Electrophysiological properties of the tongue epithelium of the toad Bufo marinus
Department of Biological Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA
* Author for correspondence (e-mail: hillyard{at}ccmail.nevada.edu )
Accepted 17 April 2002
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Summary |
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Key words: toad, Bufo marinus, tongue, lingual epithelium, K+ channel, taste
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Introduction |
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Among other vertebrate classes, the taste response of the amphibian tongue
was studied by Pumphry (1935),
who demonstrated that the application of salty or acidic solutions to the
lingual surface stimulated the glossopharyngeal nerve of frogs. Sato
(1976
) has summarized the
historical development of studies on the chemosensory responses of the
amphibian tongue, and the morphology of the tongue has been described by
Jaeger and Hillman (1976
).
Experiments to date with the isolated lingual epithelium of amphibians have
not demonstrated a transepithelial voltage difference when the tissue was
bathed on both sides with identical Ringer's solutions
(Soeda and Sakudo, 1985
).
Detailed studies of acutely dissociated taste cells from frog tongue have been
conducted by Avenet and Lindemann
(1988
), who utilized whole-cell
patch-clamp methodology to demonstrate a cationic current across these cells.
This current was inhibited by amiloride, suggesting that, like taste cells
from mammalian tongue (DeSimone et al.,
1981
,
1984
), epithelial
Na+ channels play a role in salt taste transduction. In the present
study, we tested the hypothesis that the transepithelial current across the
lingual epithelium of the toad Bufo marinus is similar to that of
mammals. We observed that, in contrast to the mammalian tongue, the
short-circuit current across the lingual epithelium of Bufo marinus
is outwardly directed and is carried by active transport of K+ from
the serosal to the mucosal surface of the tissue. Fluctuation analysis (for a
review, see Van Driessche,
1994
) of the short-circuit current demonstrates the presence of
spontaneously fluctuating channels in the apical membrane of the epithelium
that may be involved in K+ secretion.
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Materials and methods |
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Current measurements
The Isc is classically defined as the direct current
(µA cm-2) that maintains the voltage across the tissue at 0 mV
with identical Ringer bathing both sides of the tissue
(Ussing and Zerahn, 1951).
With asymmetric bathing solutions, the Isc may also
reflect passive diffusion of ions. The alternating component of the current
was filtered and amplified for fluctuation analysis. This technique is based
on the assumption that the macroscopic Isc is the sum of
currents through a population of individual ion channels that fluctuate
between an open and a closed state, thereby inducing current fluctuations of a
much smaller magnitude. Analysis of these fluctuations can be used to
characterize an ionic current as passing through channels and can provide
information about the kinetic properties of the channels. The methodology for
this procedure has recently been reviewed
(Van Driessche, 1994
) and is
briefly summarized as follows. The alternating current signal was filtered
with a fundamental frequency of 0.5 Hz and a low-pass filter of 1 kHz. The
signal was also passed through an anti-aliasing filter, amplified and
converted to a digital signal that was subject to a fast Fourier transform.
The resulting power spectrum is displayed as a double-logarithmic plot of
spectral density, Sf (A2 s cm-2)
versus frequency, f (Hz). The spectrum was fitted as the sum
of a linear and a Lorentzian function:
![]() | (1) |
The Lorentzian function includes So, which is the power
density of the Lorentzian plateau at low frequencies, and the corner frequency
(fc), which is the frequency at which the power density
has relaxed to half that of So. The presence of a
Lorentzian function in a power spectrum is the result of changes in the
conductance state of individual ion channels and is taken as evidence for a
spontaneously fluctuating population of ion channels contributing to the
Isc (Conti et al.,
1975; Lindemann and Van
Driessche, 1977
; Van
Driessche, 1994
). Equations 2-5 (see Discussion) were developed in
detail by Van Driessche
(1994
). They are presented in
this paper to facilitate the interpretation of the fluctuation analysis
measurements in terms of models that describe the kinetic properties of ion
channels.
Experimental protocols
Isc measurements were begun with NaCl Ringer's solution
bathing both mucosal and serosal surfaces of the tissue and with the command
voltage set to 0 mV. Under these conditions, there are no voltage or
concentration gradients across the tissue, and any currents that are passed by
the voltage clamp represent the active transport of ions
(Ussing and Zerahn, 1951).
Four sets of experiments were performed: cation exchange, anion exchange,
mucosal Ba2+ treatment and mucosal quinine treatment.
The cation exchange experiments involved replacing NaCl Ringer with KCl Ringer (112.5 mmol l-1 KCl, 2.5 mmol l-1 KHCO3, 1 mmol l-1 CaCl2) on the mucosal surface, re-equilibration with NaCl in the mucosal and serosal solutions and then replacement with KCl in the serosal solution. Under these conditions, passive concentration gradients for K+ are established across the tissue, and the changes in current and fluctuation analysis parameters can be compared with those obtained in the absence of a K+ gradient.
For the anion exchange experiments, the tissue was initially bathed in NaCl
Ringer. The first step was to change both mucosal and serosal solutions to
Na2SO4 Ringer (57.5 mmol l-1
Na2SO4, 2.5 mmol l-1 KHCO3, 1 mmol
l-1 CaCl2). When a stable Isc had
been obtained, a power spectrum was recorded. The mucosal and then the serosal
solutions were exchanged with K2SO4 Ringer (57.5 mmol
l-1 K2SO4, 2.5 mmol l-1
KHCO3, 1 mmol l-1 CaCl2) to duplicate the
sequence of cation substitutions performed above with KCl. Chloride ions are
known to be transported across many epithelia
(Larsen, 1991). If the
Isc across the toad tongue is the result of Cl-
transport, substitution of Cl- should eliminate that current.
Ba2+ and quinine are known blockers of K+ channels, so the inhibition of Isc by these substances would support the hypothesis that the Isc is the result of K+ transport. The effects of Ba2+ on the Isc were first recorded with NaCl Ringer bathing the mucosal and serosal surfaces of the tissue. The mucosal Ringer's solution contained, sequentially, 0.5, 1, 2, 5 and 10 mmol l-1 BaCl2. After maximal inhibition of Isc had been observed, the mucosal solution was replaced with Ringer containing no Ba2+. In a second set of experiments, the inhibition of Isc by mucosal Ba2+ was recorded from tissues bathed at the serosal surface with KCl Ringer and at the mucosal surface with NaCl Ringer.
Quinine experiments were performed with mucosal applications of 10 µmol l-1 followed by 100 µmol l-1 quinine with NaCl Ringer as the mucosal and serosal solutions. When the quinine solution had been washed out, 10 mmol l-1 BaCl2 was added to the mucosal side and then washed out. Isc was continuously recorded on a chart recorder, and noise analysis was conducted upon equilibration of Isc after each solution change described above.
All data are reported as means ±1 S.E.M.
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Results |
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Replacement of mucosal NaCl Ringer's solution with KCl Ringer resulted in significant changes in Isc, So and fc: Isc became positive, So declined and fc shifted to a higher frequency. Statistical comparisons, unless otherwise specified, were with Fisher's Protected Least Significant Difference (PLSD) test using StatView Version 4.5 for Windows. A representative power spectrum obtained with KCl Ringer as the mucosal solution is shown in Fig. 2B. The spectrum in Fig. 2B is from the same preparation as that of Fig. 2A and illustrates the decline in So and the increase in fc presented in Table 1. All these effects of mucosal KCl were reversed by changing the solution back to NaCl Ringer.
When the serosal solution was changed from NaCl to KCl, Isc became significantly more negative (Fig. 1; Table 1). This change in current was reversed by switching the serosal solution back to NaCl. With Cl- as the anion, power spectra obtained with a serosa-to-mucosa K+ gradient displayed two Lorentzian components in six of seven experiments (Table 1). One Lorentzian component had an fc value that was somewhat smaller than that obtained with NaCl Ringer on both sides of the tissue, and So was increased but was highly variable among preparations and not significantly greater than with NaCl solutions on both sides of the tissue (Table 1). The second Lorentzian component had a higher fc and lower So than the first. A representative power spectrum fitted with two Lorentzian components is shown in Fig. 2C.
Anion substitution
Substitution of NaCl Ringer's solutions with Na2SO4
Ringer in both the mucosal and serosal baths produced no significant change in
any of the three variables (Table
2). Substitution of Na2SO4 Ringer on both
sides of the tissue with mucosal K2SO4 Ringer's solution
produced results similar to those seen with mucosal KCl:
Isc increased to positive values, So
declined and fc increased, all significantly
(Table 2). As with the addition
of mucosal KCl, these results were reversed by changing the mucosal solution
back to Na2SO4. When the serosal solution was changed
from Na2SO4 to K2SO4, with
Na2SO4 Ringer bathing the mucosal surface,
Isc became significantly more negative. Thus, the general
pattern of Isc changes depicted in
Fig. 1 also apply to
experiments with SO42- as the anion. Three of the seven
power spectra obtained under these condition could be fitted with either one
or with two Lorentzian components like that shown in
Fig. 2C. The values for the
single Lorentzian fits are presented in
Table 2, with the understanding
that the higher-frequency Lorentzian component, when present, is less
prominent than that observed with Cl- as the anion.
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Ba2+ inhibition of
Isc
With NaCl Ringer in the mucosal and serosal bath solutions, the addition of
increasing concentrations of BaCl2 to the mucosal surface resulted
in a dose-dependent inhibition of the negative Isc from an
initial value of -13.00±2.22 µA cm-2 (N=6)
(Fig. 3). Typically, the
inhibition of Isc was rapid, and a stable value for each
concentration was achieved within 2-3 min. Upon removal of the
BaCl2 solution from the mucosal surface, the
Isc recovered to a level not significantly different from
its pre-treatment value, -11.87±0.82 µA cm-2
(N=6). Statistical analysis using a non-parametric paired sign test
shows the inhibition of Isc to be significant at each
concentration change (P=0.0313). The pattern of Ba2+
inhibition was further analyzed with a direct linear plot
(Eisenthal and Cornish-Bowden,
1979). This is a modification of the MichaelisMenten
equation for a pseudo-first-order inhibitor. Graphically, a family of lines is
drawn between the points representing the inhibitor concentrations, as
negative values, on the abcissa and the decrement in Isc
for each blocker concentration on the ordinate
(Fig. 4A). If the inhibitor is
binding to a single inhibitory site, the lines, extended to positive
x axis values, should intersect at a common point that describes the
inhibitory constant (Ki) as the x coordinate and
the amount of inhibitor-sensitive current as the y coordinate. It can
be seen in Fig. 4A that the
lines for inhibition by 0.5, 1 and 2 mmol l-1 Ba2+
intersect near a common point but that the lines for 5 and 10 mmol
l-1 Ba2+ intersect at points that indicate a higher
binding constant and thus a lower binding affinity.
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The inhibition of Isc by Ba2+ was also examined with KCl as the serosal solution. In these experiments (N=4), the Isc was -30.37±5.14 µA cm-2, and a similar pattern of inhibition was seen when Ba2+ was added to the mucosal solution. Specifically, the lines of the direct linear plot intersect at a common point at lower Ba2+ concentrations of 0.5-5 mmol l-1, but the point of intersection at 10 mmol l-1 Ba2+ indicates a lower-affinity blockage (Fig. 4B). If the amount of Ba2+-sensitive Isc is compared between the mean values for 10 mmol l-1 Ba2+ data points in Fig. 4A and B, it can be seen that -8.46 of -13.00 µA cm-2 (65 %) is inhibited with no K+ gradient across the tissue while 11.72 of 30.37 µA cm-2 (38.6 %) is inhibited in the presence of a serosa-to-mucosa K+ gradient, i.e. the increased negative Isc is largely blocker-insensitive.
The fluctuation analysis results obtained for Ba2+ inhibition of Isc with NaCl Ringer on both sides of the tissue showed no significant changes in either fc or So as Isc was inhibited (Fig. 3).
Quinine inhibition of Isc
The addition of 10 µmol l-1 quinine, a bitter tastant as well
as a K+ channel blocker, to the mucosal surface consistently
produced a decrease in the mean Isc value from -14.53 to
-11.62 µA cm-2 (Fig.
5). Analysis of these data with a non-parametric paired sign test
showed this to be significant (P=0.0313). Further inhibition of the
current is seen with 100 µmol l-1 quinine (P=0.0022).
Wash-out of the quinine does not produce a recovery of the initial current.
Addition of 10 mmol l-1 Ba2+ to the mucosa, after the
quinine wash-out, causes a further inhibition of the Isc
(P<0.0001), which is reversible upon its removal.
|
The fluctuation analysis experiments with quinine and Ba2+ showed that So values declined as Isc was inhibited and increased when Ba2+ was washed out. The changes in fc were very small, however, and did not correlate with the changes in Isc or So (Fig. 5).
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Discussion |
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As noted above, Soeda and Sakudo
(1985) conducted Ussing
chamber experiments with the isolated lingual epithelium of bullfrogs but
detected no spontaneous potential difference across the tissue bathed with
identical Ringer on both sides. They measured a resistance of 720±180
cm2, which is very similar to the value of 615±152
cm2 for B. marinus tongue (present study) and
576±127
cm2 for dog tongue
(DeSimone et al., 1981
). It is
generally assumed that the lingual epithelium of mammals is a low-resistance
tissue with both transcellular and paracellular pathways for ion transport and
sensory transduction (Ye et al.,
1991
; Gilbertson and Zhang,
1998
). Given the similar low resistance values for frog and toad
tongue, it appears that these epithelia also have paracellular as well as
transcellular transport pathways. Soeda and Sakudo
(1985
) also observed that the
addition of 200 mmol l-1 NaCl, 5 mmol l-1 acetic acid or
5 mmol l-1 quinine to the mucosal bath increased the potential
across the bullfrog tongue, and they suggested that the change in
transepithelial potential might be associated with a chemosensory function. We
have conducted preliminary experiments with the lingual epithelium of the frog
Rana pipiens and have noted a similar pattern of K+
secretion as was noted above for the toad tongue, i.e. an outwardly directed
Isc, Ba2+-sensitivity and a Lorentzian
component in power spectra. These observations suggest a mucosa-positive
Isc that is mediated by K+ secretion to be the
pattern for the lingual epithelium of at least the bufonid and ranid families
of the Anura. It is not clear why Soeda and Sakudo
(1985
) did not observe this
pattern of transport.
We observed no effect of 10 µmol l-1 amiloride on
Isc across the toad tongue bathed with NaCl Ringer on both
sides of the tissue. The sensory processes of taste cells make up a small
percentage of the membrane area of the taste discs and an even smaller
percentage of the membrane area of the lingual epithelium
(Jaeger and Hillman, 1976). It
is possible that a small amiloride-sensitive component to the measured
Isc that might be carried by inward Na+
transport across taste cells would not be resolved at the microampere level.
Avenet and Lindemann (1988
)
estimated the Na+ current per taste cell to vary between 10 and 700
pA when clamped to -80 mV. The minimum number of cells required to produce 1
µA of current is thus greater than 1000, assuming that toad and frog taste
cells have a similar Na+ transport capability and that the in
situ membrane potentials are of the order used in the patch-clamp study.
Without this information and a detailed histological examination of the tissue
used in specific experiments, it is not possible to speculate further. It is
noteworthy that Kitada and Mitoh
(1997
) observed no effect of
amiloride on the activity of the glossopharyngeal nerve of the frog during
exposure of the tongue to NaCl concentrations between 0.1 and 0.5 mol
l-1. DeSimone et al.
(1981
) imposed inwardly
directed Na+ gradients across the canine lingual epithelium to
obtain larger amiloride-sensitive Isc values. This
procedure might produce a sufficiently large Na+ current in toad
tongue to be detected at the microampere level. We have conducted a few
measurements of the in situ potential across the lingual epithelium
of pithed toads, and the values we obtained are similar to that observed
during the initial open-circuit voltage in the Ussing chamber experiments,
approximately 10-15 mV. Thus, the electrical properties of the tongue in
situ appear to be similar to those of the isolated tissue.
Avenet et al. (1988) have
also used patch-clamp methodology to demonstrate the presence of three types
of K+ channel in isolated frog taste cells. The direct linear plots
and the presence of two Lorentzian functions in power spectra from the present
study are consistent with the presence of multiple K+ channels in
the apical membrane of the lingual epithelium which could include both taste
and non-taste cells working in parallel.
Spontaneous current fluctuations
The presence of a single Lorentzian component in power spectra obtained
with NaCl Ringer bathing both sides of the lingual epithelium supports the
hypothesis that spontaneously active K+ channels are associated
with the outward K+ current. The reduction in
So that resulted from the addition of KCl Ringer to the
mucosal bath is predicted from a reduction in the chemical gradient for
K+ secretion across the apical membrane and a reduction in the
single-channel current, as calculated from the equation:
![]() | (2) |
![]() | (3) |
The addition of K+ Ringer to the serosal bath augmented
Isc and resulted in the appearance of a second Lorentzian
function in the power spectra in 10 of 14 experiments. This observation
suggests the presence either of two types of K+ channel or of
multiple conductance states with different kinetics and affinity for
Ba2+ inhibition. This procedure should depolarize the basolateral
membrane, which would allow a voltage clamp of the apical membrane
(Lindemann and Van Driessche,
1977). The Ba2+-sensitive Isc with
a serosa-to-mucosa K+ gradient amounted to 38.6 % of the total
compared with 65 % for K+ secretion in the absence of a
concentration gradient. Thus, the bulk of the enhanced current is
Ba2+-insensitive and may be paracellular. For this reason, there
may not be substantially larger values for I or i via a
transcellular pathway and, from equation 2, So would not
be larger in these preparations relative to the control condition with NaCl
Ringer on both sides of the tissue. In experiments with double Lorentzian
components, the variability in fc was large, and we have
not analyzed them further.
Effects of blockers on current fluctuations
The inhibition of Isc by Ba2+ occurred in
the absence of change in fc. A linear increase in
fc would have been expected if the inhibition of the
apical K+ channels obeyed pseudo-first-order kinetics and the
kinetics of a single channel type was the source of the current fluctuations.
In a two-state model of channel inhibition, channels fluctuate between an open
and a blocked state. Increasing the blocker concentration will increase
fc in a linear fashion, as described by the equation:
![]() | (4) |
![]() | (5) |
Quinine treatment produced a decrease in Isc that did
not wash out. Van Driessche and Hillyard
(1985) showed that quinidine,
a stereoisomer of quinine, inhibited K+ channels in the basolateral
membrane of larval frog skin but only when applied to the mucosal side of the
tissue. The inhibition was not readily reversible, and it was suggested that
inhibition occurred after the blocker had diffused into the cell and become
ionized at the lower pH of the cytosol and, thus, could not be entirely washed
out, as had been suggested by Yeh and Narahashi
(1976
) for experiments with
K+ channels in squid axon membranes. From these experiments, it
would appear that inhibition occurred as the result of blockage at the
cytosolic face of the membrane, in contrast with Ba2+ which binds
at the extracytoplasmic face of the channels and is readily washed out. We
cannot rule out the possibility that quinine might be blocking channels in the
basolateral as well as the apical membranes of the lingual epithelium.
The reduction in So during quinine treatment roughly paralleled the inhibition of Isc and persisted after quinine had been washed out, as would be expected from equation 5. The addition of Ba2+ further reduced Isc but not So, which is consistent with the results in Fig. 3 that also show Ba2+ to inhibit Isc without lowering So. So did increase in concert with an increase in Isc when Ba2+ was washed out. The changes in fc were small and not consistent with the two-state model described by equation 4. This also suggests that the Lorenzian function that is fitted in the power spectra represents a spontaneously fluctuating K+ channel that is blocker-insensitive. As noted above, the analysis of blocker-induced current fluctuations that are not compatible with a two-state model is beyond the scope of the present study.
Given the complex cellular structure of the toad tongue, it is premature to
describe a cellular model for K+ secretion. The simplest model
would be one like that of the distal nephron or colon in which K+
is transported into the epithelial cells by the Na+/K+
pump in the basolateral membrane and expelled from the cells through apical
K+ channels (Berne and Levy,
1988). The role of the Na+/K+ pump in
K+ secretion has been difficult to study because this enzyme in
bufonid anurans is resistant to ouabain inhibition
(Jaisser et al., 1992
),
presumably to protect the enzyme from alkaloid substances in the skin
secretions and body fluids that are used for protection from predators
(Butler et al., 1996
).
Functional significance of the K+ current
Kinnamon (1992) has
suggested that K+ channels serve a chemosensory function in the
taste cells of both mammalian and amphibian tongue epithelia. Bitter tastants
block K+ secretion and depolarize the taste cells. As noted above,
the proportion of the Isc carried by K+
transport across taste cells versus other epithelial cells cannot be
resolved from our measurements. Another source for K+ secretion
across the lingual epithelium is salivary and mucus secretion
(Jaeger and Hillman, 1976
).
The secretion of both mucus and saliva in mammalian digestive systems is
accompanied by an elevated K+ concentration in the secretion
(Berne and Levy, 1988
). A
coating of mucus on the tongue is required for the capture of insects, which
raises the question of the function of taste modalities in anurans that feed
in this manner. Specifically, insects are captured on a thick mucous layer at
the dorsal surface of the tongue, held briefly in the mouth and then swallowed
whole. Taste buds are thought to provide sensory input about the quality of
food being ingested and, as pointed out by Lindemann
(1996
), chemosensory cells
respond to relatively high concentrations of chemical tastants.
Many anuran species, including ranids and bufonids, swallow prey, primarily
insects, whole, and most nutritive components are contained within an
exoskeleton that is only partially degraded after ingestion
(Larsen, 1992). Thus, the
taste buds on the tongue would be unable to assess nutritional value. It is
possible that tastants on the cuticular surface might provide some measure of
prey selection. Mikulka et al.
(1981
) were able to show that
coating prey insects with lithium chloride elicited an aversion to feeding.
However, Larsen (1992
)
observed anecdotally that toads will swallow glass beads placed in the mouth.
The thick layer of mucus required to capture the prey and the short time that
prey are retained in the mouth would allow for minimal diffusion of tastant
molecules to taste cells and appears to be an unreliable mechanism for
evaluating food quality. Takeuchi et al.
(1994
) observed a rejection of
gelatine capsules containing 1 mmoll-1 quinine by the urodele
amphibian Ambystoma mexicanum and that the presence of 100
mmoll-1 NaCl in the capsules reduced the proportion rejected. It
was suggested that the salamanders were able to discriminate between the taste
qualities of these stimuli. The authors also noted that this salamander has a
non-distensible tongue and feeds by snapping its jaws to capture food items
dropped into the water. Thus, the feeding behavior does not require a layer of
mucus covering the taste cells, and a limited amount of mastication
occurs.
Recent studies by Sato et al.
(2000,
2001
) propose an interesting
function for salivary secretion in chemosensory transduction. They observed
that stimulation of the frog glossopharyngeal nerve elicited slow potentials
at the tongue surface and in taste cells in the fungiform papillae. This was
blocked by atropine, suggesting that the effect was mediated by
parasympathetic nerve fibers. Depolarizing slow potentials enhanced the
receptor potential response to exposure to 0.5 moll-1 NaCl, and it
was suggested that the parasympathetic stimulation had produced salivary
secretions whose ionic composition modified the liquid junction potential
across the lingual epithelium. Assuming that salivary secretion in toads
involves K+ secretion, as in mammals
(Berne and Levy, 1988
),
K+ secretion could contribute to the measured
Isc and also to the chemosensory function of the
tongue.
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Acknowledgments |
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