acid detection by taste receptor cells

7/31/2019 Acid Detection by Taste Receptor Cells

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Respiration Physiology 129 (2001) 231245

Acid detection by taste receptor cells

John A. DeSimone a,*, Vijay Lyall a, Gerard L. Heck a, George M. Feldman b

a Department of Physiology, Virginia Commonwealth Uni6ersity, Richmond, VA 23298, USAb Department of Medicine, Virginia Commonwealth Uni6ersity, Richmond, VA 23298, USA

c McGuire Veterans Affairs Center, Richmond, VA 23249, USA

Accepted 7 March 2001

Abstract

Sourness is a primary taste quality that evokes an innate rejection response in humans and many other animal

Acidic stimuli are the unique sources of sour taste so a rejection response may serve to discourage ingestion of food

spoiled by acid producing microorganisms. The investigation of mechanisms by which acids excite taste receptor ce

(TRCs) is complicated by wide species variability and within a species, apparently different mechanisms for stron

and weak acids. The problem is further complicated by the fact that the receptor cells are polarized epithelial ce

with different apical and basolateral membrane properties. The cellular mechanisms proposed for acid sensing in tas

cells include, the direct blockage of apical K+ channels by protons, an H+-gated Ca2+ channel, proton conductio

through apical amiloride-blockable Na+ channels, a Cl conductance blocked by NPPB, the activation of t

proton-gated channel, BNC-1, a member of the Na+ channel/degenerin super family, and by stimulus-evoked chang

in intracellular pH. Acid-induced intracellular pH changes appear to be similar to those reported in other mammalia

acid-sensing cells, such as type-I cells of the carotid body, and neurons found in the ventrolateral medulla, nucleuof the solitary tract, the medullary raphe, and the locus coceuleus. Like type-I carotid body cells and brainste

neurons, isolated TRCs demonstrate a linear relationship between intracellular pH (pH i) and extracellular pH (pH

with slope, DpHi/DpHo near unity. Acid-sensing cells also appear to regulate pH i when intracellular pH changes occu

under iso-extracellular pH conditions, but fail to regulate their pH when pHi changes are induced by decreasin

extracellular pH. We shall discuss the current status of proposed acid-sensing taste mechanisms, emphasizin

pH-tracking in receptor cells. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Acid base, acid detection; Amphibians, frog, salamander; Channels, ion, pH-gated; Mammals, rodents, primat

humans; Perception, taste, acid, submodalities; Receptor, taste detection; Taste transduction, perception

www.elsevier.com/locate/resphys

1. Introduction

Among the chemoreceptors in the oral cavity

a subset that respond vigorously to acids. The

are taste receptor cells (TRCs) that establish th

* Corresponding author. Tel.: +1-804-828-4489; fax: +1-

804-828-7382.

E-mail address: [email protected] (J.A. DeSimone).

0034-5687/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 0 3 4 - 5 6 8 7 ( 0 1 ) 0 0 2 9 3 - 6
mailto:[email protected]:[email protected]


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J.A. DeSimone et al. /Respiration Physiology 129 (2001) 231245232

response patterns in the peripheral gustatory sys-

tem that ultimately produce the conscious percep-

tion of sourness. Sourness is one of the primary

taste sensations (Scott and Plata-Salaman, 1991;

Smith and Frank, 1993). It evokes an innate

rejection response in infants (Beauchamp et al.,

1991) and generally remains aversive to humans

and most other animals throughout life. Aversivebehavior toward sourness is easy to rationalize

considering that acids uniquely comprise the class

of sour stimuli, and acids are potentially harmful

substances. Since each stimulus evoking sour sen-

sations produces dissociable hydrogen ions, it

would at first appear that TRCs are most likely

extracellular pH detectors. On that basis the per-

ception of sourness ought to be a graded function

of stimulus pH. However, this proves not to be

generally true. A poor correlation exists between

the perception of sourness and stimulus pH, afinding that has been repeatedly confirmed in

human psychophysical studies (Richards, 1898;

Liljestrand, 1922; Beatty and Cragg, 1935;

Ganzevles and Kroeze, 1987) and in human stud-

ies where the rate of acid-induced salivary secre-

tion has been used as the index of sourness

(Makhlouf and Blum, 1972). Recordings from

gustatory afferents in rats have also been consis-

tent in failing to show a strong correlation be-

tween stimulus pH and the neural response to

particular acidic stimuli (Beidler, 1967; Beidlerand Gross, 1971; Ogiso et al., 2000). These results

beg the question: What specifically do TRCs sense

when presented with acidic stimuli? In the follow-

ing we will review a surprising diversity of trans-

duction mechanisms that have been proposed for

the sour taste modality and an equally surprising

species variability. The reason for the plethora of

proposed transduction mechanisms is, in part, the

consequence of the high chemical reactivity of

protons. At low concentration protons affect ion

traffic across epithelia at the level of ion channelsin the apical and basolateral cell membranes and

in the paracellular shunt pathways connecting the

cells. In addition they may affect these and other

functional elements (e.g. neutral ion exchangers)

from both outside and inside the cells (Lyall and

Biber, 1994).

2. Sour perception in humans

One of the earliest observations in taste psycho

physics is that acetic acid is perceived as mo

sour than HCl at the same pH, but that HCl

more sour than acetic acid at equimolar concen

trations (Richards, 1898). It was soon recognize

that at least part of this paradox derives from th

fact that HCl is completely dissociated in solutio

while acetic acid is not. Beatty and Cragg (193

used the perceived sourness of HCl to establish

scale against which other acids could be com

pared. On that scale it required lower concentra

tions relative to HCl for the diprotic tartaric ac

to produce the same sourness. For acetic aci

higher concentrations relative to HCl were judge

equisour. They showed, in addition, that sourne

correlated better with titratable acid than wi

pH. This hypothesis was further explored b

Makhlouf and Blum (1972) who used salivaflow rate as the measure of the sour response

humans. In their study of six weak acids, the

concluded that relative potency was determine

by the amount of acid titratable to pH 56. The

also reported a poor correlation between salivar

flow rate and stimulus pH. The salivary flow ra

data were accurately represented as saturab

functions of the acid concentration. The ran

ordering of their parameters, K (acid concentr

tions giving half-maximal salivary flow rates) wa

the same as that found by Beidler (1967) in eletrophysiological recordings from rat chorda tym

pani nerves. Ganzevles and Kroeze (198

compared the perceived sourness of acetic acid

humans under two adaptation conditions. In on

case the subjects tongues were adapted to wate

before tasting acetic acid, and in the second cas

the adapting solutions were dilute HCl solution

with pH values ranging from 2.79 to 3.13. Th

test acetic acid solutions had the same pH valu

as the adapting HCl solutions. The subjects r

ported no difference in the perceived sourness oacetic acid with either water or HCl adaptatio

In addition there were no significant differences

the perceived sourness of the acetic acid solution

compared with the same concentrations of acet

acid buffered to higher pH with sodium acetat

These results indicated that extracellular pH p


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J.A. DeSimone et al. /Respiration Physiology 129 (2001) 231245 2

se could not be the sour stimulus. If it were, the

sour taste of acetic acid would have been com-

pletely eliminated by adaptation in isohydric HCl.

A remarkable speculation appeared in a 1930

paper by Taylor et al. (1930), viz. all acid

solutions which taste equally sour produce the

same pH within the interior of the cell. This is an

area that is currently being investigated and it

appears that the speculation is fundamentally cor-

rect (Feldman et al., 2000; Lyall et al., 2000)

Taylor et al. (1930) also noted a strong correla-

tion between the hydrophobicity of a fatty acid

and its ability to evoke a sensation of sourness.

This was confirmed in subsequent studies by Gar-

dener (1980). The possibility that apical epithelial

Na+ channels or an apical Na+/H+ exchanger

might be involved in human sour taste has been

investigated by evaluating the effects of amiloride

application to the tongues of subjects. In each

case amiloride had no effect on the perception ofsourness due to acid stimulation (Schiffman et al.,

1983; Tennissen and McCutcheon, 1996; Osse-

baard and Smith, 1995).

3. Species differences in sour taste mechanisms

Insights into the cellular mechanisms of ac

taste reception have been gleaned from a varie

of animal models. Surprisingly many of the pro

posed mechanisms derived from studies on

given species fail to generalize fully to other sp

cies (Table 1). For that reason we will discu

these mechanisms according to species.

3.1. Necturus

The salamander, Necturus maculosus, has un

usually large cells, a characteristic that has mad

it a favorite subject for intracellular recording

Kinnamon and Roper (1988) used an isolate

lingual epithelial preparation to make recording

from TRCs. The cells were stimulated focally nea

the taste pore by applying pressure to an extrace

lular pipette containing taste stimuli. Applicatioof KCl to the taste pore region produced a depo

larizing intracellular potential and a decrease

input resistance relative to the resting potenti

Table 1

Technique Acid-effectsPreparationReferenceSpecies

Kinnamon andNecturus Isolated lingual Microelectrodes Citric acid blocked apical K+ conductance

Roper, 1988 epithelium

Kinnamon etNecturus Isolated TRCs Acid blocked K+ channels localized in the apicalLoose-patch

recordingsal., 1988 membrane

Isolated lingual Microelectrodes HCl depolarized TRCs via apical H+ gatedMiyamoto etBullfrog

al., 1988 preparations channels permeable to Ca2+ and Na+

Okada et al., Microelectrodes DCCD blocked Acetic acid induced depolarizationBullfrog Isolated lingual

preparations1993

Patch-clampPolarized taste NPPB blocked citric acid induced depolarizationMiyamoto etMouse

bud preparationsal., 1998

In vivo CTDeSimone et Voltage-clamp CT responses to HCl are amiloride-and voltageRat

insensitiveal., 1995 recording

Acid-induced decrease in TRC pHi is amilorideTRC pHiIsolated TRCsLyall et al.,Rat

1997 insensitiveIntact lingualGilbertson et Loose patchHamster Amiloride blocked citric acid-induced current

epitheliumal., 1992 clamp transients in TRCs

Stewart et al., TRC pHiIsolated TRCs Acid-induced decrease in TRC pHi isHamster

amiloride-insensitive1998 measurement

In vivo CT Single unitHellekant et al.,Monkey/Chimpan- N-fibers respond poorly while H-fibers respond

zee 1997a,b recordings best to acid stimulationrecordings


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(50 to 60 mV) and baseline resistance, respec-

tively. When the resting potential was made slightly

hyperpolarizing by current injection, the KCl-in-

duced depolarization was often accompanied by a

single action potential. The KCl-induced depolar-

ization was blocked by bath application of TEA.

Application of citric acid focally also caused a

depolarizing potential, often with an initial burst ofaction potentials, but in this case with increasing

input resistance. TEA also blocked the citric acid

responses. In a subsequent study Kinnamon et al.

(1988) used a loose-patch recording method on

isolated Necturus TRCs to map the distribution of

voltage-gated K+ channels along the cell surface.

They concluded that the K+ conductance was

about 50-fold greater on the apical membrane

relative to the basolateral membrane, suggesting

that voltage-gated K+ channels may then have a

role in the detection of K+

ions and acids. Inside-out and outside-out patches from the apical mem-

brane confirmed the high K+-selectivity of the

apical membrane (PK/PNa=28) (Cummings and

Kinnamon, 1992). The acid block of the K+

channels occurred only when acid was added to the

bath perfusate with the channels in the outside-out

configuration. The results indicate that for Nectu-

rus the apical membrane provides a high density of

voltage-gated K+ channels that are conductive

under resting conditions. These serve as chemical

transducers for K+

and also for H+

ions becausethe latter are effective blockers of K+ channels. It

appears, however, that the apical membranes of

TRCs of many other species do not contain potas-

sium channels (Miyamoto et al., 1996, 1998; Ye et

al., 1994; Avenet and Lindemann, 1991 Furue and

Yoshii, 1997) so it is unlikely that this mechanism

generalizes extensively. It should be noted, how-

ever, that isolated TRCs from the tiger salamander

(Ambystoma tigrinum) do show evidence of

voltage-gated K+ channels that can be blocked by

citric and acetic acids (Sugimoto and Teeter, 1991)in a manner analogous to those of Necturus.

However, it is unknown if these channels also are

concentrated in the apical membranes of the taste

cells. The status of the K+-channel block mecha-

nism for the tiger salamander is, therefore, not as

clear cut as in the case of Necturus.

3.2. Frog

Miyamoto et al. (1988) made intracellul

recordings from the bullfrog (Rana catesbetana

using a preparation that permitted control of th

composition of the interstitial fluid as well as th

lingual superficial fluid. Applying HCl to th

tongue produced a dose-dependent depolarizatio

of the intracellular potential with threshold

0.01 mM. Depolarization was accompanied by

decrease in input resistance. The followin

changes in the composition of the interstitial flu

had no effect on the depolarization evoked by

mM HCl: substituting choline for Na+, removin

Ca2+, substituting choline for Na+ and removin

Ca2+ at the same time, substituting isethiona

for Cl, increasing K+ to 100 mM, or addin

tetrodotoxin. From these results they conclude

that the HCl-induced receptor potential does no

depend on changes in ionic conductances locateon the basolateral membranes of the TRCs. I

contrast to the effects of ion substitutions on th

basolateral compartment, removing Ca from th

lingual surface perfusate the amplitude of the HC

depolarizing potential was inhibited by 48%, an

removing Na reduced it by 63%. The addition

Cd2+ or Co2+ inhibited the HCl response. I

creasing the Ca2+ concentration to 10 or 20 mM

significantly increased the HCl-induced depola

ization and made further reductions in input resi

tance. Measuring the response as a function Ca2+ concentration with and without Na+ an

making the inverse measurements of response as

function of Na+ concentration with and withou

Ca2+ gave in each case competitive inhibitio

kinetics. The reversal potential for the HCl-in

duced depolarization was estimated to lie betwee

+60 and +90 mV, a range bracketing the Ca2

and Na+ equilibrium potentials. The authors con

cluded that in the frog a major part of the aci

induced depolarizing potential (putative recept

potential) occurred through proton-gated iochannels in the apical membrane permeable

Ca2+ and Na+. In a subsequent study Okada

al. (1993) showed that in the frog the depolarizin

potential due to acetic acid could be blocked wit

0.1 mM N,N%-dicyclohexylcarbodiimide (DCCD

an inhibitor of the channel component of th


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V-type proton pump (Lauger, 1991). The view

that emerges from these studies is that both a

proton-gated ion channel and a proton pump are

the membrane transducers for acids in frogs.

Frog TRCs also have apical membrane K+

channels. A 80 pS channel is found exclusively in

the apical region while a 40 pS K+ channel is

found at highest density in that region (Fujiyama

et al., 1994). Whether these channels have a role

in acid sensing is presently unclear. In a more

recent study Kolesnikov and Bobkov (2000) have

made patch clamp recordings from TRCs of Rana

temporaria. Their data suggested that extracellular

K+ may serve as a ligand activating ionic chan-

nels (potassium-activated (PA) channels). The

influence of different ions on the PA current

reversal potential indicated that the responsible

channels are mainly permeable to K+ and H+.

Relative permeabilities were estimated as PH/

PK=3600. They reported that external Kmarkedly increased the sensitivity of isolated

TRCs to bath solution pH due to the activation

of the PA channels suggesting their role in sour

transduction. This interesting possibility will

doubtlessly be further persued.

3.3. Mouse

Miyamoto et al. (1998) made patch clamp

recordings from mouse (C57BL/6 and BALN/c)

TRCs using a preparation that preserves the po-larity of the taste bud. Injection of elastase fol-

lowed by incubaton allowed for the removal of

the lingual epithelium from the underlying muscle

with the intact fungiform taste buds still attached

to the epithelial sheet at their apical poles.

Recordings were made from the basolateral mem-

brane using the whole cell path clamp method

while the taste bud was held in position by a

slight negative pressure applied to a narrow strip

of epithelial tissue. The preparation was con-

stantly perfused with Ringers solution made toflow from the submucosal side toward the mu-

cosal side. Stimulation pipettes were positioned

near the taste pore. Under pressure a small vol-

ume of stimulus could be sent toward the pore

where it could interact with the apical processes of

the TRCs before the counter flow of Ringers

solution removed it. Results showed that 43% o

the cells responded to 25 mM citric acid with

depolarization of 1030 mV from the restin

potential. The acid-induced depolarization w

partially suppressed reversibly by 5-nitro 2-(

phenylpropylamino)-benzoic acid (NPPB) a Cl

channel blocker, suggesting the participation of

Cl flux in acid detection by TRCs. A possib

role for Cl in acid detection by taste recepto

was suggested by an earlier study on the transep

ithelial short-circuit current across isolated canin

lingual epithelium (Simon and Garvin, 1985

Miyamoto et al. (1998) localized the NPPB-sens

tive site to the basolateral side of the taste bu

The NPPB-sensitive part of the current had

reversal potential of approximately 0 mV, whic

was close to the Cl reversal potential of 4

mV. The NPPB-insensitive current had a revers

potential of +35 mV. Since the citric acid solu

tions were Na+ free, this suggests that in additioto activating a basolateral Cl conductance, a

apical membrane cation-selective channel perm

able to Ca2+, H+, or K+ is also activated. Bot

NPPB-sensitive and NPPB-insensitive respons

caused an increase in conductance. However,

an earlier study in mouse where impalement m

croelectrode methods were used, two cell popul

tions emerged (Tonosaki and Funakoshi, 1984

In one cell type HCl-induced depolarization o

curred with increased conductance while in th

other the conductance decreased. The reason fothe apparent discrepancy may lie in the differen

recording methods and stimulating solution com

positions employed.

3.4. Rats

Rats have been the preferred mammalian mod

for gustatory electrophysiology. Numerou

recordings have been made from the rat anterio

lingual receptive field innervated by the chord

tympani (CT). Beidler (1967) made a comparativstudy of the CT response to 20 acids. The acid

were rank ordered according to the concentratio

required to give the same magnitude CT respon

as that obtained with 5 mM HCl. It was apparen

that the pH of the stimulus per se is not th

determining factor in the ranking. For example,


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required just 10 mM succinic acid to give the

same response as 150 mM butyric acid, but the

former had a pH of 3.08 while the latter had a pH

of 2.84. The poor correlation between CT re-

sponse and stimulus pH was further investigated

by comparing responses to 0.1 M acetic acid and

0.1 M acetic acid buffered with 0.01 M sodium

acetate. The responses were essentially the same

even though the pH of the former was 2.88 and

that of the latter 3.76. A similar study has recently

been done by Ogiso et al. (2000). They obtained

the CT response to acetic acid+Tris-acetate mix-

tures at a constant total concentration of 0.1 M

over the pH range 37. At every pH the responses

were greater than responses to HCl at the same

pH. The authors concluded that the undissociated

form of acetic acid must be a major factor in the

response to acetic acid, since the free proton

response (represented by the HCl response)

amounted to only about 25% of the total responseto acetic acid at pH 3. With impalement mi-

croelectrodes, studies of rat TRCs have yielded

evidence for acid-induced depolarization with in-

creasing conductance (Ozeki, 1971) and for depo-

larization with decreasing conductance (Sato and

Beidler, 1982), results similar to the mouse.

DeSimone et al. (1995) obtained rat CT re-

sponses to NaCl and HCl with the stimulated part

of the anterior lingual receptive field under

voltage clamp. Responses to NaCl were enhanced

at submucosa negative clamp voltages and sup-pressed at submucosa positive voltages. NaCl re-

sponses were also amiloride-sensitive. In contrast

responses to HCl were voltage and amiloride-in-

sensitive. The results support the view that taste

transduction for NaCl is mediated through apical

membrane epithelial sodium channels (ENaC)

(Heck et al., 1984; Stewart et al., 1995; Kretz et

al., 1999). However, the lack of amiloride sensitiv-

ity in the HCI responses indicated that ENaC is

not involved in acid sensing. The lack of

amiloride sensitivity in rat chorda tympani re-sponses to HCl has also been noted in single unit

recordings (Ninomiya and Funakoshi, 1988).

Moreover, the lack of voltage sensitivity suggested

that the apical membrane of the TRCs is not very

H+ permeable. Recordings of the transepithelial

potential during HCl stimulation showed an in-

creasing submucosa positive potential at the ons

of the HCl that then reached a plateau. Howeve

upon washout the potential did not return

prestimulus levels, but rather became more pos

tive than during stimulation. This was consisten

with a change in the ion-selectivity of the parace

lular shunts, the low resistance pathways throug

the tight junctions between the TRCs. The su

mucosa positive direction of the change suggeste

that upon HCl stimulation the shunts were con

verted from cation to anion selective by diffusin

protons. This, in turn, suggested that proton

stimulate TRCs from the basolateral side.

The lack of an inhibitory amiloride effect o

the chorda tympani response to acids indicat

that acid sensing in the anterior lingual taste bud

(i.e. taste buds in fungiform papillae) does n

involve H+-gated ion channels from the Na

channel/degenerin family. A study by Ugawa

al. (1998), however, demonstrates that the dgenerin, MDEG-1, is present in the circumvalla

papilla TRCs of the rat. They screened a cDN

library from rat circumvallate papillae for mem

bers of the degenerin cation channel family an

obtained 18 cDNA clones. When pooled cRN

corresponding to a mixture of these clones w

expressed in Xenopus oocytes, no amiloride sens

tive current was detected at pH 7.5 but at a

extracelluar pH of 5.5 a large inward curre

developed that was partially blocked b

amiloride. Expressing the clones individualshowed that the low pH-induced amiloride-sens

tive current was the unique property of MDEG-

Ugawa et al. (1998) also showed that MDEG

mRNA is found in circumvallate TRCs and th

MDEG-1 immunoreactivity is found only

TRCs. Mindful of the fact that taste studies sho

that acetic acid is a stronger sour stimulus tha

HCl at the same pH, they compared the inwar

currents across Xenopus oocyte membranes pr

duced by HCl and acetic acid at pH 5.0. Acet

acid produced the greater current. While thwould appear to confirm that MDEG-1 is a prob

able sour taste receptor, there remain importa

issues to be resolved. First, the H+-gated mem

bers of the Na+ channel/degenerin family a

activated by extracellular (not intracellular) p

(Waldmann et al., 1997). That being the case it


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difficult to explain why current flow through

MDEG-1 for HCl and acetic acid in the oocytes

would be different since the extracellular pH was

the same for both of them. The results are more

consistent with intracellular pH gating the current

magnitude (cf Feldman et al., 2000; Lyall et al.,

2000, and below). Second the acid stimuli were

presented in solutions having the usual concentra-

tions of salts consistent with extracellular fluid

(i.e. Na+ 140 mM) so that current carriers for

MDEG-1 would be available. However, neural

responses to acids are readily obtained in the

absence of salts (Beidler, 1967), an observation

that puts into doubt that MDEG-1 functions in

situ as a sour taste receptor protein (see also

Kinnamon et al., 2000).

3.5. Hamster

The hamster chorda tympani is more sensitiveto saccharides than that of the rat and for that

reason the hamster taste system has also been well

studied (Frank et al., 1988). For acids and salts,

however, the chorda tympani response of the

hamster is similar to that of the rat. The inte-

grated chorda lympani response of the hamster to

NaCl is strongly inhibited by amiloride, and like

the rat, the integrated neural response to HCl is

affected little, if at all, by amiloride treatment

(Herness, 1987; Hettinger and Frank, 1990; Stew-

art et al., 1998). Consistent with this, recordingsfrom neurons in the hamster nucleus of the soli-

tary tract (NST), the first postsynaptic locus in

the gustatory neuraxis, show no amiloride sensi-

tivity to lingual stimulation with HCl (Smith et

al., 1996). A detailed study of the effect of

amiloride and its analogs on hamster chorda tym-

pani responses to HC1 was made by Stewart et al.

(1998). The amiloride analog, benzamil, a more

specific blocker of ENaC than amiloride, was a

potent blocker of the chorda tympani response to

NaCl, but had no effect on the response to 1 mMHCl. Benzamils effect on responses to 10 mM

HCI were not statistically significant. However,

three preparations gave unusually large responses

to 10 mM HCl (up to twice the mean response

level). In these cases benzamil caused up to 50%

suppression, but in no cases was amiloride a

blocker of the HCl response at either 1 or 10 mM

Methylisobutylamiloride, a blocker of the Na+

H+ exchanger, had no effect on either respons

to NaCl or HCl. Stewart et al. (1998) al

recorded the responses to NaCl and HCl und

lingual voltage clamp conditions. Similar to th

rat, the chorda tympani responses to NaCl we

enhanced at submucosa negative clamp volta

(60 mV) and suppressed at positive voltag

(+60 mV). Responses to both 1 and 10 mM HC

were clamp voltage insensitive, i.e. like the ra

responses to HCl do not appear to involve mem

bers of the Na channel/degenerin family in th

apical membranes of TRCs. A reinvestigation o

the effect of amiloride on the response to HCl

the NTS indicated that amiloride had no effect o

the response in HCl-best single units (i.e. uni

firing with highest frequency to acids) (Bought

and Smith, 1998). However, in NaCl-best unit

amiloride inhibited responses to NaCl, HCl, ancitric acid. On this basis the authors suggest tha

coding for sour may involve inputs to the NS

from both HCl-best and NaCl-best afferen

(Boughter and Smith, 1998). One possible wa

that might occur is for H+ ions to stimula

receptor cells through the Na+ channel/degener

family. Other explanations involve possib

branching of chorda tympani inputs at the NS

level. A final resolution of this point awaits fu

ther research.

Evidence for a direct role for ENaC in hamsteacid taste responses was obtained by Gilbertson

al. (1992, 1993). Gilbertson et al. (1992) used

recording technique introduced by Avenet an

Lindemann (1991). This consisted of a pipett

electrode that fit over a single fungiform papil

on the dorsal surface of an excised hamst

tongue. The pipette-electrode was held in place b

weak vacuum. An agar bridge recording electrod

was placed in the posterior muscle exposed by th

cut to remove the tongue. The pipette-electrod

served as ground. Various taste stimuli were applied through an inflow tube that traveled t

within 50 mm of the patch pipette opening to th

papilla surface. Recordings were made using

standard path clamp amplifier and consisted

stimulus evoked currents with the potential b

tween the electrodes held at zero voltage-clam


8/15


NaCl and citric acid each produced rapid current

transients (named action currents). Citric acid at

8 mM (pH 2.6) produced action currents of 710

pA at a frequency of 12 Hz. The frequency

increased in a dose-dependent manner with de-

creasing pH with threshold between pH 5 and 6.

The action currents to both NaCl and citric acid

were blocked by amiloride in a dose dependent

manner (Ki for citric acid and NaCl were 2.4 and

1.9 mM, respectively). The results suggested that

perhaps both Na+ and H+ excite TRCs through

ENaC. This is difficult to determine from these

studies alone, since the biophysical interpretation

of the action currents is unclear. In a subsequent

paper, however, Gilbertson et al. (1993) used

whole cell patch clamp methods on isolated ham-

ster taste buds to observe the effects of citric acid.

They applied citric acid, using a pressure injection

pipette, to the isolated taste buds in a Na+ and

K+-free medium. Citric acid application gave aninward current at clamp voltages between 80

mV and +20 mV. The reversal potential was

estimated to be near the equilibrium potential for

H+ at extracellular pH 4.5. The calculation of the

H+ equilibrium potential assumed a constant in-

tracellular pH of 7.2. However, in a medium of

pH 4.5 the intracellular pH will not remain at 7.2,

but will assume a much lower pH (as much as

12 pH units lower) due to the pH-tracking prop-

erty of TRCs (Lyall et al., 1997; Stewart et al.,

1998). The H+ equilibrium potential may be,therefore, much lower than the reversal potential,

suggesting that H+ ion may not be the sole

current carrier. The inward currents were, how-

ever, blocked by 30 mM amiloride, suggesting a

role for the Na+ channel/degenerin family in acid

detection in isolated taste cells. However, chorda

tympani responses to acids are not amiloride sen-

sitive. This suggests that the putative proton-con-

ducting channels are present in the basolateral

membranes of TRCs. If these channels are func-

tional in situ, they may not be blocked by topicalapplication of amiloride on the lingual surface,

since amiloride will have to reach basolateral

membrane sites through the paracellular pathway

where amiloride transport is likely to be restricted

by low permeability. The functionality of the

channels would depend on the acid stimuli reach-

ing them by the paracellular route, a possibili

suggested by the behavior of the transepitheli

potential in the presence of acids recorded in sit

(cf DeSimone et al., 1995; Stewart et al., 1998)

3.6. Rhesus monkey and chimpanzee

Not surprisingly primates appear to be the be

animal model for human taste. Hellekant et a

(1997a) have recorded taste single unit activity

both the chorda tympani and glossopharynge

nerves of the rhesus monkey (Macaca mulatta

Of the chorda tympani units investigated, 15 uni

gave best responses to NaCl and were designate

the N cluster. However, N cluster fibers al

responded moderately well to citric acid and a

partic acid. Eight of the fibers, designated the

cluster, gave best responses to citric acid an

aspartic acid. The 16 S cluster fibers designated a

S fibers, responded best to sweet-tasting stimuwhich included saccharides (sucrose, fructose, glu

cose, and galactose), and many compounds suc

as aspartame, saccharine, dulcin, acesulfame-K

D-tryptophan and others which are also swe

tasting to humans. The S cluster also responde

well to aspartic acid and citric acid. Four fiber

designated the Q cluster, gave best responses

quinine and other substances with some bitt

attributes. These fibers did not respond well t

acids. It is in the near congruence of the patter

of sweet sensitivity between many primates anhumans that the primate model excels. Therefor

it appears that neural input evoked by acids fro

the chorda tympani to the NST is carried main

by H-cluster fibers with some input from bot

N-and S-cluster fibers, but with virtually no inpu

from the Q-cluster. A similar analysis b

Hellekant et al. (1997a,b) on the taste respons

from the glossopharyngeal nerve revealed a M

cluster that gave largest responses to monosodiu

glutamate with more moderate responses to citr

acid, and aspartic acid, a Q cluster that respondewell to quinine and other bitter tasting com

pounds, but not to acids, and a S-cluster th

responded to sweeteners but not to acids. So tas

information about acids from posterior lingu

taste receptive fields travels with fibers that al

respond well to glutamate.


9/15


Hellekant et al. (1997b) have also made single

unit recordings from the chorda tympani of the

chimpanzee (Pan troglodytes). Of 49 units sur-

veyed, only 2 gave best responses to acids. Similar

to the monkey a large proportion of the units

(47%) were found to be sucrose-best. The S-clus-

ter did not respond to acids. Twenty-nine percent

of fibers were NaCl-best units. This N-cluster was

itself composed of three subclusters, (1) a Na-sub-

cluster; (2) a NaK subcluster; and; (3) a M-sub-

cluster. The Na-subcluster responded well to

NaCl and LiCl, but not to KCl and acids. The

Na+ and Li+ responses were strongly inhibited

by amiloride. The NaK subcluster responded

about equally to NaCl and KCl, and also gave

small responses to acids. The M-subcluster re-

sponded well to monosodium glutamate and gave

low responses to acids and some sweet-tasting

stimuli. Quinine-best fibers accounted for 20% of

the population. The Q-cluster fibers were, how-ever, more broadly tuned than NaCl-best or su-

crose-best fibers and included robust responses to

acids as well as bitter-tasting compounds. Unlike

the rhesus monkey, the chimpanzee chorda tym-

pani lacks a H-cluster, so most of the acid-evoked

responses appear to travel with the Q-cluster.

This, therefore, contrasts sharply with the monkey

in which the Q-cluster was essentially acid-

insensitive.

4. Evidence supporting a role for intracellular pH

decrease in acid taste

Undissociated acid molecules as well as free

protons interact directly with the apical mem-

branes of TRCs. This interaction docs not seem to

require specific H+ receptors on the apical mem-

branes of TRCs. This conclusion is based on the

observation that CT nerve responses to acids are

not influenced by pretreatment of the tongue sur-

face with protease (Ogiso et al., 2000) whereasthat to sucrose are reduced effectively (Hiji, 1975).

This implies that unlike sucrose sensing, mem-

brane receptors or other surface proteins are not

involved in acid detection. However, acid-induced

effects on TRCs may not be restricted to the

apical cell membrane. DeSimone et al. (1995)

have presented data suggesting that protons pa

through the paracellular pathway and a

buffered by the fixed anionic sites as they do s

This raises the possibility that stimulus proton

may also interact with the basolateral membrane

of TRCs.

It is not clear whether TRCs respond directly t

a change in extracellular pH (pHo) or if a chang

in intracellular pH (pHi) of TRCs is involved i

acid sensing. Several studies have identified dire

effects of acids on TRC membrane conductance

(Miyamoto et al., 1988, 1998; Gilbertson et a

1992, 1993; Ugawa et al., 1998; Kolesnikov an

Bobkov, 2000; Kinnamon et al., 2000; Steven

and Lindemann, 2000). The underlying hypothes

in these studies is that pH-gated changes in mem

brane conductances depolarize the receptor poten

tial in TRCs leading to the release

neurotransmitter(s) and excitation of the tas

nerves.As discussed in Section 2, the perception

sour in the case of weak organic acids is not

graded function of pHo. This has led to the h

pothesis that weak organic acids can penetra

TRC membranes in the undissociated form an

may dissociate intracellularly leading to cytopla

mic acidification. This hypothesis is supported b

the observation that a strong correlation exis

between lipid solubility (i.e. membrane permeabi

ity) and sourness of weak acids (Gardener, 1980

In addition, cytoplasmic acidification has beereported to reduce the electrical coupling betwee

TRCs in Necturus (Bigiani and Roper, 1994) an

to impair electrical communications via gap jun

tions (Spray and Bennett, 1985; Spray et a

1981).

To investigate the possibility that changes

TRC pHi are indeed involved in acid sensin

Lyall et al. (1997) and Stewart et al. (1998) mon

tored the relationship between pHo and pHiisolated rat and hamster circumvallate and fung

form taste bud fragments and single TRCs. Isingle isolated TRCs altering pHo by changes

external H+ ion concentration caused parall

changes in TRC pHi (Fig. 1). The changes in TR

pHi were independent of the anion concentratio

in the external buffer system. For example,

stimulus solutions containing the CO2/HCO


10/15


Fig. 1. Pseudo-color ratio images of a single TRC loaded with BCECF. (A) Shows an optical section through a single rat fungifor

TRC. Scale bar, 5 mm. (B) The same optical section is shown as a BCECF fluorescence image, excited at the pH-sensiti

wavelength, 490 nm. Note that the dye distribution pattern is a reflection of the irregular shape of the TRC. The same optical secti

is shown as a ratio of BCECF fluorescence pseudo-color images when excited alternately at 490 and 440 nm at the steady state pH

of 6.6 (C) and 7.7(D). Note that a decrease in pHo induced a parallel decrease in pHi both in the soma and apical process. Th

figure is reprinted from Lyall et al. (1997) with permission from The American Physiological Society.

buffer system, the changes in TRC pHi demon-strated a linear relationship to pHo whether pHowas altered by varying the PCO

2at constant exter-

nal HCO3, concentration or if pHo was altered

by varying the HCO3 concentration at constant

PCO2. The changes in TRC pHi were rapid and

sustained (Fig. 2). No spontaneous recovery of

pHi was observed as long as pHo was maintaineat acidic pH. The relationship between pHo an

TRC pHi was observed to be linear with a slop

near unity. Thus isolated TRCs demonstrate fou

main characteristics of a pH sensory cell. Firs

changes in pHo induce a proportionate respon

in pHi. Second, the changes in TRC pHi a


11/15


rapid. Third, the changes in TRC pHi are main-

tained during the acidic stimulus. Fourth, the

changes in pHi were observed in all regions of

interest in the taste bud fragment suggesting that

a significant number of TRCs in a taste bud are

involved in pH tracking (Stewart et al., 1998). The

sustained changes in pHi seem to be a common

characteristic of pH-chemosensory cells (Buckleret al., 1991; Ritucci et al., 1997).

The above studies were made in isolated non-

polarized TRCs. In these experiments both apical

as well as basolateral membranes of TRCs were

exposed to changes in pHo. However, in the intact

lingual epithelium, TRCs maintain their natural

polarity and are only exposed to acidic stimuli o

the apical side. Therefore, before a firm conclu

sion can be made regarding pHi as the signal fo

acid transduction, measurement of TRC pHi mu

be made in polarized TRC preparations in whic

the apical and basolateral membranes remain se

regated and the TRCs are exposed to acidic stim

uli only on the apical membrane. In ou

preliminary studies (Feldman et al., 2000; Lyall

al., 2000) using an isolated lingual epitheliu

preparation mounted in a special microscop

chamber (Chu et al., 1995) apical application o

acidic stimuli induced sustained decreases in TR

pHi. These data support the conclusion that

decrease in TRC pHi is a first essential step

sour taste transduction.

The studies of TRCs in the CO2/HCO3 buff

system indicate that to achieve a decrease in TR

pHi it is not necessary to have changes in pHo.

is the entry of acid equivalents that decreasTRC pHi. For example increasing the concentr

tion of both CO2 and HCO3, simultaneous

while maintaining constant pHo decreased th

TRC pHi (Lyall et al., 1997). Further a chang

from a HEPES-buffered solution (pH 7.4) to

solution buffered with 72 mM HCO3; and 10

CO2 (pH 7.4) caused a decrease in TRC pH

(Lyall et al., unpublished observations). In TRC

the hydration of CO2 to form H2CO3 is catalyze

by intracellular carbonic anhydrase (Komai et a

1994; Daikoku et al., 1999; Goto et al., 2000). CO2-induced decrease in pHi has been shown t

be the primary signal for the activation

chemosensory neurons during hypercapnia (Ri

ucci et al., 1997, 1998; Wiemann et al., 199

Dean et al., 1990; Pineda and Aghajanian, 199

Buckler and Vaughan-Jones, 1994).

The sustained changes in TRC pHi do n

imply that TRCs are incapable of regulating pH

under resting conditions. Lyall et al. (1997), Stew

art et al. (1998) reported that at constant pH

exposing the cells to short pulses of Na-acetatNa-propionate, NH4Cl or PCO

2acidified TRC

However, under these conditions TRCs demo

strated rapid spontaneous recovery of pHi to the

resting values (Fig. 3). These results suggest th

under resting conditions TRCs behave like man

other cells and regulate pHi. Consequently, lik

Fig. 2. Effect of changing pHo on pHi. (A) A circumvallate

TBF was perfused with HEPES-buffered Tyrode solution ofpH 7.4, 6.7, and 7.9. The top horizontal bar represents the

periods during which the TBF was perfused with solutions of

different pHs. (B) The steady-state relationship between pHoand pHi in five TBFs similar to that of (A). The line of best fit

determined by linear regression (S=0.9790.02; r=0.9969

0.002). This figure is reprinted from Lyall et al. (1997) with

permission from The American Physiological Society.


12/15


Fig. 3. Effect of weak acids. A fungi form TBF initially

perfused with HEPES-buffered Tyrode solution was perfused

with a similar Tyrode solution containing 30 mM sodium

propionate (Na Pro). The top horizontal bar represents the

period during which the TBF was exposed to sodium propi-

onate. This figure is reprinted from Lyall et al. (1997) with

permission from The American Physiological Society.

5. Summary

In summary there is a remarkable diversity o

mechanisms that appear to have a role in th

transduction of the sour taste stimulus at the lev

of the TRCs. In some cases the mechanisms ap

pear to be species specific. Most of these mech

nisms involve putative cell membrane ent

pathways for H+ ions or ion pathways modulate

by H+ ions. However, weak organic acids ca

permeate cell membranes as undissociate

molecules, suggesting that intracellular p

changes may also be important in acid taste recep

tion. This view is supported by psychophysic

studies that demonstrate a poor correlation b

tween extracellular pH and the perception

sourness in general. Imaging studies on isolate

taste buds show that intracellular pH follow

changes in extracellular pH according to a linea

relation that resembles the characteristics of acisensing cells in the brain and carotid bodie

Recent studies on taste buds still attached to th

lingual epitheliurn, i.e. with maintained epith

lium polarity, show that pH-tracking can be ob

served from the apical side. In the case

mammals further studies are needed to identi

the specific entry pathways for H+ ions in tas

receptor cell membranes that are involved

transduction. While it appears that a decrease

intracellular pH is an important early step

transduction, subsequent steps in the process leading to neurotransmitter release are still obscu

and await additional research for clarification.

Acknowledgements

Supported by NIH grants DC-02422 and DC

00122 and the Department of Veterans Affairs.

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