Transcellular and paracellular pathways in lingual epithelia and their influence in taste...

MICROSCOPY RESEARCH AND TECHNIQUE 26:196-208 (1993)

Transcellular and Paracellular Pathways in Lingual Epithelia and Their Influence in Taste Transduction SIDNEY A. SIMON, VIRGIL F. HOLLAND, DALE J. BENOS, AND GUIDO A. ZAMPIGHI Department of Neurobiology, Duke University, Durham, North Carolina 27710 (S.A.S., V.F.H.); Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294 (D.J.B.); Department of Anatomy and Cell Blology, UCLA School of Medicine, Los Angeles, California 90024 (G.A.Z.)

KEY WORDS

ABSTRACT The lingual epithelium is innervated by special sensory (taste) and general sensory (trigeminal) nerves that transmit information about chemical stimuli introduced into the mouth to the higher brain centers. Understanding the cellular mechanisms involved in eliciting responses from these nerves requires a detailed understanding of the contributions of both the paracellular and transcellular pathways. In this paper we focus on the contribution of these 2 pathways to the responses of salts containing sodium and various organic anions in the presence and absence of amiloride. Electrophysiological recordings from trigeminal nerves, chorda tympani nerves, and isolated lingual epithelia were combined with morphological studies investigating the location (and permeability) of tight junctions, the localization of amiloride-inhibitable channels, and Na-K-ATPase in taste and epithelial cells. Based on these measurements, we conclude that diffusion across tight junctions can modulate chorda tympani and trigeminal responses to sodium- containing salts and rationalize the enhancement of taste responses to saccharides by NaC1.

Tight junctions, Amiloride, Na-K-ATPase, Trigeminal, Chorda tympani

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INTRODUCTION The dorsal surface of the lingual epithelium is rou-

tinely subjected to a wide range of chemical stimuli at different temperatures, osmolalities, and pHs. Re- sponses to these stimuli are transmitted to the CNS via special sensory and general sensory nerves. These nerves provide information about the quantity and quality of the stimuli so that the organism may decide whether particular stimuli will be permitted to enter its digestive system. The special sensory (or taste) pathways involved in the initial events of taste transduction include taste cells located in fungiform, circumvallate, and foliate papillae; branches of the facial, glossopharyngeal, and vagal nerves that synapse with the taste cells, and tight junctions between the cells (Murray, 1971). The general sensory pathway for the anterior of the tongue consists of the lingual epithelium and the lingual branch of the trigeminal nerve.

The paradigm for the initial events in special sensory taste transduction is that chemical stimuli interact either with receptors or channels in microvillar membranes of taste cells, resulting in changes of membrane potential or the concentration of second messenger(s). After a cascade of steps, transmitter(s1 are exocytosed from taste cells and interact with sensory fibers, thus changing their activity. This paradigm is supported by recordings showing that both protons and saccharin inhibit potassium channels on apical taste cells (Behe et al., 1990; Kinnamon et al., 1988a). It is also likely to be strictly correct for taste transduction involving sweet-tasting proteins of several thousand molecular weight since, by their size alone, these compounds are too large to diffuse into taste cells or across tight junctions (Kim et al., 1988; Wright and Diamond, 1969).

However, when considering the response to high concentrations of salts and small nonelectrolytes (e.g., NaC1, glucose, urea, glycerol), the above paradigm should be augmented to include the influence of paracellular pathways. In many tissues, paracellular pathways are the primary route of entry of small electro- lytes and nonelectrolytes into the extracellular space (Diamond and Wright, 1969; Wright and Diamond, 1977). Compounds entering epithelia via this pathway can, in principle, influence taste by interacting directly with taste fibers, altering transport pathways on basolateral membranes, or by altering the resistance of paracellular pathways (Elliott and Simon, 1990). Thus a detailed understanding of taste transduction requires knowledge of both transcellular and paracellular pathways.

Lingual epithelia are innervated by general sensory fibers that may also respond to chemical stimuli. Gen- eral sensory fibers terminate in lingual epithelia, either as “free” nerve endings or in specialized terminals associated with mechanoreceptors (Kruger and Man- tyh, 1989). Trigeminal fibers respond to a wide variety of chemical stimuli, spanning the range from hydro- phobic to hydrophilic (Silver et al., 1986; Sostman and Simon, 1991). It is not fully understood how chemicals introduced into the mouth elicit responses from these fibers, especially since these fibers do not appear to form synapses with epithelial cells. To obtain a better

Received March 1, 1991; accepted in revised form June 1, 1991. Address reprint requests to Dr. Sidney A. Simon, Department of Neurobiology,

Duke University, Durham, NC 27710.

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INFLUENCE OF TIGHT JUNCTIONS IN TASTE 197

understanding of special and general sensory taste transduction, i t is necessary to identify the transcellular and paracellular pathways in and between taste and epithelial cells.

Transcellular pathways are composed of plasma membranes and transport proteins that regulate the entry and exist of compounds into and out of cells. Paracellular pathways consist of tight junctions and the extracellular space between adjacent cells. Infor- mation about these separate pathways has been gathered by several experimental approaches. Patch-clamp- ing taste cells provided information about transport proteins involved with salt, sweet, bitter, and sour taste (Avenet and Lindemann, 1989a,b; Behe et al., 1990). Transport measurements across isolated lingual epithelia provide information about the type and selectivity of trans- and paracellular pathways. Immunocy- tochemical studies have identified a few of the transport proteins and transmitters involved in taste transduction (Finger, 1986; Finger et al., 1990; Kruger and Mantyh, 1989; Silverman and Kruger, 1990; Si- mon et al., 1989a, 1991; Yamasaki et al., 1984). These studies, together with structural studies of nerve-taste cell interactions, permeability of tight junctions, and distribution of gap junctions have lead to a primitive, but integrated, picture of the initial events in taste (Roper, 1989).

There is mounting evidence to suggest that the responses of lingual epithelia to salts containing Na+ with different organic anions is modulated by both trans- and paracellular pathways (Elliott and Simon, 1990; Sostman and Simon, 1991). The transcellular pathways involved in the responses to NaCl have been studied in great detail. Application of the epithelial sodium channel ir:hibitor, amiloride, to the dorsal surface of mammalian tongue decreases the perceived intensity of NaCl about 50% (Schiffman et al., 1983). Recordings from chorda tympani fibers show that amiloride inhibits about 60% of the NaCl response (DeSimone and Ferrell, 1985), thus demonstrating the presence of at least 2 distinct pathways. Evidence obtained from transport across lingual epithelia and chorda tympani responses suggests that Na + enters taste cells through amiloride-sensitive channels and is extruded from them by Na-K-ATPase (Heck et al., 1989). Finally, amiloride-inhibitable currents and channels have been identified in ra t taste cells (Behe et al., 1989, 1990). Despite the vast literature on the electrophysiology of NaCl responses, 2 transport proteins clearly involved in the response of taste cells to NaCl have only recently been identified in lingual epithelia (Simon et al., 1989a, 1991). Here we present immunocytochemical studies that localize amiloride-inhibitable channels and Na-K-ATPase in lingual epithelium.

As described above, the Na' transport pathways across taste cells are now fairly well understood. How- ever, little is known about the C1- transport pathways and the influence of C1- in the responses to NaC1. In- formation regarding the role of the anion in the NaCl response may be obtained by substituting other anions for C1-. For sodium salts containing organic anions, virtually the entire chorda tympani (tonic) response is

inhibited by amiloride (Elliott and Simon, 1990; For- maker and Hill, 1988; Nakamura and Kurihara, 1988). We will present evidence that shows these data are consistent with a paradigm for taste transduction for NaCl requiring amiloride-sensitive channels, Na-K- ATPase, and the diffusion of anions across paracellular pathways between taste cells. We will also show that the stimulation of trigeminal fibers by NaCl arises as a consequence of i t diffusing across tight junctions between epithelial cells.

MATERIALS AND METHODS Integrated Responses From Chorda Tympani

and Trigeminal Nerves Recording. Adult male and female Sprague-Daw-

ley and Zivic-Miller rats (300-350 g) were used. Ani- mals were anesthetized with Na pentobarbital injected IP (initial dose = 50 mg/kg, maintenance dose was one third of the initial dose every hour). Anesthetized an- imals were placed on an animal warmer throughout the experiment, and their body temperature, monitored with a rectal thermistor, was maintained at 36- 38°C.

Chorda Tympani. The chorda tympani nerve was exposed by dissection of the overlying bone and muscle. The nerve was cut where it entered the tympanic bulla, desheathed, and placed on a hook electrode of platinum wire. A stainless-steel syringe needle placed in a nearby tissue served as a reference electrode, and nerve activity was recorded differentially using a Grass P-15 preamplifier. Signals were displayed on a Tektronix oscilloscope, stored on tape using a TEAC audiotape recorder, and played back through an inte- grator (RC circuit with time constant of 0.5 s) onto a Gould chart recorder.

Trigeminal. Following removal of overlying tissue, the lingual nerve was exposed for about 5 mm distal to its exit from the foramen ovale, cut proximally, desheathed, and placed over a silver-wire electrode. The preparation was then covered with Vaseline@ and mineral oil. An indifferent electrode was placed in nearly muscle, and grounding was provided through the headholder. Neural activity was amplified with a differential amplifier (Grass P-151, monitored with an oscilloscope, and stored on tape for later integration (Coulbourn S76-01; time constant, 0.5 s) and display on a Gould chart recorder.

Stimulation (Chorda Tympani). The tongue was drawn by gentle suction through a latex membrane into a glass tongue holder through which stimulus solutions flowed by gravity from an elevated reservoir as described previously (Elliott and Simon, 1990). The flow rate was approximately 2 ml/s. Solutions were maintained at 30°C. The adapting solution was 5 mM HEPES at pH 7.4.

The response to a given stimulus solution was recorded by first adapting the tongue to 50 ml of the adaptation medium, then flowing 50 ml of the stimulus solution over the tongue, and finally rinsing the tongue with 50 ml of adaptation medium. Responses to the next stimulus were recorded after an interval of no less than 2 min. When a stimulus containing 0.1 mM amiloride was being tested, the tongue was adapted to

198 S.A. SIMON ET AL.

50 ml of the adaptation medium, followed by 15 ml of 0.1 mM amiloride in adaptation medium (giving a pre- exposure to amiloride of about 10 s), and finally by 50 ml of the test stimulus plus 0.1 mM amiloride. The tongue was then rinsed in an adaptation medium followed by several hundred milliliters of distilled, deion- ized water. This protocol usually gave recovery of chorda tympani responses to preamiloride levels within 15 min.

The application of 0.5 M NH4C1 elicits a robust amiloride-insensitive response (Formaker and Hill, 1988) and was used as an indicator of general chorda tympani nerve health and quality of recording. The amiloride insensitivity was confirmed in our experiments for concentrations of NH4C1 from 0.1 to 2.0 M. Responses to test stimuli were measured in arbitrary units of amplitude of the integrated response a t 20 s after stimulus onset. Responses were then reported as the ratio between this 20 s value for the test stimulus and the 20 s value for the most recent response to 0.5 M NH4C1. Responses to a range of concentrations of various salts were measured in the absence and presence of 0.1 mM amiloride. During stimulation the tongue was exposed to amiloride for about 40 s to minimize irreversible effects (or a t least those with very long recovery times) of amiloride. Each application of amiloride was followed by 15-20 min of rinsing before testing the next salt concentration in the series.

Stimulation (Trigeminal). Stimulus solutions were gravity-fed to the tongue of the rat. The tongue was inserted through a latex dam into the base of a Y-shaped glass holder. The flow rate was approximately 2 ml/s, as described previously (Sostman and Simon, 1990). In order to control for the mechano- and thermoreceptor activity elicited by flow onset and ther- mal change a t the tongue surface, stimulus sequences involved application of a 50 ml adapting stimulus of distilled water, followed immediately by 50 ml of the test solution. Sequences of 2 successive 50 ml applica- tions of distilled water provided rinsing and also served as additional controls for comparison with the test sequences. These were interspersed at least twice between test stimuli, which, in turn, were separated by a t least 3 min to avoid any sequential response habitua- tion. Solutions were kept a t 29.5 -3O.O0C, the temperature a t which chorda tympani responses to NaCl are maximal (Nakamura and Kurihara, 1988).

Transport Across Isolated Lingual Epithelia Adult dogs were killed by intravenous injection of Na

pentobarbital (70 mg/kg), and the anterior two thirds of their tongues were removed. An excised section of tongue, in which the muscle layer had been dissected away, was placed in an Ussing chamber (area 3.1 cm2) between symmetrical solutions of a modified Krebs- Henseleit (KH) solution a t 35 2 1"C, as described previously (Simon et al., 198913). The composition of the KH solution was 118 mM NaC1, 6 mM KC1, 5.6 mM D-glucose, 1.2 mM MgSO,, 2.0 mM CaCl,, 25 mM NaHCO,, and 1.3 mM NaH2P04. When equilibrated with 95% 02-5% CO,, the pH was 7.4. Experiments using 0.05 M NaCl in 5 mM HEPES were adjusted to pH 7.4. Modulators of transport pathways, such as

100 r I 0.1 mM Amiloride

Time (min)

Fig. 1. Stimulation of short circuit current (Isc) across isolated canine epithelium by 0.5 M NaC1. The lingual epithelium was initially bathed with 0.05 M NaCl, 2 mM HEPES at pH 7.4 on its mucosal side and Krebs-Henseleit buffer on its serosal side. At the indicated time a volume of the mucosal solution was removed and replaced with the same volume of a concentrated NaCl solution to obtain a final concentration of 0.5 M NaCl. After steady state was attained, 0.1 mM amiloride was added to the mucosal solution (arrow), and after some time 1.0 mM ouabain (arrow) was added to the serosal solution.

amiloride, LaCl,, and ouabain, were added from concentrated stock solutions.

Electrical Measurements Measurements of the short circuit current (Isc) and

the open circuit potential (Voc) have been described previously (Simon et al., 1988). Voc is defined with respect to the mucosal solution and Isc has a negative sign when cations flow from the mucosal to serosal solutions or when anions flow from the serosal to mucosal solutions. Voc is measured with calomel electrodes in saturated KC1 solutions interfaced to 1-2% agar bridges containing 0.15 M NaC1. Platinum wires are used as current passing electrodes. These 4 electrodes are connected to a voltage clamp circuit that compensates for the series resistance arising from the electrodes and the KH solutions. The transepithelial resistance (Rt) is obtained by dividing Voc by Isc. Val- ues for Isc or Voc were recorded on a strip chart recorder. The chart recording was subsequently digitized for ease of presentation (Fig. 1).

Electron Microscopy Lanthanum Penetration. The lingual epithelium

and underlying connective tissues were dissected from the anterior two thirds of the tongue and mounted in an Ussing chamber containing symmetrical solutions of bicarbonate-free Krebs-Henseleit buffer at 36°C as described above. After steady-state Isc was reached, 5.0 mM LaC1, was added to either the mucosal or serosal solutions. After steady state was attained, epithelia were removed from the Ussing chambers and incu-


bated in 0.1 M Na cacodylate buffer containing 10 mM Na,CO, for 30 min a t room temperature. Lanthanum is precipitated by this solution into large electron- dense deposits that are easily distinguishable in thin sections. In this manner, electrophysiological measurements regarding information about tight junctional resistance and information about their permeability were obtained from the same specimen. We recognize that by using these methods some organelles of the tissue will not be as well preserved as when the animal is perfused. Tight junctions between epithelial cells nevertheless remain impermeable to lanthanum during these procedures (Holland et al., 1990).

Thin Sections. A single fungiform papilla was dissected from the epithelium and fixed by immersion for 30 min in the following fixative solutions: 1) 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.4 and 0.3% H,O,; b) 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.4; and c) 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.4 containing 1% tannic acid (Zampighi et al., 1988). This primary fixation protocol preserves intercellular junctions, although not necessarily other structures, such as nerve fibers and mitochondria.

Postfixation was carried out in 1% osmium tetroxide in 0.1 M Na cacodylate, pH 7.4, for 90 min at room temperature. The papillae were subsequently rinsed in 0.1 M Na acetate at pH 5.5 and stained in block for 12 hours in 1% uranyl acetate in 0.1 M Na acetate at 4°C. The papillae were then dehydrated in ethanollwater mixtures, passed through 100% ethanol and propylene oxide, and embedded in Polybed 812. Sections having silver-to-gray interference colors were collected on Formvar-coated, carbon-backed, single-hole grids and stained with uranyl acetate and lead citrate solutions. Electron microscopy was performed a t 60 kV in a JEOL 1OOCX electron microscope.

Immunocytochemistry Na-K-ATPase and Amiloride-Sensitive Sodium

Channel Antibodies. Two monoclonal antibodies (#2F and #5), grown in mouse to the catalytic (a) subunit of the Na-K-ATPase, were provided by Dr. D. Fambrough (Department of Biology, The Johns Hop- kins University). These antibodies were prepared against the a-1 isoform of chicken (K.J. Renaud, un- published observations). Given the extremely tight conservation of amino acid sequence between avian and mammalian Na-K-ATPase a-subunit isoforms (Takeyasu et al., 1990), #2F presumably crossreacts only with the mammalian a-1 isoform. Monoclonal antibody #5 (Lebovitz e t al., 1988) binds to all avian a-subunit isoforms (Takeyasu et al., 1989) and is pre- sumed to bind to all mammalian a isoforms. No differences were seen in the behavior of these 2 antibodies in terms of their staining characteristics of canine lingual epithelia. A polyclonal antibody raised in rabbits against purified bovine kidney Na + channel (Tousson et al., 1989) was used to localize the amiloride-sensitive channel.

taste buds in these papillae. Freshly dissected, unfixed circumvallate papillae were immersed in cold PBS buffer containing 20% sucrose, transferred to plastic molds containing Tissue-Tek freezing medium, and fro- zen in liquid nitrogen. Alternatively, circumvallate papillae were initially fixed for 1-3 hours in 4% parafor- maldehyde plus up to 0.1% glutaraldehyde in PBS buffer, and then prepared as in described above. Sec- tions of 7-10 pm thickness were cut in a Reichert model 2800 FrigoCut cryostat. They were retrieved on subbed slides and stored at -80°C until processed.

Staining. Slides were unfixed or fixed tissue sections were dried in air. Slides with unfixed tissue were washed in PBS buffer for 10 min and either processed directly or fixed in acetone for 5-10 min and washed in PBS buffer for 10 min. To reduce nonspecific back- ground staining, the sections were incubated for 30 rnin with 1-10% horse serum and 2% BSA in PBS buffer. The blocking solution was blotted and the sections were incubated with primary antibody a t dilu- tions up to 1:1,000 in PBS buffer in 2% BSA at room temperature for 30-60 min. The sections were washed with PBS and incubated for 30 min (at room temperature) with a solution containing biotinylated secondary antibody at a dilution of 1:lOO (Vector Laboratories, Burlingame, CAI. The sections were again washed with PBS buffer and again incubated for 30 min in a solution containing biotin-avidin peroxidase complex at a 1:lOO dilution (Vector Laboratories Burlingame, CA). After washing for 10 min in PBS buffer, sections were treated for 6-10 min (in the dark) with 0.1% di- aminobenzidine tetrahydrochloride (DAB), 15 ml fresh 3% H,O,, and 5 ml PBS buffer (pH 7.4). This procedure was followed by a 10 min wash in distilled water and counterstaining for 30-60 min with methyl green. The sections were subsequently washed in distilled water and dehydrated in ethanol. The alcohol was replaced by xylene through a series of exchanges, and the tissues were mounted under coverslips with mounting medium. The same procedures were followed as described for the Na-K-ATPase, except that a FITC-labeled anti- rabbit secondary antibody obtained from Sigma Chem- ical Company was used to visualize the primary antibody.

Controls For the localization of Na-K-ATPase, negative con-

trols consisted of substituting the primary antibody for BSA or horse serum, or omitting the secondary antibody. Dog kidney proximal tubules were used as a positive control to judge the efficacy of the primary antibody and the reliability of the experimental procedure. For the amiloride-sensitive channel, negative controls consisted of substituting for the primary antibody BSA or goat serum, or omitting the secondary antibody. Dog kidney collecting ducts were used as positive controls.

RESULTS Electrophysiological Studies of Amiloride- and

-0uabaii-Inhibitable Pathways Tissue Preparation for Immunocytochemistry.

For immunocytochemical studies dog circumvallate papillae were used because of the high concentration of

Isolated dorsal canine lingual epithelium adapted to 0.05 M NaCl produces a large and rapid increase in inward current upon the addition of hypo-osmotic con-


NaCl Na acetate Before +Aniiloride A l l e t . Before +Amiluride After

- lmin

Fig. 2. Chorda tympani responses to NaCl and Na acetate. Inte- grated responses from the whole chorda tympani nerve to 0.5 M NaCl and 0.5 M Na acetate at pH 7.4 are illustrated. The left-hand panels show the controls, the middle panels show the response after incubation of the tongue with 0.1 mM amiloride. and the right-hand panels show the reversibility of the responses. (Adapted from Elliott and Simon, 1990.)

centrations of NaCl to the mucosal solution (Fig. 1; DeSimone et al., 1984; Simon and Garvin, 1985). This current is inhibited by about 60% upon the addition of 0.1 mM amiloride to the mucosal solution and is further inhibited by adding l mM ouabain to serosal solution (Fig. l). Isotopic flux studies show that the increase in Isc induced by NaCl is mostly accounted for by an increased influx of Na+ through an amiloride- sensitive pathway (DeSimone et al., 1984). Epithelial responses to hyperosmotic concentrations of NaCl are also inhibited by amiloride and ouabain in isolated rat lingual epithelium (Mierson et al., 1988a; Simon et al., 1988). These pharmacological studies show that amiloride-inhibitable pathways are present on the apical membranes of taste and/or epithelial cells and that Na-K-ATPase is present on the basolateral membranes of taste and/or epithelial cells.

Integrated responses to 0.5 M NaCl elicited from rat chorda tympani fibers are also inhibited 60-70% by amiloride (Fig. 2). Both the phasic (rapidly rising) and tonic (steady-state, adapting) components of the chorda tympani responses are inhibited by amiloride (Brand et al., 1985; DeSimone and Ferrell, 1985; Hill and Bour, 1985; Schiffman et al., 1983). These studies show that amiloride-inhibitable pathways must be either present in taste cells in fungiform papillae, or in the special sensory fibers synapsed to them.

In contrast to the partial inhibition of the chorda tympani response to NaCl response by amiloride, the tonic component of the chorda tympani response to 0.5 M Na acetate (pH 7.4) is completely inhibited by 0.1 mM amiloride (Fig. 2; Formaker and Hill, 1988). One possible explanation for this difference could be that the uncharged form of acetate diffused into taste cells and lowered their intracellular pH and, by some un- known mechanism, increased the sensitivity of the amiloride-inhibitable channel to amiloride. At pH 7.4 (almost 3 log units above the pKa of 4.75 for acetate; Weast, 19791, the concentration of undissociated acetic acid in a solution of 0.5 M Na acetate is about 0.5 mM. This concentration is sufficient to lower the intracellular pH about 1 pH unit (Zampighi et al., 1988). We tested this possibility by increasing the pH of the ex-

tracellular solution to 9.2. This decreases the concentration of undissociated acetic acid in 0.5 M Na acetate to about 5 pM, a concentration too small to alter the intracellular pH over the time scale of these experiments (Zampighi et al., 1988). We found that even a t pH 9.2, 0.1 mM amiloride completely inhibited the tonic portion of the response to 0.5 M Na acetate (El- liott and Simon, 1990). On the other hand, the response to 0.5 M NaCl in the absence and in the presence of amiloride remained the same at pH 9.2 as at 7.4 (El- liott and Simon, 1990).

We measured the chorda tympani responses as a function of concentration for NaC1, Na isethionate, Na acetate, Na methanesulfonate, and Na, tartrate in the presence and absence of 0.1 mM amiloride (Fig. 3). Re- sponses of the different sodium salts increased until the same maximal value was attained. All the sodium salts tested reached the same maximal response (relative to 0.5 M NH,Cl). However, for other Na' concentrations, sodium salts with organic anions produced smaller responses compared with those elicited by NaCl. The amiloride-insensitive components of the responses to organic sodium salts were all smaller than the amiloride-insensitive response to NaC1. Since amiloride inhibits transport across transcellular pathways, it follows that when the chorda tympani response is completely inhibited by amiloride, there is compara- tively little transport occurring across the paracellular pathways.

To further investigate differences in chorda tympani responses between NaCl and sodium salts containing organic anions, we tested whether well-established inhibitors of anion transport would shift the NaCl response to higher concentrations or increase the percentage of amiloride inhibition. Neither furosamide, bumetanide, SITS, DIDS, 9-anthracene carboxylic acid, nor an antibody demonstrated to inhibit chloride transport pathways in mammalian and amphibian epithelial cells (Finn et al., 1989) affected the NaCl response (Elliott and Simon, 1990).

Localization of Amiloride-Sensitive Channels and Na-K-ATPase in Lingual Epithelia

Antibodies raised against amiloride-inhibitable sodium channels specifically labeled both taste and epithelial cells in canine circumvallate papillae (Fig. 4A). Similar results were observed with canine fungiform papillae. In taste cells, labeling appears on both apical (arrow) and basolateral membranes. However, not all taste cells stained with the same intensity, suggesting that the distribution of amiloride-sensitive channels may not be uniform in all cells of the taste bud.

Antibodies raised against Na-K-ATPase labeled taste and epithelial cells of canine circumvallate papillae (Fig. 4B). In taste cells, specific labeling was observed on basolateral membranes. Thus in taste cells this enzyme appears to be located on basolateral membranes, as it is in most polarized epithelial cells (Rod- riguez-Boulan and Nelson, 1989; Sweadner, 1989). Na- K-ATPase is also present in the basal and lower spinosal cell layers of the epithelium but is absent from the uppermost layers. Thus the concentration of Na-K- ATPase, as indicated by the density of stain, dimin-

1 0

0.8

0.6

=*

2 s

F 0.4

0.2

0.0

INFLUENCE OF TIGHT JUNCTIONS IN TASTE

I I

201

0 0.2 0.4

Fig. 3. Chorda tympani responses to sodium salts. Chorda tympani response ratios of the tonic component of 5 sodium salts taken with respect the response of 0.5 M NH,CI. The salts include NaCl(o) , Na isethionate (A), Na gluconate (EE), Na, tartrate (O), and Na acetate

ishes as epithelial cells age and migrate to the mucosal surface. In canine fungiform papillae the distribution of stain is similar to that in circumvallate papillae (Si- mon et al., 1991).

Electrophysiology of Trigeminal Responses to NaCl

The importance of paracellular pathways on trigeminal responses to salts can also be inferred from measurements of transepithelial resistance, the location and permeability of epithelial tight junctions, and recordings from trigeminal fibers. One method of deter- mining the location of tight junctions is to use lanthanum salts. Lanthanum diffuses poorly across tight junctions and thus increases their resistance (Machen et al., 1972). Also because the electron density of lanthanum is large, it can be used to visualize tight junctions in thin-section electron microscopy (Hashimoto, 1971).

Electrophysiological measurements from isolated canine lingual epithelia showed that transcellular resistance pathways are larger than paracellular resistance pathways (Rp), and hence the total epithelial resistance (Rt) is approximately equal to Rp (Simon and Garvin, 1985). The addition of 5 mM LaC1, to isolated canine lingual epithelia bathed in symmetrical Krebs- Henseleit buffer increases Rt (Rp) from 1019 2 122 ohm-cm2 to 1764 f 326 ohm-cm2 (N = 5; Holland et al., 1989, 1991). Since tight junctions are found at the pe- rimeters of epithelial cells and since epithelial cells comprise more than 99% of the total perimeter of the

0.6 0.8 1 .o

(0). Open symbols represent the responses in the absence of amiloride, and filled symbols represent the responses in the presence of 0.1 mM amiloride. (Adapted from Elliott and Simon, 1990.)

epithelial surface, it follows that Rp is an approximate measure of the junctional resistance between epithelial cells (Holland et al., 1989,1991). Should the trigeminal responses to NaCl arise from its diffusion across epithelial tight junctions, then these responses should be inhibited by lanthanum but not by transcellular Na’ pathway inhibitors, such as amiloride and TTX (Hille, 1984). To this point, amiloride-inhibitable and TTX- inhibitable sodium channels have been identified in taste cells (Akabas et al., 1988; Behe et al., 1989,1990).

Responses from rat trigeminal nerves to 2.5 M NaCl were reversibly inhibited by 2.5 mM LaC1, (Fig. 5). These responses were not, however, inhibited by amiloride or TTX (Sostman and Simon, 1991). Trigem- inal responses were not elicited by 1.0 M LaCl,, demonstrating that the NaCl response was not simply a consequence of changes in ionic strength or osmolality of the solution. Also, 2.5 M Na isethionate failed to elicit a response, demonstrating that the trigeminal response to NaCl has a component that depends on the nature of the anion (Sostman and Simon, 1991).

Localization of Epithelial Tight Junctions In thin sections, tight junctions appeared as punctate

regions of closely apposed membranes, having an over- all thickness of 11-12 nm. Lanthanum added to the mucosal solution showed electron-dense deposits in the extracellular space at all levels in the stratum corneum and at the interface between the stratum corneum and stratum granulosum (Fig. 6). Lanthanum deposits were never observed to penetrate beyond this

S.A. SIMON ET AL.

A

B

Fig. 4. Immunohistochemical studies of lingual epithelia. A: Lo- calization of amiloride-sensitive channels in canine circumvallate papillae. This micrograph is from a section along the trench of the circumvallate papillae showing 3 taste buds. The white lines represent the fluorescently labeled secondary antibody associated with the primary antibody raised against amiloride-sensitive epithelial sodium channels. Note that the label is visualized on both the apical (arrow) and the basolateral membranes of taste cells. Bar = 10 km. B: Lo-

calization of Na-K-ATPase in canine circumvallate papillae. This micrograph is from a section along the trench of the circumvallate papillae showing 2 taste buds (TB). Note that the stain in the taste buds does not extend to the apical surface. The epithelial cells surrounding the taste buds are only stained in the lower epithelial layers. Note that the stain completely follows the contour of the cells, suggesting that it is in the plasma membrane. Bar = 10 km.

INFLUENCE OF TIGHT JUNCTIONS IN TASTE

2 .5M NaCl 2 . 5 m M LaCl3 Recovery

20 sec

Fig, 5. Inhibition of trigeminal nerve responses to NaCl by LaCI,. Integrated response from whole trigeminal nerves to 2.5 M NaCI. Traces were recorded under control conditions (left), immediately fol-

interface into the living cell layers of the epithelium when added to the mucosal solution. Lanthanum added to the serosal solution diffused up to, but never beyond, the interface between the stratum granulosum and stratum corneum (Holland et al., 1989,1991). Thus, in lingual epithelia the barrier to diffusion of small hydrophilic compounds through paracellular pathways is at the interface between the stratum granulosum and stratum corneum.

In taste buds, tight junctions are found beneath the microvilli of taste cells (Akisaka and Oda, 1978; Jahnke and Baur, 1979; Holland et al., 1990). Lantha- num added from the serosal solution showed electron- dense deposits beyond the region of the tight junctions into the taste pore (Holland et al., 1991). Thus, tight junctions between taste cells appear to be more permeable to lanthanum than are those between epithelial cells. In this regard they behave as tight junctions between “leaky” epithelia.

DISCUSSION Paracellular Pathways Between Epithelial Cells

When introduced into the oral cavity, hydrophilic compounds, such as salts, can enter lingual epithelia and stimulate trigeminal fibers by diffusing across tight junctions at the interface between the stratum corneum and stratum granulosum. Three types of experiments support this conclusion: a) Morphological studies show lanthanum deposits accumulate at tight junctions at the interface between the stratum corneum and granulosum when added from the mucosal solution (Fig. 6); b) recordings from trigeminal fibers show that lanthanum reversibly inhibits responses elicited by NaCl and other salts (Fig. 5; Sostman and Simon, 1991); and c) measurements of transport across isolated lingual epithelia show that the addition of lanthanum increases the resistance of the paracellular pathway. Further evidence, albeit less direct, is that 2.5 M Na isethionate, in contrast with 2.5 M NaC1, does not elicit trigeminal responses. One interpretation of this observation is that the large isethionate anion is relatively impermeant to epithelial tight junctions. Thus, Na+ diffusion into the epithelium would be di-

203

lowing a 30-s incubation with 2.5 mM LaCI, (center), and 20-40 min after LaCI, incubation (right). Salt stimulation is indicated by the solid line. (Adapted from Sostman and Simon, 1991.)

minished because liquid-junction potentials would be generated because of the relatively low permeability of isethionate anions (Harper, 1987).

Transcellular Pathways in Taste Cells Electrophysiological recordings from sensory fibers

(Fig. 2) and patch clamp studies from rat taste cells (Behe et al., 1989, 1990) indicate they contain amiloride-sensitive channels. These channels are im- portant in salt (NaC1) and possibly sweet taste (Heck et al., 1984, 1989; Mierson et al., 198813; Simon et al., 1988, 1989a). Immunohistochemical studies show that amiloride-sensitive channels are present on the apical membranes of some taste cells (Fig. 41, where they might play a role in taste transduction. Morphological studies show there are several morphologically distinct types of taste cells (Kinnamon, 1987; Kinnamon et al., 1985). It remains to be demonstrated whether all taste cells express this channel or whether these channels are present only in specific types of taste cells. Such information will be necessary to explain the origin of the chemical selectivity of individual taste fibers, since such fibers receive synapses from several taste cells in the same taste bud and with taste cells in other taste buds (Beidler, 1969; Kinnamon et al., 1988b; Miller, 1988). Patch clamp studies using taste cells in intact taste buds excised from rat fungiform papillae show that taste cells exhibit amiloride-sensitive currents on both their apical and basolateral membranes (Behe et al., 1989,1990). These data are entirely consistent with the immunohistochemical data presented here, where both the apical and basolateral membranes of taste cells are labeled (Fig. 4B). However, in intact taste cells the amiloride-inhibitable channels on the basolat- era1 membranes would have to be either closed, inac- tivated or desensitized, given the relatively large sodium concentration in the extracellular space. Con- sequently, there may be more than one type of amiloride-inhibitable channel in taste cells, since it is extremely unusual to have the exact same channel located on both the apical and basolateral membranes of polarized epithelial cells (Rodriguez-Boulan and Nel- son, 1989).


Fig. 6. Lanthanum penetration into lingual epithelium. Trans- mission electron micrograph of an unstained longitudinal section from canine fungiform papillae that was infiltrated with lanthanum from the mucosal solution. The electron-dense deposits are precipi- tates of lanthanum. In the upper figure lanthanum penetrated across the stratum disjunctum and accumulated only in the extracellular space between the first and second layers of the stratum corneum (SC). Bar = 1.0 pm. Inset: Experiment showing lanthanum

penetration to the interface between the stratum corneum and stratum granulosum (SG), where it was stopped by a tight junction. Two modified desmosomes (D) indicated by the arrowheads. Bar 0.5 pm. The tight junction (TJ), which prevented lanthanum from diffusing into the stratum granulosum, is shown a t a higher magnification in the inset in the lower right-hand corner. Bar = 100 nM. (Reproduced from Holland et al., 1989, with permission of John Wiley & Sons, Inc.)


Information obtained by measuring amiloride-inhibitable currents across isolated lingual epithelia (Fig. 1) cannot provide definitive information about whether amiloride-sensitive channels are in taste cells. The current can arise from amiloride-sensitive channels in taste cells, epithelial cells, or both cell types. However, immunocytochemical localization of sodium channels in taste cells, together with simultaneous measurements of the short-circuit current and chorda tympani in rat tongue, strongly support the idea that some of the current arises from transport across taste cells (Heck et al., 1989).

The 2 functions attributed to Na-K-ATPase in taste cells are first to generate and maintain the electrochemical gradients across the cells and second to repo- larize them after they become depolarized by chemical stimuli (Heck et al., 1989). Taste cells become depolarized by Na+ influx through amiloride-sensitive channels in their apical membranes. The resulting increase in intracellular sodium stimulates Na-K-ATPase, thus repolarizing the cells. The immunohistochemical study presented here (Fig. 4A), wh:l-h localizes Na-K-ATPase to the basolateral membranes of taste cells, is consistent with these roles for this enzyme.

Paracellular Pathways Between Taste Cells Two findings that cannot be fully explained by Na'

entering taste cells through amiloride-sensitive channels and exiting them via the Na-K-ATPase are the shifts of the chorda tympani response profiles to higher Na' for sodium salts with organic anions and the anion dependence of the fraction of the amiloride-inhibitable response (Figs. 2 and 3). These findings also cannot be rationalized by C1- influx or efflux into taste cells because different inhibitors of chloride transport had no effect on the response to NaCl or the percentage of amiloride inhibition (Elliott and Simon, 1990). Low- ering of the intracellular pH also cannot explain these results, since the chorda tympani responses were the same for Na acetate at pHs 7.4 and 9.2. The intracellular pH should not be lowered at pH 9.2 because at this pH acetate is completely ionized and thus relatively membrane impermeant (Walter et al., 1982). The shifts in the chorda tympani responses also cannot be rationalized by invoking differences in the ionic strengths, osmolalities, or activity coefficients of the solutions. Solutions of 0.12 M NaCl and 0.12 M Na acetate have the same ionic strength and approximately the same osmotic pressures, and yet give sig- nificantly different chorda tympani responses (Fig. 3). Finally, at 0.3 M, where the responses to NaCl and Na acetate differ by 40%, the mean molal activity coefficients for NaCl and Na acetate differ only by 5% (Par- sons, 1959). Thus, explanations involving transcellular pathways cannot easily explain these data.

The single factor that could rationalize the chorda tympani data would be if substitution of organic anions for C1F hyperpolarized taste cells. If this were the case then the chorda tympani responses for Na+ salts having organic anions would be shifted to higher Na+ concentrations compared with the response of NaC1, as is indeed the case (Fig. 3). Moreover, amiloride, being

positively charged at pH 7.4, exhibits voltage-dependent blockage of amiloride-sensitive channels (Palmer, 1984, 19851, meaning that amiloride will block more effectively at hyperpolarizing potentials and thus inhibit a greater percentage of the chorda tympani response in the presence of organic anions. The problem, then, is to demonstrate how alterations in the resistance of the paracellular pathway, Rp, can result in changes in the potential of taste cells (see Note Added in Proof).

For simple epithelia, the transepithelial potential (Vms) is equal to the sum of the potentials across the apical (Vmc) and basolateral (Vcs) membranes (Schultz, 1979); that is, Vms = Vmc + Vcs, where, m, c, and s refer to the mucosal, serosal, and intracellular compartments, respectively. In intracellular recordings from taste cells, the ground electrode is placed in the extracellular space and consequently the resting potential is equal to Vcs. By analyzing an equivalent circuit for coupled taste cells that form a simple epithelia (i.e., extend from the basement membrane to the apical surface), it can be shown that changes in Rp change Vms. Let Ra and Rb represent the resistances of apical and basolateral membranes, respectively. For simple epithelia the apical and basolateral membranes are connected in series with each other and in parallel with the paracellular resistance, Rp. If the electromo- tive forces generated at the apical, basolateral, and paracellular pathways are Ea, Eb, and Ep, respectively, then Vms = Rp(Eb - Ea) + Ep(Ra + Rb)/(Ra + Rb + Rp) (Reuss, 1979). Thus changing R p alters V m s and hence the resting potential, Vcs. For symmetrical salt solutions (Ep = 0) and in mammalian tongue where Ra -t Rb >> Rp, the above equation simplifies to Vms = Rp(Eb-Ea)/(Ra + Rb). Thus the greater Rp, the greater Vms. Analysis of gallbladder epithelium using the above equivalent circuit, together with experimental data gathered on this tissue, shows that increasing Rp results in hyperpolarization of the gallbladder cells (see Fig. 6 in Schultz, 1979). Using Nec- turus urinary bladder, Demarest and Finn (1987) found that the apical membrane potential became 40 mV more negative (i.e., hyperpolarized) upon doubling the paracellular resistance. In summary, the potential profiles across epithelial cells depends on the paracellular pathway(s) resistance and selectivity (Reuss, 1979; Schultz, 1979).

For taste cells, the information necessary to compute the change in Vcs for a change in Rp cannot be performed, since the necessary measurements of intracellular ion concentration have not yet been made. How- ever, a recent analysis of NaCl transport across rat taste cells undertaken to quantitate the shapes of the chorda tympani responses as a function of NaCl had all the C1- passively diffusing across the tight junctions between taste cells (Fidelman and Mierson, 1989). Taste cells are joined at their apical surface by tight junctions (Akisaka and Oda, 1978; Holland et al., 1990, 1991; Jahnke and Baur, 1979). The ion selectivity and permeability of these tight junctions are extremely dif- ficult to measure, since they constitute a very small percentage of the lingual surface (Mistretta, 1971) and also because they are either below the taste pores in


fungiform papillae or in trenches in circumvallate and foliate papillae. Moreover, they cannot be investigated in isolated taste cells, nor in isolated taste buds, since one can never be certain that their permeability properties are unchanged after removal from the tissue. Consequently, their characteristics can only be inferred by evidence such as that presented here and with anal- ogy to other epithelia.

To understand the chorda tympani responses to sodium salts with organic anions in terms of the junctional resistance, it must be assumed that the tight junctions between taste cells are permeable to C1-, an assumption supported by studies on every tight junction investigated, including those from “tight” epithelia (see Table 3 in Schultz, 1979). Therefore, it is rea- sonable to assume that tight junctions between taste cells are also permeable to C1-. Tight junctions between taste cells, like all other tight junctions, discrim- inate among ions on the basis of their charge density and size. The larger the ion and/or the greater its charge density, the more slowly it diffuses across tight junctions (Diamond and Wright, 1969; Wright and Di- amond, 1977). The more slowly the ion diffuses across tight junctional pathways, the larger the junctional resistance and the more hyperpolarized the taste cells will be. Thus, it is predicted that the extent of shift of the dose-response curves for sodium salts (Fig. 4) should be in the order of their ability to increase the junctional resistance. In other words, the shift of the responses (relative to NaC1) should be greatest for sodium salts having divalent anions, such as tartrate and sulfate, and smallest for salts such as NaBr. Experi- mentally, this is the case (Fig. 4; Beidler, 1967; Elliott and Simon, 1990; Formaker and Hill, 1988; Nakamura and Kurihara, 1988).

Physiological Significance of Tight Junctions in Taste

The anion selectivity of tight junctions between taste cells may explain why different sodium salts (and acids) exhibit different tastes (Beidler, 1967; Pfaffmann, 1971; Schiffman, 1980). Consider the responses of taste cells to sodium salts. Na+ enters taste cells via the amiloride-inhibitable channels, leading to responses associated only with Na’ . However, because the resistance of the paracellular pathway depends on both the concentration and type of anions, these compounds can change the membrane potential across all taste cells and thus produce a broad spectrum of tastes. In this manner, different organic salts (and acids) could elicit markedly different tastes depending on the permeability and ion selectivity of the tight junctions. Thus, the unique taste of NaCl depends on both Na’ and C1- transport across trans- and paracellular pathways.

Tight junctions may also modulate the enhancement of NaCl of saccharide or sweet taste responses (Ku- mazawa and Kurihara, 1990; Simon et al., 1989b). The addition of NaCl (to 0.1 M) to solutions containing only glucose or sucrose increases the current across isolated canine lingual epithelia (Simon et al., 1989b) and responses from canine chorda tympani fibers (Kumazawa and Kurihara, 1990). In a model of saccharide taste

transduction involving tight junctions, the addition of NaCl to salt-free saccharide solutions would decrease the resistance of the paracellular pathway, and hence taste cells would respond as if the saccharide were increased. The addition of other less permeable salts to saccharide solutions would yield other chorda tympani responses (Kumazawa and Kurihara, 1990).

Thus, the detailed understanding of the separate contributions of the transcellular and paracellular pathways provides a far more complete picture of events modulating chemical transduction.

ACKNOWLEDGMENTS We thank Drs. Ellen Elliott and Ann Sostman for

their many contributions to this work, Mr. Lee Hall for general assistance, and SAS for typing this manuscript. This study was supported by NIH grants DC01065 and DK37206, and the Smokeless Tobacco Research Council.

NOTE ADDED IN PROOF After this manuscript was submitted it was demon-

strated that tight junctions between taste cells in rat fungiform papillae indeed modulate chorda tympani responses to sodium salts (Ye et al., 1991).

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