest that there may be two sub-compartments, a cytosolic compartment and a synaptic compartment in taste cells in which changes in [Ca2+]i play different roles in taste reception. While changes in [Ca2+]i in the synaptic compartment in taste cells play a role in neurotransmitter release, changes in [Ca2+]i in the cytosolic compartment play a regulatory role in modulating the activity of ion channels, transporters and other downstream intracellular signals in transduction
A specific enhancement in the Bz-sensitive salt taste sensitivity by ENaC enhancers may contribute to lower Na+ intake. Alternately, a specific inhibition in the Bz-sensitive salt taste sensitivity by ENaC inhibitors may contribute to increased Na+ intake. Inhibiting ENaC activity by amiloride appears to render NaCl qualitatively indistinguishable from KCl
In contrast to the studies in rodents
In summary, our results demonstrate that enhancing ENaC activity by Compound 1, Compound 2, 8-CPT-cAMP, BAPTA-AM and alkaline pH specifically increases the magnitude of the Bz-sensitive NaCl CT response, apical membrane Na+ conductance and apical Na+ flux across the apical membrane of a subset of fungiform taste bud cells. Unlike the studies on hENaC expressed in oocytes
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