Modulation of Rat Chorda Tympani NaCl Responses and Intracellular Na+ Activity in Polarized Taste Receptor Cells by pH

Abstract

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pHi) and Na+ activity ([Na+]i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pHo). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pHo to 2.0 or 10.3. At constant pHo, buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO3 −/CO2 inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO3 −/CO2 buffer, suggesting a regulatory role for pHi. In polarized TRCs step changes in apical pHo from 10.3 to 2.0 induced a linear decrease in pHi that remained within the physiological range (slope = 0.035; r2 = 0.98). At constant pHo, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO3 −/CO2 decreased resting TRC pHi, and MK-507 or MK-417 attenuated the decrease in pHi in TRCs perfused with HCO3 −/CO2 buffer. In parallel experiments, TRC [Na+]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na+, and (d) acid loading the cells with NH4Cl or sodium acetate at constant pHo. Diethylpyrocarbonate and Zn2+, modification reagents for histidine residues in proteins, attenuated the CO2-induced inhibition of NaCl CT responses and the pHi-induced inhibition of apical Na+ influx in TRCs. We conclude that TRC pHi regulates Na+-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Na+-H+Exchange Activity in Taste Receptor Cells

“…Consistent with our previous observations (Lyall et al 2002b), immediately following the perfusion of Ringer solution containing 30 mM NaA (RNaA; Table 1) in the basolateral compartment, pH i rapidly acidified (a to b), presumably due to the entry of membrane-permeable undissociated acetic acid and its conversion to free intracellular H ϩ ions plus acetate anion (Roos and Boron 1981). The intracellular acidification was transient and was followed by a spontaneous recovery of pH i to baseline (b to c).…”

“…During acid stimulation, a decrease in TRC pH i , rather than a decrease in extracellular pH (pH o ), is the stimulus intensity variable that correlates specifically with increased chorda tympani (CT) taste nerve activity. Inhibiting acid-induced TRC acidification also inhibits the acid-evoked CT response (Lyall et al , 2002b. These results indicate that a decrease in TRC pH i is the proximate stimulus for sour taste.…”

“…For the measurement of pH i , TRCs within the taste bud were loaded with BCECF, and for Na ϩ measurement ([Na ϩ ] i ), the taste cells were loaded with either 1,3-benzenedicarboxylic acid, 4,4Ј- [1,4,10-trioxa-7,13-diazacyclopentadecane-7, 13-diylbis(5-methoxy-6,12-benzofurandiyl]bis-, tetrakis[(acetyloxy) methyl] ester (SBFI) or Na-green. The detailed methods for the measurement of pH i using BCECF (DeSimone et al 2001b;Lyall et al , 2002a and the measurement of [Na ϩ ] I using SBFI or Na-green have been described earlier (Lyall et al 2002b). In brief, TRCs in the taste bud were visualized from the basolateral side through a 40ϫ objective (Zeiss; 0.9 NA) with a Zeiss Axioskop 2 plus upright fluorescence microscope and imaged with a set up consisting of: a cooled CCD camera (Imago, TILL Photonics, Applied Scientific Instrumentation, Eugene, OR) attached to an image intensifier (VS4 -1845, Videoscope, Washington, DC), an epifluorescent light source (TILL Photonics Polychrome IV), dichroic beam splitters, and emission filters for BCECF and fura-2 (Omega Optical).…”

“…For this reason, SBFI is not suitable for measuring changes in TRC [Na ϩ ] i in the presence of these drugs. Accordingly, these experiments were performed with the single wavelength excitation dye, Na-green (Lyall et al 2002b). …”

Taste Receptor Cells Express pH-Sensitive Leak K+ Channels

“…Acid modification may occur by direct interaction of H ϩ on ion channels that function as taste transducers, such as the epithelial Na ϩ channel (ENaCs). Protons modulate its activity and salt sensation by interaction with either the extracellular (Zhang et al 1999) or intracellular sides of the channel (Lyall et al 2002), or the H ϩ modification may be indirectly altering activities of ion channels and proteins that influence cell excitability. The degree of modification depends on acid concentrations and the H ϩ sensitivity of these components.…”