1Department of Oral Biology, College of Dentistry, Ohio State University, Columbus, Ohio 43210; and 2Department of Pharmacology, Pharmacia & UpJohn Incorporated, Kalamazoo, Michigan 49007
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ABSTRACT |
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Herness, M. Scott and
Xiao-Dong Sun.
Characterization of Chloride Currents and Their Noradrenergic
Modulation in Rat Taste Receptor Cells.
J. Neurophysiol. 82: 260-271, 1999.
Taste receptor
cells contain a heterogeneous array of voltage-dependent ion
conductances that are essential components for the transduction of
gustatory stimuli. Although mechanistic roles have been proposed for
several cationic conductances, the understanding of anionic currents is
rudimentary. This study characterizes biophysical and pharmacological
properties of chloride currents in rat posterior taste cells using
whole cell patch-clamp recording technique. Taste cells express a
heterogeneous array of chloride currents that displayed strong outward
rectification, contained both calcium-dependent and
calcium-independent components, and achieved a maximal conductance of
almost 1 nS. Reversal potentials altered predictably with changes in
chloride concentration. Currents were sensitive to inhibition by the
chloride channel pharmacological agents DIDS, SITS, and niflumic acid
but were insensitive to 9-AC. Adrenergic enhancement of chloride
currents, present in other cell types, was tested on taste cells with
the -adrenergic agonist isoproterenol (ISP). ISP enhanced the
outwardly rectifying portion of the chloride current. This enhancement
was calcium dependent and was blocked by the
-adrenergic antagonist
propranolol. Collectively these observations suggest that chloride
currents may participate not only in usually ascribed functions such as
stabilization of the membrane potential and volume regulation but
additionally play active modulatory roles in the transduction of
gustatory stimuli.
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INTRODUCTION |
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Taste receptor cells are differentiated epithelial
cells that detect the presence of sapid stimuli within the oral cavity and relay this information to the CNS via sensory afferent nerves. Taste cells are electrically excitable and utilize a diverse array of
voltage-gated ion channels to elicit either receptor potentials or
action potentials in response to appropriate stimulation. The detailed
characterization of these ion channels is prerequisite to elucidating
the variety of transduction mechanisms employed by these cells. Many of
these currents have been fully or partially characterized. In the
mammalian taste receptor cell, these include voltage-dependent sodium
channels (Doolin and Gilbertson 1996; Herness and
Sun 1995
), delayed-rectifier, transient, and calcium-activated outward potassium currents (Béhé et al.
1990
; Chen et al. 1996
), inwardly rectifying
potassium currents (Sun and Herness 1996b
), and calcium
currents (Béhé et al. 1990
; Chen and
Herness 1997
). In rat posterior taste cells, these channels
display an obvious diversity in expression across cells. For example,
many taste cells do not express voltage-dependent sodium channels and
are hence incapable of eliciting action potentials. Potassium channel expression is also very heterogeneous and strongly correlated with
action potential duration. The differences in distribution of ion
channels parallel well-documented differences in chemosensitivity that
also occurs across taste cells. In studies of taste cells, one major
class of channel, chloride channels, has yet to be characterized.
Membrane conductance to chloride, once dismissed as artifact and
generically termed "leak," is now recognized to participate in a
variety of physiological roles that include stabilization of the
membrane potential, volume regulation, and salt transport. These
diverse physiological roles are matched by the broad heterogeneity of
this channel type. Presently, at least nine genes are recognized that
encode chloride channel proteins (CLC) that function as either chloride
channels or putative chloride channels (Jentsch 1996). There are several hypothetical reasons to suggest possible roles of
chloride currents in the normal physiology of taste cells. First, fluid
and electrolyte secretion is known to be coupled to vectorial chloride
movement (for reviews see Anderson et al. 1992
;
Frizzell and Morris 1994
) and because taste cells are
implicated in salt transport across the tongue (DeSimone et al.
1981
, 1984
; Mierson et al. 1996
),
chloride channels are likely to be involved. Second, a major chloride
conductance, cystic fibrosis transmembrane conductance regulator
(CFTR), is cAMP dependent (e.g., Gadsby et al. 1995
) and
cAMP is an important intracellular messenger in posterior taste
receptor cells (Herness et al. 1997
). Third, chloride
channels play essential roles in regulatory volume decrease produced by
changes in osmolarity (Nilius et al. 1996
), and taste cells are routinely subjected to osmotic extremes from water to hyperosmotic foodstuffs. Finally, chloride currents are sometimes active participants in electrical responses of excitable cells (e.g.,
Harvey 1996
) and hence may play yet unrecognized roles in gustatory transduction.
Some preliminary characterizations of chloride currents in amphibians
taste cells have been reported. A calcium-dependent outward chloride
current has been reported in Necturus taste cells (Mcbride and Roper 1991). With the use of intracellular
techniques, it was observed that calcium influx during the action
potential was sufficient to stimulate a calcium-dependent chloride
current. These channels appear to be on both apical and basolateral
membranes. Their function was postulated as twofold, to help
discriminate chloride and nonchloride salts, and to help shape the
receptor potential responses initiated by taste stimuli. It was later
reported (Taylor and Roper 1994
) with patch-clamp
techniques, that this current is sensitive to SITS and DIDS and that it
may play a role in adaptation, by terminating a long depolarizing
receptor potential.
The present study was designed to investigate the nature of the chloride conductance in mammalian taste receptor cells using the whole cell configuration of the patch-clamp recording technique. Biophysical and pharmacological evidence is presented that taste receptor cells likely possess multiple chloride conductances.
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METHODS |
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All experiments were performed on isolated taste receptor cells dissociated from circumvallate and foliate papillae of the rat tongue using standard patch-clamp procedures in the whole cell recording mode.
Dissociation procedure
Taste receptor cells were dissociated from excised regions of
the posterior rat tongue as previously described (Herness
1989). Lingual tissue was excised from an animal that had
previously reached a surgical level of anesthesia with an intramuscular
injection of 0.09 ml/100 gm body wt Ketamine/Acepromazine mixture (91 mg/ml Ketamine, Fort Dodge Laboratories; 0.09 mg/ml Acepromazine,
Butler Laboratories). Papillae were blocked from tongue tissue and
incubated in a cysteine-activated (1 mg/ml) Papain/divalent-free
bicarbonate-buffered solution (14 U/ml) for several hours at 32°C in
5% CO2-95% air. Cells were dissociated in a
pseudo-extracellular fluid (ECF) by mild agitation. Some papillae were
maintained in ice-cold ECF solution for later dissociation. Dissociated
taste receptor cells were easily identified by their characteristic
morphology. However, in the dissociated state it is not always possible
to distinguish apical from basolateral domains.
Solutions
The divalent-free solution for enzymatic incubation was composed of (in mM) 65 NaCl, 20 KCl, 26 NaHCO3, 2.5 NaH2PO4 · H2O, 20 D-glucose, and 1 EDTA. The standard ECF solution used for the dissociation procedure included (in mM) 126 NaCl, 1.25 NaH2PO4 · H2O, 5 KCl, 5 NaHEPES, 2 MgCl2, 2 CaCl2, and 10 glucose; it was pH adjusted to 7.4 with NaOH.
The composition of intracellular fluid (ICF) used for for filling the recording pipette consisted of (in mM) 24 CsCl, 116 methanesulfonic acid (cesium salt), 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, and 4 ATP (magnesium salt); this solution was adjusted to a final pH of 7.2 with CsOH and yielded a final chloride concentration of 30 mM. In some cases, a high chloride ICF was used with a final chloride concentration of 146 mM; its composition was identical to the previous ICF except that the CsCl concentration was 140 mM and methanesulfonic acid was omitted. A potassium-free extracellular solution was employed for the bath solution; it consisted of (in mM) 126 NaCl, 5 HEPES, 2 CaCl2, 2 MgCl2, and 10 glucose with a final chloride concentration of 134 mM. For some experiments, this solution may have additionally contained 1 BaCl2 and/or 0.5 or 1 CdCl2 to inhibit either inwardly rectifying potassium currents or calcium currents, respectively. The extracellular recording solution was pH adjusted to 7.4 with Tris base.
For ion substitution experiments, where either intracellular or extracellular chloride concentration was manipulated, chloride was substituted with the cesium salt of methanesulfonic acid for ICF solutions or the sodium salt of isethionic acid for ECF solutions. For pharmacological analysis of chloride currents, agents were dissolved in the superfusing solution. These included two stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, Sigma) and 4-acteamido-4'-isothiocyanastilbene-2,2'-disuldonic acid (SITS, Sigma), each at 500 µM, niflumic acid (Sigma) tested at 500 or 1,000 µM, or 9-anthracene carboxylic acid (9-AC, Sigma) at 500 µM.
Whole cell electrophysiological recording
Micropipettes were pulled on a gas-cooled multistage puller from
1.5-mm OD borosilicate glass (World Precision Instruments, Sarasota,
FL) and were fire polished. Resistances were typically 2-4 M when
filled with ICF and measured in ECF. Junction potentials were corrected
before the electrode contacted the cell. [Although small junction
potentials are introduced when using silver:silver chloride electrodes
during changes of chloride concentration, an agar bridge was not
employed during experiments with altered extracellular chloride (Figs.
2 and 7) because these measurements were largely qualitative and agar
bridge are, at best, imperfect solutions (Neher 1992
).
Nevertheless, artifacts of several millivolts may have been introduced
during the experiments with altered chloride concentrations for this
reason.] The pipette tip was positioned to contact the cell membrane,
and negative pressure was applied to its interior to facilitate
gigaseal formation. Seal resistances were on the order of several
decades of gigaohms. Further negative pressure was applied to enter
whole cell recording mode.
Fast and slow capacitance compensation was employed as necessary with
amplifier controls. Cell membrane capacitance and uncompensated series
resistance were adjusted to produce optimal transient balancing. Membrane capacitance was 3-6 pF; series resistance averaged 10 M.
Low-pass filtering due to resistance-capacitance coupling was
considered minimal. The product of these factors produces a time
constant of 30-60 µs or a cutoff frequency (1/2
RC) of 2.6-5.3 kHz.
Data were acquired with a high-impedance amplifier and a
high-resistance feedback headstage (Axopatch 200A; Axon Instruments), a
486 computer equipped with a 12-bit 330-kHz A/D converter (Digidata 1200; Axon Instruments), and a commercially available software program
(pCLAMP, version 6.0.3; Axon Instruments). Membrane currents were
acquired after low-pass filtering with a cutoff frequency of 5 kHz (at
3 dB). A software-driven D/A converter generated the voltage
protocols. In some situations, currents were measured with voltage
protocols using standard step potentials with a holding potential of 0 mV and a series of 80-ms command potentials applied in 20-mV increments
from final potentials ranging
140 to +120 mV and acquired with a
sampling rate of 195 µs. Most data were acquired with the use of a
ramp protocol that clamped the membrane potential from
130 to +120 mV
over a period of 3 s (83.3 mV/s) using a sampling rate of 2 ms.
Leak subtraction was not employed. Experiments with a P/4 leak
subtraction protocol resulted in current profile that was outwardly
rectifying; however, much of the linear component was removed. Because
this linear portion of the total current is present when chloride is
the major conducting ion and is inhibited by chloride channel blockers,
the leak subtraction protocol was concluded to inappropriately remove a
linear component of the total chloride current. Moreover, in addition
to confounding current-voltage (I-V) relationships, leak
subtraction would complicate interpretations of zero-current potentials
and steady-state current amplitudes.
In general, obvious rundown of currents was not observed. The findings that chloride currents responded appropriately to manipulations of the bath solutions during the recording session, including altering chloride concentrations, altering calcium currents, and combinations of isoproterenol followed by a second manipulation (either low chloride, cadmium, or propranolol), collectively argue that current was behaving as expected during the recording session without obvious signs of rundown.
Recordings were made at room temperature. Positive currents reflect the inward flux of chloride; negative currents represent an outward chloride flux.
Data analysis
Data were analyzed with a combination of off-line software programs that included a software acquisition suite (pCLAMP, Axon Instruments) and a technical graphics/analysis program (Origin; MicroCal Software).
Theoretical equilibrium potentials were calculated according to the
Nernst equation using chloride concentrations of intracellular and
extracellular solutions. Whole cell chloride conductance was calculated
as
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RESULTS |
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Chloride currents were recorded from all cells that previously satisfied criteria for whole cell recording. These currents were considered to be conducted by chloride ions because they were dependent on the presence of chloride in either the bathing or pipette solutions, the currents reversed at a predicted Nernstian potential, and they demonstrated a pharmacology consistent with that of chloride channels.
Isolation of chloride currents
A series of inward and outward currents was recorded from
dissociated taste receptor cells in response to hyperpolarizing and
depolarizing voltage steps delivered from a holding potential of 0 mV.
Currents were recorded in potassium-free ECF and ICF solutions that
established a chloride gradient of 134/30 mM (outside/inside). Data
from a representative cell are presented in Fig.
1A; the I-V plot
for this cell, obtained from steady-state values, is illustrated in
Fig. 1B. Evident in both the current traces and the
I-V plot is a prominent outward rectification with little time dependence to the current over the 80-ms pulse. (We use the standard convention of referring to positive currents as outward currents, although they literally represent an inward movement of
chloride ions.) Additionally, these currents often produced a
distinctive tail current at the break of either large hyperpolarizing pulses (greater than 80 mV) or large depolarizing pulses (>70 mV).
Tail currents appear to be complex, receiving contributions from a
variety of voltage-dependent conductances; they will not be further
considered in this communication. Currents reversed at a predicted
Nernstian reversal potential for the chloride gradient used for these
recording, between
35 and
40 mV. The predicted potential for a
chloride gradient of 134 mM external and 30 mM internal chloride is
38 mV.
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A ramp command potential was also employed to evoke membrane currents.
Data from 12 cells, using a ramp protocol, are presented in Fig.
1C. Current was ramped from 130 to 120 mV from a holding potential of 0 mV (inset of Fig. 1C) using a rate
(dV/dt) of 83 mV/s. The ramp potential
facilitated data acquisition by evoking a broad membrane range over a
short period of time. Additionally, the combined use of a 0-mV holding
potential, the ionic composition of the recording solutions, and more
gradual change of the clamped membrane potential (compared with abrupt
step changes) helped to ensure inactivation of other membrane
conductances. This protocol was used for all subsequent experiments. As
with currents evoked by step potentials, the data displayed pronounced
outward rectification and reversed at the appropriate membrane
potential. The dotted line represents extrapolated data using the
linear portion of the outward current. The linear regression of this
area of the I-V curve produced a slope conductance of 0.82 nS (r = 0.9997). Chord conductance (Fig. 1D)
plateaued at 0.7 nS and was adequately described by a Boltzmann
relationship with a half-maximal potential of
3.5 mV and a steepness
coefficient of 21 mV per e-fold. The steepness coefficient
produced an equivalent gating charge of 1.2. This value agrees quite
well with the typically modest voltage dependence of the equivalent
gating charge of 1.2-2.0 that are ascribed to chloride channels
(Hille 1992
). As a comparison with other membrane
conductances in these cells, we have previously measured in posterior
taste receptor cells a voltage-dependent sodium conductance of 7.3 nS
(Herness and Sun 1995
) and total outward potassium
conductance of 23 nS (Chen et al. 1996
). Although little
current is evoked at membrane potentials that correspond to the resting
potential in these cells, a measurable conductance does occur. When
considered with the high-input resistant of these cells (5-20 G
),
it is likely that these currents can contribute to total membrane
conductance at zero-current potentials.
That currents isolated under these conditions were in fact conducted by
chloride was confirmed by ionic substitution of either external or
internal chloride concentrations. Because the majority of current was
outward (hence due to an inward flow of chloride ion), these currents
would be expected to be reduced in a sizable and predictable amount by
reduction of extracellular chloride. In Fig.
2A, currents were recorded
using 134, 30, or 8 mM extracellular chloride while keeping
intracellular chloride constant at 30 mM. Current records represent the
mean of 14 cells (134 mM), 5 cells (30 mM), or 8 cells (8 mM). As
expected, reduction of external chloride reduced the outwardly
rectifying current. Data are summarized in histogram form in Fig.
2C for current magnitudes resulting from +120-mV membrane
depolarization. However, inward currents responded enigmatically to
reduction of extracellular chloride. Current magnitudes, expected to be
somewhat enhanced, were slightly reduced in magnitude and the reversal
potential, expected to be shifted in the depolarizing direction, was
shifted in the hyperpolarizing direction. Additionally, these inward
currents subsequently proved to be insensitive to most manipulations of
the chloride conductance subsequently described. Thus it is likely that
these small inward currents could result from a contaminating
conductance, such as a cation leak current. Along this line,
Doolin and Gilbertson (1996), using rat posterior taste
receptor cells, recorded a leak inward current at these potentials that
was partially sodium selective. However, it seems unlikely that the
leak current recorded in their study is contributing to the inward
current in the present study. Doolin and Gilberston
(1996)
recorded an inward current of about
120 pA at a
holding potential of
70 mV using usual compositions of ECF and ICF.
Substituting a sodium-free ECF, this steady inward current was reduced
to about
40 pA (or ~80 pA of sodium-dependent leak current). The
present study differed considerably in both the holding potential (0 mV) and compositions of ECF and ICF. Using a ramp potential, the total
inward current, at
70 mV, was only about
10 pA (rather than
120
pA) and was essentially the same in sodium-free ECF (Fig.
3). This suggests that the combination of
ionic composition and holding potential used in our experiments substantially eliminated any sodium leak current even before sodium substitution. Additionally, a sodium leak current would be expected to
shift the reversal potential in the depolarizing direction (toward the
sodium equilibrium potential of about +50 mV), whereas the actual
reversal potential shifted in the hyperpolarizing direction. Taken
together, the magnitude of the inward current, its insensitivity to the
reduction of extracellular sodium, and the direction of the reversal
potential shift make sodium an unlikely candidate for any putative leak
conductance. Although several potassium-blocking agents were present
during the recording, a small inwardly rectifying potassium conductance
might possess more appropriate biophysical characteristics as a leak
current candidate than sodium. As well, it is not unusual for chloride
conductances to deviate from strict Nerstian predictions. Unlike cation
channels, anion channels have appreciable cation conductance (e.g.,
Hille 1992
) and in low chloride solutions cation
permeability becomes larger and significantly affects the reversal
potential (Franciolini and Nonner 1987
; Gelband et al. 1996
). Taken together, these observations suggest that these inward currents are not an ideally chloride-selective conductance but do not eliminate the possibility that channels with differing biophysical properties and/or leak current may carry the inward currents.
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Modulation of intracellular chloride concentration was also
performed. Figure 2B presents mean data using two
intracellular chloride concentrations of either 30 mM
(n = 12 cells) or 146 mM (n = 20 cells)
while keeping extracellular chloride constant at 134 mM. In these
experiments the expectations of enhanced inward currents, shift of the
reversal potential to close to zero, and diminished outward currents
were all observed. However, above membrane potentials of about +50 mV,
elevated intracellular chloride concentrations actually enhanced the
recorded current. Although the basis of this enhanced current is
presently unknown, a recent report that high intracellular chloride
concentrations can act to depress G protein modulated ionic
conductances may be germane (Lenz et al. 1997). We have
studied the G protein cAMP mediated inhibition of potassium currents in
these cells (Herness et al. 1997
). Depression of
baseline inhibition of these potassium currents could result in
measurable enhancement of the outward current. Because potassium
currents in these cells are on the order of magnitude of ~8,000 pA at
+120 mV, even a small contribution of potassium current to the outward
current could account for the enhancement to 200 pA.
In another set of experiments, chloride currents were recorded in the absence of extracellular sodium to test for potential contributions of the electrogenic Na/K/Cl pump, known to occur in some tissues, or alternatively for contributions of a leak sodium current to the recorded currents. In these experiments, extracellular sodium was replaced with choline while maintaining extracellular chloride at 134 mM. Data are presented in Fig. 3. There was little, if any, difference between current records produced with ramp potentials in these two recording solutions (n = 4 cells; Fig. 3A). A small but consistent reduction in the current (~4%) was measured at potential more positive than +50 mV. At +120 mV the current was 142 ± 32 pA in normal sodium ECF and 136 ± 36 pA in choline-replaced ECF (Fig. 3B). Currents were identical at potentials more negative to +50 mV. This observation suggests that sodium contributes little if at all to the total outward current recorded.
Calcium dependence of chloride currents
To test whether chloride currents recorded from taste receptor cells contained a calcium-dependent component, current was evoked before and during presentation of the calcium-channel blockers cadmium (a blocker of a wide variety of calcium channels) or nifedipine (a blocker more specific for L-type calcium channels). Data are presented in Fig. 4. Both calcium channel blockers were effective in reducing the magnitude of chloride currents, suggesting that these currents contain a calcium-dependent component. Cadmium, tested at 1 mM, effectively reduced the outwardly rectifying portion of the current. Inward chloride current was unaffected by the presence of cadmium. This would be expected because calcium currents would not be active at these potentials. Similarly, the reversal potential was unchanged in the presence of cadmium. Outward currents, however, were reduced up to 48% (P = 0.019). Data with nifedipine were similar to those obtained with cadmium. Only the outwardly rectifying portion of the current was affected by nifedipine, tested at 100 µM (Fig. 4B). Data are the mean value of three cells. Neither the inward chloride current nor the reversal potential was affected. Outward current was reduced by up to 21% (P = 0.015). Data (mean ± SE) are summarized in Fig. 4C in histogram form for a test pulse to +120 mV. Cadmium reduced the current at this potential from 153 ± 8.8 pA to 80 ± 12.5 pA, whereas nifedipine reduced the current from 157 ± 40 pA to 123 ± 29 pA.
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Pharmacological analysis of chloride currents
Chloride current was further characterized by examining the effect of several chloride channel blockers. Four agents were tested: the two stilbene derivatives DIDS and SITS, the fenamate blocker niflumic acid, and the chloride channel blocker 9-anthracene carboxylic acid (9-AC).
When tested at 500 µM, DIDS effectively inhibited the outward portion
of the chloride current without noticeable effects on the inward
portion (Fig. 5A). The effect
could be partially washed out, with removal of DIDS from the bathing
solution, but recovery was never complete, in agreement with the
observations of others (Ackerman et al. 1994;
Ullrich and Sontheimer 1996
). Peak current, at +120 mV,
was inhibited from 116 ± 0.9 pA to 70 ± 0.7 pA, or about a
40% inhibition (Fig. 5C, mean ± SE of 5 cells;
P = 0.0014). Inhibition was most effective at positive
depolarization potentials, whereas it was ineffective at negative
membrane potentials. DIDS was similarly effective in inhibiting the
outward current in the presence of 0.5 mM cadmium
([Cl
]in/[Cl
]out = 30/134 mM; n = 5, data not shown), again with no
effect on the inward portion of the current. This suggests that at
least some of the current inhibited by DIDS is calcium independent. SITS, another distilbene inhibitor, was less effective than DIDS in
reducing the magnitude of the evoked current. When tested at 500 µM
in K-free ECF, outward, but not inward, currents were inhibited (Fig.
5B, n = 3 cells). Outward current was
inhibited from 155 ± 0.9 pA to 126 ± 0.2 pA, or about a
19% inhibition (Fig. 5C; P = 0.046). The
reversal potential was unaltered.
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Niflumic acid was an effective inhibitor of the outwardly rectifying portion of the chloride currents. Data are presented as both 500 and 1,000 µM (Fig. 6, A and B). At a test pulse of +120 mV (Fig. 6C), current was inhibited by 42 ± 9.5% by 500 µM niflumic acid (n = 5 cells; P = 0.024) and by 40 ± 6.1% at 1,000 µM (n = 7 cells; P = 0.00038). Data for 1,000 µM niflumic acid was obtained using a chloride gradient of 134/146 (out/in), which explains why the baseline current is larger in Fig. 6B than in 6A (as was also observed in Fig. 2B).
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Finally, the agent 9-AC appeared to be ineffective on these currents.
9-AC was tested at both 100 and 500 µM without effect. Data are
summarized in Fig. 6C (n = 2 cells). Mean
response during the presence of 9-AC was 99.8 ± 0.02%
(n = 2 cells). Because this agent required
solublization in 0.01% DMSO before preparation in solution, DMSO alone
was tested (0.01%) and also determined to be without effect on these
currents. We have similarly determined that DMSO by itself at this
concentration does not affect outward potassium currents in these cells
(Herness et al. 1997).
Isoproterenol enhancement of outward chloride currents
In several cell types, such as cardiac myocytes (Harvey
1996) and amphibian epithelium (Larsen et al.
1995
; Willumsen et al. 1992
), chloride currents
are known to be positively modulated by noradrenergic stimulation,
generally via
-receptor activation. We tested this possibility in
taste receptor cells using the
-receptor agonist isoproterenol (ISP)
and subsequently with the
-receptor antagonist propranolol. When
tested at 100 µM, ISP effectively enhanced the outward portion of the
chloride current in a total of 15 tested cells (Figs.
7A, 8A,
and 9A). Currents were
measured as previously described by a ramp protocol before and during
application of 100 µM ISP (Fig. 7A, n = 8 cells; P = 0.000016). Peak current, measured at +120
mV, was enhanced to 128 ± 10.7% of control values (Fig.
7C, n = 8 cells). Enhancement was only
observed for the outwardly rectifying portion of the current. Inward
chloride current remained unaffected by application of ISP.
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To verify that the enhanced conductance was carried by chloride ions, an ion substitution experiment was performed. Extracellular chloride was lowered from 134 to 8 mM. The reduction of extracellular chloride inhibited outward and, to a lesser degree, inward chloride currents, essentially replicating the data presented in Fig. 2A (42% inhibition; P = 0.006). Subsequently, ISP was presented in low extracellular chloride and now failed to significantly modify the magnitude of the evoked current (Fig. 7B, n = 4 cells; P = 0.0085). Current, at 120 mV was 90 ± 4.5 pA in low chloride and 76 ± 4.7 pA in low chloride plus ISP (Fig. 7D).
Experiments were performed to test for the calcium dependence of the ISP enhancement of chloride currents. In two separate sets of experiments, cadmium was added to ISP either after ISP administration (Fig. 8A) or before its administration (Fig. 8B). With the use of the former protocol, ISP was first demonstrated to be effective in enhancing the outward currents (Fig. 8A; n = 4 cells). However, when cadmium (1 mM) was added to the bathing solution in the sustained presence of ISP, the currents were diminished to below control levels. These data are consistent with those presented in Fig. 4A, where cadmium similarly reduced chloride current magnitude. Similar results were obtained when cadmium administration was applied before ISP administration. Here cadmium administration eliminated the calcium-activated component of the chloride current (Fig. 8B; 32% inhibition; P = 0.012). When ISP was subsequently applied, no enhancement was observed (38% inhibition of control currents; P = 0.016). The reductions of the control current were to 100 ± 21 pA for cadmium and 95 ± 20 pA for cadmium in the presence of ISP. These data suggest that the ISP enhancement is calcium dependent.
In a final set of experiments, the specificity of receptor
mechanism of the ISP enhancement was tested. ISP enhancement, if operating through -receptors, should be prevented by prior exposure to a
-receptor antagonist. Propranolol, a commonly used adrenergic antagonist specific for
-receptors, was employed to test this hypothesis. Data are presented in Fig. 9. Cells were first exposed to
100 µM ISP to measure enhancement of the current then subsequently exposed to 10 µM propranolol followed by an ISP/propranolol mixture. In two cells, ISP application was tested again after propranolol exposure, if recording conditions permitted. In the tested cells, ISP
enhanced the current as previously observed. (1.40 ± 0.87%, n = 4; P = 0.016; data normalized to
preexposure control current). However, in the presence of propranolol,
ISP was ineffective in altering the magnitude of the evoked current
(0.98 ± 0.022) suggesting the ISP effect to occur through
-receptors. Control current and ISP/propranolol current were
essentially indistinguishable. These normalized data are summarized in
Fig. 9B. In two cells, the recording session lasted long
enough to allow a second application of ISP. In both of these cells,
the second ISP application increased the evoked current, although not
to the original degree (1.12 ± 0.02). Both rundown and residual
binding of propranolol may contribute to the diminished effect of the
second ISP exposure.
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DISCUSSION |
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Gustatory transduction mechanisms require the complex coordination
of intracellular signaling cascades that ultimately influence cellular
excitability by modulating a variety of ion channels. Fundamental to
the detailed understanding of these mechanisms is the careful
examination of individual ionic conductances expressed in taste cells.
In our studies of posterior taste receptor cells, we have examined a
variety of cationic conductances, such as voltage-dependent sodium
currents (Herness and Sun 1995), outward potassium
currents, including delayed-rectifier, transient, and calcium-activated currents (Chen et al. 1996
), and inwardly rectifying
potassium currents (Sun and Herness 1996b
). With the
exception of inwardly rectifying potassium currents, these currents are
heterogeneous in their distribution across taste cells, just as the
chemical sensitivity of individual taste cells varies on a cell-by-cell basis. Anionic currents, however, have not been described. Because chloride currents subserve diverse functions in other cell types, including the modulation of cellular excitability, their examination is
essential toward understanding the physiology of taste receptor cells.
Types of chloride currents in posterior taste receptor cells
Taste receptor cells, which are differentiated epithelial cells, appear to possess a heterogeneous array of chloride conductances that are typical to many epithelia. Prominent features of the whole cell chloride conductance in taste cells include pronounced outward rectification, calcium dependence of a portion of the current, measurable conductance near the resting potential, and putative modulation by adrenergic agents.
Epithelia typically possess multiple chloride conductances, several of
which are outwardly rectifying and/or calcium dependent (e.g.,
Frizzell and Morris 1994; Valverde et al.
1995
). Three major chloride conductances in epithelia include a
cAMP-dependent current, a calcium-dependent current, and a
volume-sensitive current important in regulatory volume decrease (RVD).
Each display some unique biophysical properties. For example, the
cAMP-dependent current is mostly linear in its I-V
properties, is not calcium dependent, and is DIDS insensitive. This
current in epithelia is often carried by the CFTR channel or an isoform
of it (Anderson et al. 1992
). Conversely, the
calcium-dependent and volume-sensitive currents are both outwardly
rectifying; however, the former often displays a time-dependent
increase in current magnitude with maintained depolarization, whereas
the latter displays a time-dependent decrease in current magnitude.
In taste receptor cells, most of the chloride conductance is outwardly rectifying, that is likely composed of linear and rectifying components. A portion of this current is calcium dependent, as indicated in Fig. 4. In the presence of either calcium inhibitors or chloride channel blockers, such as DIDS, the current loses much of its rectification, leaving a remainder current more linear in nature. The linear nature of the DIDS-insensitive current, and the adrenergic enhancement of the whole cell current (next section) are suggestive, but not conclusive, evidence for a cAMP-dependent current.
Adrenergic modulation of chloride currents
Data obtained with the -adrenergic agonist ISP and antagonist
propranolol suggest that an adrenergic enhancement of chloride current
may be occurring in taste receptor cells. This observation, which is a
novel observation to taste receptor cells, is similar to the
well-documented adrenergic modulation in other systems such as cardiac
myocytes and amphibian skin.
In cardiac myocytes, two chloride currents are activated or enhanced by
-stimulation (Harvey 1996
): a cAMP-regulated current similar to CFTR, which is directly activated by cAMP, requires protein
kinase A (PKA)-dependent phosphorylation, and acts to shorten the
action potential, and a calcium-dependent chloride current, which is
likely indirectly activated by an enhancement of an L-type calcium
current. Adrenergic enhancement of chloride current also occurs in
amphibian epithelium. Two cell types (the principal cells and the
mitochondria-rich cells) work in concert to transport salt. The
mitochondria-rich cells transport chloride specifically, and this
chloride transport is enhanced by ISP in a manner mediated by cAMP
(Larsen et al. 1995
; Willumsen et al. 1992
).
The underlying mechanism of adrenergic enhancement of chloride current
in taste cells remains to be elucidated. It is presumed, but presently
untested, that such enhancement would be cAMP dependent, because ISP is
a -receptor agonist and
-receptors operate through G protein
stimulation of adenylate cyclase. Alternatively, because the
ISP-enhanced current was determined to be calcium dependent, ISP could
enhance calcium currents and secondarily enhance the calcium-dependent
chloride currents. Such enhancement of calcium currents by ISP was
recently reported in the amgydala (Huang et al. 1998
).
ISP has other actions on different ionic conductances in taste cells.
In taste cells we have observed that ISP also inhibits outward
potassium currents. This mechanism appears to be mediated by cAMP and
PKA phosphorylation (Herness et al. 1997; Sun and Herness 1996a
). Additionally, recent evidence suggests that
cAMP inhibits voltage-dependent sodium currents (Herness
1997
). Thus ISP-mediated elevations of cAMP might be expected
to additionally modulation sodium channels. These additional actions of
ISP on other currents are also observed in cardiac tissue. In response to sympathetic stimulation of the heart,
-adrenoceptor activation also stimulates an L-type Ca current, the pacemaker current, delayed rectifier current, and inhibits sodium current (Harvey
1996
).
The source of norepinephrine within the taste bud is presently unknown.
There is some evidence that adrenergic nerve fibers surround taste buds
(i.e., perigemmal nerve fibers) (Paparelli et al. 1986).
The density of these fibers seems to vary from species to species, and
they may be branches from perivascular axons. Additionally, stimulating
the sympathetic nerve supply to the tongue or systemic injections of
epinephrine was shown to enhance taste responses recorded in the chorda
tympani nerve in rats (Kimura 1961
). In the frog,
norepinephrine (but not dopamine) injected in the lingual artery was
reported to enhance glossopharyngeal nerve responses to gustatory
stimuli (Morimoto and Sato 1982
; Nagahama and
Kurihara 1985
).
In general, actions of neurotransmitters on taste receptor cells are
not yet well studied. Evidence thus far suggests that their actions may
be complex. Along these lines, we have observed that another
neurotransmitter, serotonin, actually decreases the calcium-activated
potassium current and that this inhibition is probably due to
5HT1A receptors (Herness and Chen
1997). Similarly, it is not yet known if the action of
serotonin is directly on calcium-activated potassium current or if it
acts indirectly through calcium currents.
Physiological role for chloride currents
Chloride currents likely contribute to multiple physiological
roles in taste receptor cells that include housekeeping functions such
as volume regulation, maintenance of the resting potential, and
contributions to cellular excitability. Precise roles in taste receptor
cells remain to be elucidated. One of the most alluring is the
possibility that chloride currents may play active roles in taste
transduction processes. As a comparison, chloride currents are known to
play an integral role in transduction of olfactory stimuli (e.g.,
Kurahashi and Yau 1994).
Because taste receptor cells and epithelial cells appear to share
similar distributions of chloride channels, their functions may also be
similar. Chloride currents play integral roles in salt transport across
epithelia. Similar transport across the lingual epithelium is known
(DeSimone et al. 1981, 1984
;
Mierson et al. 1996
), and the participation of chloride
conductance has been suggested (Wladkowski et al. 1998
).
Additionally, the apical ends of taste cells are routinely subjected to
both hypotonic and hypertonic extremes of osmotic pressure. RVD is
present in both excitable and inexcitable cells (reviewed in
Nilius et al. 1996
; Okada 1997
).
Interestingly, the volume activated can be blocked by quinine
(Voets et al. 1996
), a commonly employed tastant prototypic for bitter stimuli. However, an important difference between
taste receptor cells and other epithelia cells is that taste receptor
cells are electrically excitable. Here, taste receptor cells may be
more similar to neurons and cardiac cells where calcium-activated chloride channels participate in modulation of action potential (Harvey 1996
; Scott et al. 1995
). We have
observed that the shape of the gustatory action potential is modulated
by DIDS (personal observation), suggesting that chloride currents may
contribute to the repertoire of voltage-dependent conductances
underling the action potential. DIDS treatment broadened the duration
of the action potential and reduced the magnitude of the
afterhyperpolarization. Collectively, these observations suggest that
chloride channels may be multifunctional in taste receptor cells.
The ultimate effect that adrenergic modulation of chloride currents
would have on the electrical state of the cell is complex and would be
influenced by the dynamic state of the membrane potential. For example,
adrenergic modulation of a taste cell at rest would be expected to
bring the membrane potential closer to the chloride equilibrium
potential and likely act to stabilize the membrane potential. Under
these conditions, the increase of cAMP due to activation of
-receptors would not alter potassium conductances because, at rest,
most of these conductances are closed. We have observed that cAMP does
not depolarize the resting potential of posterior taste cells
(Herness et al. 1997
), consistent with the notions that
there is no closure of a resting potassium conductance and that
putative enhancement of the chloride current might be stabilizing the
membrane potential. An additional complication lies in defining the
resting potential of taste cell and establishing the true chloride
reversal potential, which is altered in the experimental situation by
the choice of extracellular and intracellular chloride ion
concentrations. The conventional values of 134 and 30 mM for external
and internal chloride establishes a reversal potential of about
35
mV, which would likely be a depolarizing influence for many taste cells
if the resting chloride conductance were enhanced. However, we have
observed mean zero-current potentials for posterior taste cells of
37
mV with a range of 0 to
85 mV (Herness and Sun 1995
,
Table 1). On the other hand, if the cell were in an active state, i.e.,
firing an action potential, norepinephrine release would be expected to
decrease an outward potassium current and increase an outward chloride
current in those taste receptor cells expressing
-receptors. Such
actions would result in a prolonged duration of the action potential,
due to inhibited outward potassium currents, and possibly an
exaggerated afterhyperpolarization, due to the contribution of chloride
currents, causing an adaptation of the firing rate. In short, the
interactions of currents regulating the electrical excitability of the
cell are complex, and such dynamic factors as the active state of the
cell and the temporal phase of the membrane potential must be
considered to understand influences of modulatory actions. Given the
heterogeneity of membrane properties and the diversity of
chemosensitivity of taste receptor cells, generalizations regarding the
properties of membrane conductances are of limited value. Correlations
of chemosensitivity, ion channel repertoire, and expression of
transmitters and their receptors within single cells will be necessary
to fully understand the roles of neurotransmitters within the taste bud.
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ACKNOWLEDGMENTS |
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This work was supported by Grant DC-00401 from the National Institute of Deafness and Other Communications Disorders.
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FOOTNOTES |
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Address for reprint requests: S. Herness, Dept. of Oral Biology, School of Dentistry, Ohio State University, 305 West 12th Ave., Columbus, OH 43210.
The 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.
Received 6 July 1998; accepted in final form 1 March 1999.
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REFERENCES |
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