A Novel Pharmacological Probe Links the Amiloride-Insensitive NaCl, KCl, and NH₄Cl Chorda Tympani Taste Responses

Abstract

Chorda tympani taste nerve responses to NaCl can be dissected pharmacologically into amiloride-sensitive and -insensitive components. It is now established that the amiloride-sensitive, epithelial sodium channel acts as a sodium-specific ion detector in taste receptor cells (TRCs). Much less is known regarding the cellular origin of the amiloride-insensitive component, but its anion dependence indicates an important role for paracellular shunts in the determination of its magnitude. However, this has not precluded the possibility that undetected apical membrane ion pathways in TRCs may also contribute to its origin. Progress toward making such a determination has suffered from lack of a pharmacological probe for an apical amiloride-insensitive taste pathway. We present data here showing that, depending on the concentration used, cetylpyridinium chloride (CPC) can either enhance or inhibit the amiloride-insensitive response to NaCl. The CPC concentration giving maximal enhancement was 250 microM. At 2 mM, CPC inhibited the entire amiloride-insensitive part of the NaCl response. The NaCl response is, therefore, composed entirely of amiloride- and CPC-sensitive components. The magnitude of the maximally enhanced CPC-sensitive component varied with the NaCl concentration and was half-maximal at [NaCl] = 62 +/- 11 (SE) mM. This was significantly less than the corresponding parameter for the amiloride-sensitive component (268 +/- 71 mM). CPC had similar effects on KCl and NH(4)Cl responses except that in these cases, after inhibition with 2 mM CPC, a significant CPC-insensitive response remained. CPC (2 mM) inhibited intracellular acidification of TRCs due to apically presented NH(4)Cl, suggesting that CPC acts on an apical membrane nonselective cation pathway.

Na+-H+Exchange Activity in Taste Receptor Cells

“…The lingual epithelium was isolated by collagenase treatment (Lyall et al 1997;Stewart et al 1998). A small piece of the anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber (Chu et al 1995) as described before (DeSimone et al 2001b;Lyall et al 2001Lyall et al , 2002a. 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.…”

“…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).…”

“…To block NH 4 ϩ flux via the K ϩ channels, 10 mM tetraethylammonium acetate (TEAA) was added to the solutions. In these experiments, the apical solutions also contained, in addition, 2 mM cetylpyridinium chloride (CPC) to block the NH 4 ϩ flux via the amiloride-insensitive CPC-sensitive cation channels in the apical membranes of fungiform TRCs (DeSimone et al 2001b). Different concentrations of NH 4 ϩ were obtained by mixing the R0Na0Cl and R(NH 4 ) 2 SO 4 (Table 2) solutions.…”

“…The data suggest that apical NHE-3 is quiescent and does not contribute to the overall Na ϩ flux across the apical membranes of TRCs. We have recently (Lyall et al 2002b) shown that apical Na ϩ flux occurs, in part, via the amiloride (or benzamil)-sensitive epithelial Na ϩ channels (ENaCs) and by an amiloride (or benzamil)-insensitive pathway, which is modulated by cetylpyridinium chloride (CPC) (DeSimone et al 2001b).…”

“…10). It is well established that Na ϩ enters TRCs across the apical membrane via amiloride-sensitive ENaCs (Herness and Gilbertson 1999;Lindemann 2001;Lyall et al 2002b;Stewart et al 1997) and by an amiloride-insensitive CPC-sensitive conductive pathway (DeSimone et al 2001b).…”