Chem. Senses 26: 965-969,
2001
© Oxford University Press 2001
Amiloride Blocks Salt Taste Transduction of the Glossopharyngeal Nerve in Metamorphosed Salamanders
Department of Physiology, Teikyo University School of Medicine, Tokyo 173-8605, Japan 1 Department of Biology and Geoscience, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
Correspondence to be sent to: Takatoshi Nagai, Department of Biology, Keio University School of Medicine, Hiyoshi, Yokohama 223-8521, Japan. e-mail: takatosh{at}hc.cc.keio.ac.jp
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Abstract |
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Studies in the last two decades have shown that amiloride-sensitive Na+ channels play a role in NaCl transduction in rat taste receptors. However, this role is not readily generalized for salt taste transduction in vertebrates, because functional expression of these channels varies across species and also in development in a species. Glossopharyngeal nerve responses to sodium and potassium salts were recorded in larval and metamorphosed salamanders and compared before and after the oral floor was exposed to amiloride, a blocker of Na+ channels known to be responsible for epithelial ion transport. Pre-exposure to amiloride (100 µM) did not affect salt taste responses in both axolotls (Ambystoma mexicanum) and larval Ezo salamanders (Hynobius retardatus). In contrast, in metamorphosed Ezo salamanders the nerve responses to NaCl were significantly reduced by amiloride. In amphibians amiloride-sensitive components in salt taste transduction seem to develop during metamorphosis.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Salt taste transduction is mediated primarily by entry of Na+ ions into taste receptor cells, because amiloride, a blocker of epithelial Na+ channels (ENaC) (Kleyman and Cragoe, 1988
), reduces the intensity of NaCl taste perceived
by humans (Schiffman et al.,
1983; Smith and Ossebaard,
1995) and the response of the chorda tympani nerve to sodium salts
in rats (Heck et al.,
1984). Whole cell clamp studies show that amiloride-sensitive
(blockable) sodium channels (ASSCs) are present in taste receptor cells
(Avenet and Lindemann, 1988;
Doolin and Gilbertson, 1996).
Furthermore, antibodies directed against ENaC in rats successfully localized
the channels in the taste cell membrane
(Stewart et al.,
1995; Lin et al.,
1999). When the lingual epithelium is exposed to amiloride
solution a reduction in taste nerve responses to sodium salts occurs in
species other than rats, such as hamster
(Hettinger and Frank, 1990)
and monkeys (Hellekant et al.,
1988). However, transduction mediated by ASSCs is not readily
generalized for salt taste transduction in vertebrates [for a review see
Halpern (Halpern, 1998)],
because the effect of amiloride is not specific to sodium salts in some
species, such as dog (Nakamura and
Kurihara, 1990), and amiloride does not reduce taste nerve
responses to sodium salts in some strains of laboratory mice
(Gannon and Contreras, 1995;
Ninomiya et al.,
1996). Furthermore, involvement of ASSCs in salt taste
transduction is less clear in non-mammalian vertebrates.
In amphibia salt solutions induce responses in the glossopharyngeal nerve,
which innervates a majority of taste receptor cells in the lingual epithelium
(Akaike et al., 1976;
Samanen and Bernard, 1981;
Takeuchi et al.,
1994). However, salt responses in the nerve are not affected by
amiloride in mudpuppies (Necturus maculosus)
(McPheeters and Roper, 1985)
and frogs (Rana catesbeiana)
(Okada et al., 1991;
Kitada and Mitoh, 1998). In
contrast to these studies, whole cell clamp studies of isolated amphibian
taste cells show that ASSCs are present in tiger salamanders (Ambystoma
tigrinum) (Sugimoto and Teeter,
1991) and frogs (Rana esculentalridibunda)
(Avenet and Lindemann, 1988).
Whether such opposing results derive from a difference in species or
electrophysiological methods of measurement is not clear.
In the present study we determined the effects of amiloride on
glossopharyngeal nerve responses in two species of amphibians, axolotls
(Ambystoma mexicanum) and Ezo salamanders (Hynobius
retardatus). In Ezo salamanders we examined not only larval animals but
also metamorphosed adults, because susceptibility of taste nerve responses to
amiloride may develop with growth, as has been reported in the mammalian taste
system (Hill and Bour, 1985;
Hill and Mistretta, 1990). A
preliminary account of this study has appeared in abstract from
(Nii et al.,
1998).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Animals
Two species of salamanders, axolotls (A. mexicanum) and Ezo
salamanders (H. retardatus), were used. Axolotl larvae of the wild
and white strains were obtained from the Indiana University Axolotl Colony and
raised until 
1 year of age (adult) in our laboratory (Shizuoka
University). Neotenic axolotls do not metamorphose under natural conditions
whereas Ezo salamanders do. Pre-metamorphic and post-metamorphic salamanders
were selected from animals raised in our laboratory. Larval Ezo salamanders
were younger than phase 0 and metamorphosed ones were older than phase 5. The
phases are defined by our previous study on the metamorphosis of the taste
buds in Ezo salamanders (Takeuchi et
al., 1997).
Electrophysiological recordings
Neural activity of the glossopharyngeal nerve in response to chemical
stimuli was recorded in axolotls and larval and metamorphosed Ezo salamanders.
Methods of neural recordings and stimulation are described in detail in our
physiological study on the glossopharyngeal nerve in axolotls
(Takeuchi et al.,
1994). The same methods were applied to obtain recordings from Ezo
salamanders. Briefly, in anesthetized animals the peripheral end of the
glossopharyngeal nerve was exposed in the caudal end of the external
mandibular levator and hooked onto bipolar platinum wire electrodes. The
overall activity of the nerve was differentially amplified and stored on a
digital magnetic tape recorder (PC204AX; Sony, Tokyo, Japan), together with an
electrical signal and voice cues marking the onset of stimulation. Taste
stimuli (for 10-15 s) and distilled water rinses (for 100 s) were alternately
presented to the rostral part of the oral floor through a peristaltic pump
with a flow rate of 6 ml/min. In the mudpuppy
(Samanen and Bernard, 1981;
McPheeters and Roper, 1985)
and the axolotl (Takeuchi et al.,
1994) gustatory response in the glossopharyngeal nerve is known to
vary over time during chemical stimulation. Therefore, the number of
stimulations in a stimulus series was set to a minimum: stimulation by the 0.5
M KCl standard was followed by between two and four concentrations of NaCl and
then the 0.5 M KCl standard again. When the response to the second 0.5 M KCl
was reduced by > 30%, data were discarded from the subsequent quantitative
analysis. The effect of amiloride on the response to NaCl and KCl solutions
was evaluated after the oral floor had been exposed for 10 min to 100 µM or
1 mM amiloride (Sigma, St Louis, MO) dissolved in distilled water. After
exposure to amiloride, the post-stimulation rinse was with amiloride solution.
The chemical stimuli were distilled water solutions of reagent grade NaCl and
KCl. Stored neural activities were played back and subjected to subsequent
data analyses. The activities obtained from axolotls and Ezo salamanders were
fed to an integrator (time constant 0.3 s). Some of those from metamorphosed
Ezo salamanders were fed to a spike counter (ET-612J; Nihon Koden, Tokyo,
Japan), because in some animals the whole nerve recordings consisted of
relatively few units (see Figure
4) and thus adequate neural integration could not be carried out
for these recordings. What caused such a difference in neural recordings is
not known, but morphological changes in the oral floor that accompany the
change in innervation during metamorphosis are possibly involved
(Takeuchi et al.,
1997). Action potentials from the nerve and integrated nerve
responses were recorded on a thermal array recorder (RTA-1200M; Nihon Koden).
The neural responses were quantified by measuring the area under the output
from the integrator with a computer-assisted digitizer or from the digital
output of the spike counter. The spontaneous ongoing activity just before
stimulation was subtracted from the activity induced for 10 s after the onset
of stimulation. All responses were normalized by comparing each response with
the response obtained to the standard solution (0.5 M KCl) recorded prior to
the test solutions and are shown as relative magnitudes of responses.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Effect of amiloride on salt taste responses in axolotls
In axolotls amiloride did not reduce the glossopharyngeal nerve responses
to NaCl solutions (Figure 1).
The response to 0.5 M KCl was also not affected by amiloride and thus was
adopted as the standard to normalize the responses to NaCl. The
intensity—response curves of NaCl solutions did not show any significant
reduction after the oral floor was exposed to 100 µM amiloride (0.1 M NaCl,
P = 0.3459; 0.3 M NaCl, P = 0.1880; 1.0 M NaCl, P =
0.2612; 0.5 M KCl, P = 0.3750; not significant at P >
0.05 by two-tailed t-test; Figure
2). Amiloride at 100 µM is thought to be sufficient to suppress
NaCl taste responses of the chorda tympani nerve in rats
(Brand et al., 1985),
but the effective concentration may differ between species. When a much higher
concentration of amiloride (1 mM) was used a significant reduction in response
was induced at 0.5 M NaCl (reduced by 40%, P = 0.0193, significant at
P < 0.05 by two-tailed t-test), but not at lower and
higher concentrations (0.1 M NaCl, reduced by 23%, P = 0.0621; 1.0 M
NaCl, reduced by 15%, P = 0.5297; not significant at P >
0.05 by two-tailed t-test). A lack of reduction in the responses at
lower concentrations of NaCl may show that amiloride at such a high
concentration did not actually block ASSCs, but exerted some other effects on
the oral floor. In axolotls the ASSCs involved in NaCl taste transduction are
few and of very low affinity, if any, for amiloride.
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) in axolotls. The
responses were normalized to the standard (0.5 M KCl) response obtained in the
control. Data points are means ± SEM of the normalized responses for
three concentrations of NaCl. The number of measurements is shown in
parentheses.
Salt taste responses in larval and metamorphosed Ezo salamanders
A similar lack of sensitivity to amiloride was seen in larval Ezo salamanders (neural data not shown). Amiloride did not significantly reduce the neural responses to 0.5 and 1.0 M NaCl and 0.5 M KCl (0.5 M NaCl, P = 0.1225; 1.0 M NaCl, P = 0.1129; 0.5 M KCl, P = 0.6339; not significant at P > 0.05 by two-tailed t-test; Figure 3).
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, 
) Ezo salamanders. For each animal the curves for
NaCl in the control (filled symbols) and after exposure of the oral floor to
100 µM amiloride (open symbols) are shown. The responses were normalized to
the standard (0.5 M KCl) response obtained in the control. Data points are
means ± SEM of the normalized responses for two (larvae) and four
(metamorphosed salamanders) concentrations of NaCl. Asterisk shows
statistically significant difference in the mean values. The number of
measurements is shown in parentheses.In contrast to axolotls and larval Ezo salamanders, amiloride reduced the nerve responses to NaCl in metamorphosed Ezo salamanders, leaving those to KCl unaffected (Figure 4). Intensity—response curves for NaCl were shifted to the right, showing a reduction in NaCl responses at all concentrations tested (Figure 3). The reductions in the responses to NaCl were statistically very significant (0.3 M NaCl, P = 0.0071; 0.5 M NaCl, P = 0.0018; 1.0 M NaCl, P = 0.0058; significant at P < 0.01 by two-tailed t-test). The responses at 0.1 M NaCl were not analyzed statistically due to the small sample size (n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We previously studied the morphology of taste organs on the tongue in two species of salamanders, axolotl and Ezo salamander, and found that salamanders undergo morphological changes of the taste organs during metamorphosis (Takeuchi et al., 1997
). The axolotls used in the present study were larvae and had
a taste bud morphology similar to that of larval Ezo salamanders (i.e.
barrel-shaped taste buds). In both axolotls and larval Ezo salamanders neural
responses to NaCl recorded from the glossopharyngeal nerve were not reduced by
pre-exposure of the lingual epithelium to 100 µM amiloride (Figures
1,2,3),
a specific blocker of ENaC. In contrast, in metamorphosed Ezo salamanders
these responses were reduced by pre-exposure to amiloride (Figures
3 and
4). Therefore, larval
salamanders lack amiloride-sensitive components in glossopharyngeal nerve
responses to NaCl, but it is likely that the nerve acquires such components
during metamorphosis.
In mammals neural responses to NaCl increase during development of the
gustatory system (Hill and Mistretta,
1990). The increase in NaCl response is attributed to ASSCs, added
to taste cell membranes during development
(Hill and Bour, 1985). The
present study on salamanders suggests that addition of ASSCs during
development also occurs in non-mammalian species. In mudpuppies recordings
from the glossopharyngeal nerve failed to show that these channels are
involved in transduction of NaCl
(McPheeters and Roper, 1985).
However, this does not necessarily mean innate absence of the channels in the
mudpuppy taste cell. We speculate that mudpuppies would develop functional
ASSCs in the taste cell if they metamorphosed, although neotenic mudpuppies do
not metamorphose in their natural environment. In support of this speculation
our preliminary experiment using axolotls artificially induced to metamorphose
by thyroxin administration showed a reduction in NaCl responses by amiloride
in the glossopharyngeal nerve (a single experiment; unpublished observation).
Although axolotls artificially induced to metamorphose are a good model for
studying the development of ASSCs in amphibians, axolotls administered
thyroxin do not survive well so that a few metamorphosed animals were amenable
only to morphological examination
(Takeuchi et al.
1997). As an alternative we have chosen naturally metamorphosed
Ezo salamanders to study the issue by a physiological approach. Ezo
salamanders acquire amiloride-sensitive components in the glossopharyngeal
nerve responses to NaCl during development. However, such acquisition may be
attributable to adaptation from an aquatic to a terrestrial environment rather
than to developmental change per se.
Absence of amiloride-sensitive components in the glosso-pharyngeal nerve
responses to NaCl is also shown by frogs
(Okada et al., 1991;
Kitada and Mitoh, 1998).
However, whole cell clamp recordings of isolated taste cells in frogs
(Avenet and Lindemann, 1988)
show the presence of ASSCs in the taste cell membrane. The same channels are
also present in larval tiger salamanders
(Sugimoto and Teeter, 1991).
However, these single cell studies do not necessarily argue against the
glossopharyngeal nerve recordings, because the studies did not clarify whether
ASSCs are distributed in the apical membrane of taste cells or in the basal
membrane. If the channels are distributed in the basal membrane of taste cells
amiloride, which is impermeable through tight junctions
(Briggman et al.,
1983; Ye et al.,
1993), would be unable to prevent the flow of Na+ ions
into taste cells through the basal membrane, while Na+ ions would
be able to pass into the intercellular space facing the basal cell membrane by
means of the paracellular pathway (Ye
et al., 1991). Therefore, absence of suppression by
amiloride in the glossopharyngeal nerve responses simply suggests absence of
ASSCs in the apical taste cell membrane, leaving the possibility that these
channels are present in the basal membrane. In fact, a recent
immunohistochemical study shows that ASSCs are present in both the apical and
basal portions of taste cells in rats (Lin
et al., 1999). Furthermore, ASSCs are expressed in a very
early stage of development (post-natal day 2) in rat taste buds
(Kossel et al.,
1997). Therefore, these channels may be present in the basal
portion of the taste cell in larval salamanders to mediate the
amiloride-insensitive part of salt taste transduction. On the other hand,
metamorphosed Ezo salamanders may have developed ASSCs in the apical portion
of the taste cell, as expression of ASSCs in the apical region of rat taste
buds can be induced by aldosterone (Lin
et al., 1999) and aldosterone participates in regulating
ASSCs in frog skin, where the channels play a role in active Na+
transport. Takada et al. showed that ASSCs develop when the skin of
tadpoles is cultured with aldosterone
(Takada et al.,
1995). Furthermore, development of these channels is suppressed by
an as yet unknown mechanism(s) in tadpole skin in vivo, but
suppression seems to be removed by an increase in endogenous thyroid hormone
during development (Takada et
al., 1999). Similar hormonal regulation may operate in the
development of ASSCs in the lingual epithelium of salamanders, including taste
buds. Metamorphosed salamanders develop taste buds with a morphology not seen
in larval salamanders (Takeuchi et
al., 1997). These `adult type' taste buds, particularly with
respect to the apical region, may be the site at which the ASSCs are
expressed. Immunohistological demonstration of these channels in amphibian
taste buds will clarify the point.
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Acknowledgments |
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We thank Indiana University Axolotl Colony for the continuous supply of axolotls. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (T.N.) and by a grant from the Saneyoshi Scholarship Foundation (H.-A.T.).
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Accepted May 1, 2001
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