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A Novel Pharmacological Probe Links the Amiloride-Insensitive
NaCl, KCl, and NH4Cl Chorda Tympani Taste Responses
JOHN A. DESIMONE,1 VIJAY LYALL,1 GERARD L. HECK,1 TAM-HAO T. PHAN,1 RAMMY I. ALAM,1
GEORGE M. FELDMAN,1,2 AND R. MICHAEL BUCH3
1
Department of Physiology, Virginia Commonwealth University, Richmond 23298-0551; 2McGuire Veterans Affairs Medical
Center, Richmond, Virginia 23249; and 3GlaxoSmithKline, Parsippany, New Jersey 07054
Received 25 May 2001; accepted in final form 31 July 2001
INTRODUCTION
Amiloride inhibits part of the chorda tympani (CT) response to
NaCl, an observation that led to the identification of the epithelial
sodium channel (ENaC) as a transducer in NaCl taste (Stewart et al.
1997). CT recordings to Na salts also show an amiloride-insensitive
(AI) response that is anion dependent (Elliott and Simon 1990;
Formaker and Hill 1988). It was proposed that anions exert their
influence through modulation of transepithelial potentials set up
across paracellular shunts in the taste buds (Elliott and Simon 1990),
and this was proved by obtaining the CT responses to Na salts under
tissue voltage clamp (Ye et al. 1994).
Address for reprint requests: J. A. DeSimone, Dept. of Physiology, Virginia
Commonwealth University, Sanger Hall 3-002, PO Box 980551, Richmond,
VA 23298-0551 (E-mail: [email protected]).
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CT responses to potassium salts and ammonium salts show
only slight amiloride sensitivity and also give CT responses that
are anion dependent (Kloub et al. 1997; Ye et al. 1994). That K
salt responses in mammals might be mediated by apical membrane K channels has generally not been supported by pharmacological studies (Ye et al. 1994). For these reasons, it has been
proposed that the AI response to Na salts and responses to K and
NH4 salts might be mediated by basolateral membrane ion channels accessible to taste stimuli by diffusion through paracellular
shunts (Kloub et al. 1997; Ye et al. 1994). A role for basolateral
ENaC and basolateral K⫹ channels is supported by the observation of ENaC immunoreactivity in the basolateral domain of taste
receptor cells (TRCs) (Kretz et al. 1999) and the presence of K⫹
channels on the basolateral membranes of TRCs (Furue and
Yoshii 1997). However, this does not preclude the existence of
additional apical membrane ion pathways (Doolin and Gilbertson
1996; Miyamoto et al. 2001).
Single-unit studies show that the amiloride-sensitive (AS)
responses are associated with N fibers that respond specifically
to Na salts. AI responses are associated with H fibers that
respond to Na⫹, K⫹, NH⫹
4 , and other cations (Frank et al.
1983). N fiber responses can be ascribed to TRCs containing
ENaCs. The generalist properties of H fibers suggest that TRCs
might also contain an apical nonselective cation pathway that
serves as the transducer for generalist cation taste responses. In
pursuit of such a possibility, we have discovered that cetylpyridinium chloride (CPC) has, depending on concentration, pharmacological actions that either reversibly amplify the AI component of the CT response to NaCl or inhibit it. At inhibiting
concentrations, it reversibly blocks the entire AI NaCl response, demonstrating that the NaCl CT response is composed
exclusively of amiloride- and CPC-sensitive components. The
effects of CPC on K⫹ and NH⫹
4 responses are consistent with
those expected for a cation generalist transduction pathway.
METHODS
CT nerve recordings
Sprague-Dawley rats (150 –200 g) were prepared for recording as
previously described (Ye et al. 1994). Neural responses were ampliThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society
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DeSimone, John A., Vijay Lyall, Gerard L. Heck, Tam-Hao T.
Phan, Rammy I. Alam, George M. Feldman, and R. Michael
Buch. A novel pharmacological probe links the amiloride-insensitive
NaCl, KCl, and NH4Cl chorda tympani taste responses. J Neurophysiol 86: 2638 –2641, 2001. Chorda tympani taste nerve responses
to NaCl can be dissected pharmacologically into amiloride-sensitive
and -insensitive components. It is now established that the amiloridesensitive, 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 ␮M. At 2 mM, CPC inhibited the entire amilorideinsensitive 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 similiar effects on KCl and NH4Cl responses except that in
these cases, after inhibition with 2 mM CPC, a significant CPCinsensitive response remained. CPC (2 mM) inhibited intracellular
acidification of TRCs due to apically presented NH4Cl, suggesting
that CPC acts on an apical membrane nonselective cation pathway.
PROBE FOR SALT TASTE RESPONSES
fied, filtered, full-wave rectified, and integrated with a time constant of
1 s. The rinse solution was 10 mM KCl. Typically stimulus solutions
remained on the tongue for 2 min. Control stimuli consisting of 300
mM NaCl and 300 mM NH4Cl, applied at the beginning and at the end
of an experiment, were used to assess preparation stability. Stimuli
consisted of NaCl solutions ranging from 20 to 500 mM, 300 mM
KCl, and 100 mM NH4Cl. The cetylpyridinium chloride (CPC,
Sigma, St. Louis, MO) dose versus CT response relation was obtained
using responses to 100 mM NaCl as baseline. CPC concentrations
were (in ␮M): 50, 100, 250, 500, 1,000, and 2,000. The data were
digitized and analyzed off-line. Responses were taken as the area
under the response curve over the first minute of stimulation. The
displayed CT responses in Figs. 1 and 2 are representative results from
at least four separate experiments.
Intracellular pH measurement
in pHi were monitored following the exposure of the apical membrane
to a similar solution containing 150 mM NH4Cl at pH 7.4 in the
presence and absence of 2 mM CPC.
RESULTS
Figure 1C shows that CPC caused the NaCl response to
increase between 50 and 250 ␮M. Beyond 250 ␮M CPC, NaCl
responses decreased reaching control level at ⬃700 ␮M. At 1
and 2 mM CPC, NaCl responses were less than control values.
Figure 1A shows the effect of 250 ␮M CPC on the response to
0.3 M NaCl. When NaCl was displaced by NaCl ⫹ 250 ␮M
CPC, the response increased rapidly to a higher level until
rinsed from the tongue. Following a second NaCl stimulation,
the AS response was eliminated in the presence of 100 ␮M
amiloride. Adding 250 ␮M CPC in the presence of amiloride
gave the same magnitude enhancement observed without
amiloride, indicating that the AS and CPC-sensitive pathways
are independent. Figure 1B shows the effect of 2 mM CPC on
the response to 0.3 M NaCl. The presence of 2 mM CPC
suppressed the response by 20%. Amiloride suppressed a second NaCl stimulation by 80%. Addition of 2 mM CPC reduced
the response to baseline levels indistinguishable from the rinse
FIG. 1. A: the effect of 250 ␮M cetylpyridinium chloride (CPC) on the integrated chorda tympani response to 300 mM NaCl
and 300 mM NaCl ⫹ 100 ␮M amiloride. CPC enhanced the response by the same magnitude in each case. B: the effect of 2 mM
CPC on the integrated chorda tympani response to 300 mM NaCl and 300 mM NaCl ⫹ 100 ␮M amiloride. CPC suppressed the
entire amiloride-insensitive (AI) part of the response. C: the effect of increasing CPC concentration on the response to 100 mM
NaCl. Rcpc is the response to 100 mM NaCl containing a given concentration of CPC, R0.1 M NaCl is the response to 100 mM NaCl.
Rcpc/R0.1 M NaCl ⬎ 1 indicates an enhanced response, Rcpc/R0.1 M NaCl ⬍ 1 indicates a suppressed response. Values represent the
means ⫾ SE (n ⫽ 4)
J Neurophysiol • VOL
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To monitor the flux of NH⫹
4 ions across the apical membranes of
polarized TRCs we used a separate in vitro preparation of a single
fungiform taste bud (Lyall et al. 2001). Changes in intracellular pH
(pHi) were measured by imaging TRCs using the fluoroprobe, BCECF
(Lyall et al. 2001). A decrease in pHi indirectly indicates the apical
entry of NH⫹
4 ions into TRCs. The TRCs were perfused on both sides
with control solution containing (in mM) 150 NaCl, 5 KCl, 1 CaCl2,
1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. The temporal changes
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DESIMONE, LYALL, HECK, PHAN, ALAM, FELDMAN, AND BUCH
response level, i.e., the NaCl response is composed entirely of
AS and CPC-sensitive components.
The response to 300 mM KCl was increased by 50% in the
presence of 250 ␮M CPC and decreased by 35% by 2 mM CPC
(Fig. 2A). The response to 100 mM NH4Cl was increased by
40% in the presence of 250 ␮M CPC and decreased by 30% by
2 mM CPC (Fig. 2B). In each case, a significant part of the
response was CPC insensitive.
The influx of NH⫹
4 ions into TRCs from the apical side was
observed in polarized TRCs. When the TRCs were bathed
symmetrically in control solution, the mean pHi was 7.48 (cf.
Fig. 2C). The pH response to NH4Cl depends on the relative
permeability of NH⫹
4 and its conjugate base NH3. Replacement
of the apical control solution by the isosmotic 150 mM NH4Cl
caused pHi to decrease, indicating that the acidic form, NH⫹
4,
can enter TRCs faster from the apical side than the base NH3.
⫹
Inside the TRCs NH⫹
and forms NH3, which
4 gives up H
escapes. In contrast, when 150 mM NH4Cl ⫹ 2 mM CPC was
then placed on the apical side, pHi increased rapidly indicating
a decrease in the apical NH⫹
4 permeability relative to NH3.
The CPC-enhanced NaCl response was studied over a range
of NaCl concentrations (cf. Fig. 3). NaCl ⫹ 250 ␮M CPC was
a saturating function of NaCl concentration with Km ⫽ 185 ⫾
35 mM. The CPC-sensitive component, obtained with NaCl ⫹
100 ␮M amiloride ⫹ 250 ␮M CPC, had Km ⫽ 62 ⫾ 11 mM.
The AS component had Km ⫽ 268 ⫾ 71 mM.
J Neurophysiol • VOL
DISCUSSION
Single units in the CT that respond nonselectively to various
cations may imply TRCs that use nonselective cation channels
as transducers. Investigation of this hypothesis has been im-
FIG. 3. Normalized chorda tympani (CT) response as a function of NaCl
concentration for: NaCl ⫹ 250 ␮M CPC (●), NaCl ⫹ 100 ␮M amiloride ⫹250
␮M CPC (■), and their difference representing the AS part of the response (Œ).
In each experiment, the data were normalized to the response to 100 mM
NaCl ⫹ 100 ␮M amiloride ⫹ 250 ␮M CPC. Values represent the means ⫾
SE (n ⫽ 5). —, the least-squares fit to: CTmax [NaCl]/(Km ⫹ [NaCl]).
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FIG. 2. A: the enhancing effect of 250 ␮M CPC and suppressing effect of 2 mM CPC on the integrated chorda tympani response
to 300 mM KCl. B: similar effects of CPC on the response to 100 mM NH4Cl. C: 150 mM NH4Cl causes taste receptor cells (TRCs)
to acidify when placed on the apical side of a taste bud. In the presence of 2 mM CPC, NH⫹
4 uptake is inhibited, resulting in TRC
alkalinization.
PROBE FOR SALT TASTE RESPONSES
This work was supported by National Institute on Deafness and Other
Communication Disorders Grant DC-02422 and by a grant from GlaxoSmithKline.
J Neurophysiol • VOL
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peded, however, by the lack of an effective pharmacological
probe for such TRCs. CPC acts on the AI part of the CT
response to NaCl, where it either reversibly enhances or suppresses the response. At the suppressing concentration of 2
mM, the blocking effect of CPC is additive with that of
amiloride, indicating that NaCl responses are composed of two
pharmacologically independent inputs. CPC has essentially
similar effects on responses to KCl and NH4Cl. In the latter
case, CPC was shown to reduce the apical influx of NH⫹
4 into
TRCs. This along with the rapidity and reversibility of CPC
action suggest that the CPC-sensitive pathway is probably an
apical membrane nonselective cation conductance. For KCl
and NH4Cl, there remain significant CPC-insensitive transduction pathways. We note that both pharmacological actions of
CPC occur at concentrations above the CPC critical micelle
concentration (Simonc̆ic̆ and Špan 1998), so the CPC actions
reported here cannot be attributed to monomer-micelle transformations occurring within that concentration range.
A comparison of the parameters of the AS and CPC-sensitive parts of the NaCl response in Fig. 3 show the former to be
a high-capacity, low-affinity system and the latter to be a
low-capacity, high-affinity system similar respectively to Nand H-fiber types. Estimates of Km values for N and H fibers
are 220 and 81 mM, respectively (Frank et al. 1983), which
compare well with 268 ⫾ 71 and 62 ⫾ 11 mM found here,
respectively, for the AS and CPC-sensitive parts of the NaCl
response. The CPC-sensitive component, like the H-fiber response, has an impact on the low concentration NaCl response
and therefore has a role in determining thresholds.
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