ARTICLE |
Correspondence to: Bernd Lindemann, Physiologie II, Bau 58, Universität des Saarlandes, D-66421 Homburg, Germany. e-mail: phblin@med-rz.uni-sb.de.
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Summary |
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Salt taste signals from the rat anterior tongue are probably transduced via epithelial sodium channels (ENaCs) residing in the apical cellular pole of taste cells. The signals are blocked by mucosal amiloride in low µM concentrations. In contrast, the rat vallate papilla does not contribute to amiloride-blockable salt taste. Two approaches were used to probe for the three subunits of ENaC in the anterior and posterior tongue of the rats in sodium balance. (a) Immunohistochemistry with antibodies against ENaC subunits and against amiloride binding sites. In the anterior tongue, reactivity for -, ß-, and
-subunits was present in taste buds and lingual epithelium. In the posterior tongue vallate papilla, reactivity for
-subunit and for amiloride binding sites was easily demonstrable, whereas that for ß-subunit and especially for
-subunit was weaker than in the anterior tongue. (b) RT-PCR techniques were used to probe for the presence of ENaC subunit mRNA. In isolated taste buds of the anterior tongue, mRNA of all three subunits was found, whereas in isolated taste buds of the vallate papilla only mRNA of the
-subunit was easily detectable. That of ß- and
-subunits was much less abundant. RNA of all three subunits was abundant only in taste buds of the anterior tongue. Therefore, subsets of elongated taste cells do express ENaC, but regional differences exist in the transcription and expression of subunits. The regional differences suggest that amiloride-sensitive salt taste, which requires all three subunits, is present in the anterior but not the posterior tongue of rats, as functional studies indicate. (J Histochem Cytochem 47:5164, 1999)
Key Words: taste, amiloride, ENaC, immunohistochemistry, RT-PCR
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Introduction |
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In rodents, the salt taste which is mediated by the anterior tongue is specific for Na+ and Li+ ions and is sensitive to amiloride in low µM concentrations (-gustducin was used as a reference molecule, providing an additional way of discriminating taste tissue from nontaste tissue (
Testing for the presence of Na+ channel RNA with a reverse transcriptase-PCR protocol, we found that RNA of -, ß-, and
-subunits of ENaC was present in taste buds of fungiform papillae (anterior tongue), whereas predominantly
-ENaC was found in the vallate papilla (posterior tongue). In addition, immunoreactivity for
-, ß- and
-subunits was found in anterior tongue taste buds and reactivity for
- and ß-subunits in the foliate and vallate papillae. Therefore the results obtained with the two independent methods point to differences in RNA abundance and in expression of the subunit proteins when areas of the tongue are compared.
A portion of our results concerning the vallate papilla was presented at the 1997 ISOT XII Symposium in San Diego (
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Materials and Methods |
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Isolation of Taste Tissues
Tongues were isolated from freshly sacrificed rats (SpragueDawley, 180240 g, in sodium balance) and stored in ice-cold Tyrode's solution for at least 10 min. Three hundred µl of Tyrode's solution [in mM: NaCl 140, KCl 5, CaCl2 1, MgCl2 1, HEPES 10 (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), adjusted to pH 7.4 with NaOH] containing 1 mg dispase I/ml (Boehringer; Mannheim, Germany), 1 mg collagenase A/ml (Boehringer), and 1 mg trypsin inhibitor/ml (Sigma; Deisenhofen, Germany) was injected under the anterior lingual epithelium (aLE), the vallate papilla (VP), and the surrounding epithelium of the posterior tongues (pLE), using 1-ml insulin syringes with needles of 0.4-mm diameter. Tongues were incubated at room temperature (RT) for 25 min. When the epithelia had loosened from the tongue muscle layer, they were peeled with fine forceps and washed in Tyrode's solution.
Isolation of Taste Buds
Anterior Tongue.
We followed the published procedure (
Vallate Papilla.
The epithelium around the VP was removed, yielding the epithelial preparation pLE. From the VP, the superficial part, which has only few taste buds, was cut off and discarded. It is known that the number of buds present in a rat vallate papilla increases from 260 (3 weeks old) to 610 (13 weeks old) (
Counting and Lysis of Buds. Using a binocular microscope and a regular light microscope, buds were inspected and those that appeared fragmented were picked out with a transfer pipette (having an opening of 100 µm) and discarded. The remainder were counted, individually transferred onto a clean piece of parafilm, and counted again. Excess medium was removed from the paraffin with the transfer pipette and the buds covered with 100 µl of lysis buffer (4 M guanidine thiocyanate, 25 mM tri-sodium citrate, pH 7.0, 0.5% lauryl-sarcosyl, 0.72% 2-mercaptoethanol). Using a 200-µl pipette, the lysate was transferred to Eppendorf tubes, vortexed, frozen, and stored at -20C.
RNA Preparation
Yeast transfer RNA (1 µg) was used as a carrier. Sequentially, 0.1 volume of 2 M Na-acetate, pH 4.0, 1 vol of acidic phenol, and 0.2 vol of chloroform was added, the mixture being vortexed after each addition. After incubation at 4C for 15 min and centrifugation at high speed, the aqueous phase was recovered and RNA was precipitated with 1 vol of isopropanol. After centrifugation, the pellet was resuspended in 100 µl of lysis buffer and RNA was reprecipitated with isopropanol. After two washings with 75% cold ethanol, the pellet was dried, dissolved in water, and either stored at -20C or used directly for RT-PRC. At the end of the purification, pLE, and VPt RNA, available in higher amounts, were quantified by optical density and the integrity was checked on a 1% agarose gel.
Polymerase Chain Reaction
For the lingual epithelium, 100 ng of total RNA was used for RT, performed either with mouse Moloney virus reverse transcriptase (Pharmacia; Uppsala, Sweden), or RT Superscript II (Gibco; Gaithersburg, MD). To compare the signal in fungiform and vallate papillae, PCR reactions were carried out with an equivalent of one taste bud per reaction (one VPt contained about 280 taste buds; see above). Genomic DNA did not contribute to the signal as suggested by two protocols. In the first, RNA was treated in parallel in the presence and absence of reverse transcriptase, and the material was then used for PCR. In the absence of reverse transcriptase there was only minimal amplification of fragments having the expected size. Subsequently, information about the genomic organization of the human genes encoding -, ß-, and
-ENaC revealed that the three human genes are characterized by a very similar intronexon organization (
The primers used for DNA amplification were as follows.
For the -subunit.
AR1306sens: 5'-ATTACGGCGACTGTACTGAGAA-3', located in exon 7, and AR1618asns: 5'-CAATCCTGGGACTTCACAGATG-3', located at the junction between exon 10 and exon 11. This pairing amplified a 312-BP fragment (as the anti-sense oligonucleotide AR1618asns, located in the middle of an intronexon boundary, was unable to amplify genomic DNA at the annealing temperature used).
For the ß-subunit. BR1197sens: 5'-CAACACGACCTATTCCATCC-3') was located in exon 7. BR1663sens: 5'-TCGGTGCTGTGCCTCATTG-3' and BR1921asns: 5'-AGCCTCAGGGAGTCATAGT-3' were both located in exon 13. The pairing (BR1663sens-BR1921asns) amplified a 258-BP fragment but could not be used when contamination by genomic DNA was observed, because both sequences were located in the same exon. The pairing (BR1197sensBR1921asns) amplified a single 724-BP fragment specific for the cDNA. No genomic DNA could be amplified under the experimental conditions used here, owing to the large intronic sequences present between the two primers.
For the -subunit.
GR1616sens: 5'-ACAAAGACCTGAACCAAAG-3' was located in exon 12, and GR1902asns: 5'-GCATAGCAGAGGTAAAAGT-3' was located in exon 13. They were amplifying a 286-BP fragment of cDNA and a 1200-BP fragment of genomic DNA.
For ß-actin. ACTINsens: 5'-TCCTAGCACCATGAAG-ATC-3' and ACTINasns: 5'-AAACGCAGCTCAGTAACAG-3' amplified a 313-BP genomic fragment and one fragment of about 200 BP transcribed from RNA.
For -gustducin.
GU401sens: 5'-CAATCCGAGAAGTAGAGAGG-3' and GU869asns: 5'-GCTGTTGAAGAGGTGAAGAC-3'. They were amplifying a 468 BP fragment of cDNA (
After reverse transcription, samples equivalent to one taste bud (about 1 ng total RNA), or to 100 buds (for vallate tissue or epithelium) were denatured for 120 sec at 95C. Twenty-five, 30, 35, or 40 cycles of amplification were performed with the following parameters: denaturation at 95C for 30 sec; annealing at 5560C for 60 sec; extension at 72C for 60 sec. Reactions were performed in the presence of 300 ng of each oligonucleotide and 0.08 U of Taq DNA polymerase (Promega; Madison, WI). Fifty percent of the amplified product was analyzed on a 2% agarose gel, either by Southern blot hybridization with specific probes or by direct ethidium bromide staining. The amplified PCR products were sequenced and the expected partial sequences of -, ß-, and
-ENaC were found.
Semiquantitative PCR
During the exponential phase of a PCR experiment, the signal measured after n cycles is given by
where A0 represents the initial amount of template and E the efficacy of the amplification reaction (with 0<E<1). In preliminary experiments performed with pure plasmids, it was checked that the different pairings used for -, ß-, and
-ENaC were characterized by similar efficacies. E was estimated to 0.69 ± 0.10, 0.64 ± 0.11, and 0.82 ± 0.10 for
-, ß-, and
-ENaC, respectively. No statistical difference was found among the three values. Therefore, under the experimental conditions used, the relative abundance of the three species of RNA in a given tissue could be calculated from the signal measured during any cycle of the exponential phase of the PCR reaction.
Southern blot hybridizations were quantified on a Fuji phosphor imager, using analytical software developed by the company. Quantification of some experiments by direct ethidium bromide staining was also performed. In that case, a standardized protocol was developed to ensure that the same volume of agarose was cast, that an equal amount of ethidium bromide was added to the gel, and that a similar time of migration on the gels and of exposure of the films was used. Quantification was then performed using the NIH Image software (http://rsb.info.nih.gov/nih-image/download. html).
Detection of Splice Variants of ENaC
To probe for the splice variant described for rat vallate papilla (
Detection of Other Members of the ENaC Gene Superfamily
The presence of two other members of the ENaCFaNaChdegenerin gene superfamily was probed by PCR using the following pairs of oligonucleotides. For ASIC1-7S: 5'-ATT-GCTCTTCCCATCTCTAT-3' and for ASIC1-10AS: 5'-TTC-AAGGCCCATACCTAAGT-3' (
Immunohistochemistry in Sections
Tongues were removed and tissue containing taste buds (anterior tongue, foliate papillae, circumvallate papilla) was excised and immersed in Bouin's fixative (prepared freshly). After 3 days of fixation at 4C, tissues were dehydrated and embedded in paraffin (Vogel; Giessen, Germany) of 60C and were sectioned to a thickness of 7 µm with a standard microtome. Incubation with primary antibody was overnight. Polyclonal antibodies were raised in rabbits against peptides of sequences specific for one of the subunits of ENaC. The sequence Q44G58 was used for -rENaC (
-rENaC (
In addition to the three anti-peptide antibodies targeting ENaC subunits, we also used an antibody directed against -gustducin, generously provided by Dr. R.F. Margolskee (
The primary antibodies were used at the following final dilution: anti--rENaC 1:100, anti-ß-hENaC 1:400, anti-
-rENaC 1:400, RA6.3 1:50, anti
-gustducin 1:1000, and anti-histaminase 1:100.
Double Staining.
Sections were first incubated overnight with the polyclonal antibody directed against -rENaC. The staining sequence was continued (above) using goat anti-rabbit linked to alkaline phosphatase as the secondary antibody and Fast Blue RR (Sigma) as the chromogen. The sections were covered with gelatin and coverglass and the distribution of blue precipitate was imaged. Then the coverglass and gelatin were removed and the section was exposed overnight to the monoclonal antibody RA6.3 directed against amiloride binding sites. The staining sequence was continued, using goat anti-mouse linked to peroxidase as the secondary antibody. The new staining pattern, i.e., the brown color of the peroxidaseDAB reaction, was also imaged. Because the two primary antibodies used for double labeling were from different species, crossreactions of secondary antibodies were not a problem. The two primary antibodies were occasionally added simultaneously. In this case, too, the two staining reactions were executed in sequence, the peroxidase reaction being the last of the two. Inhibition of the phosphatase reaction by substrates of the peroxidase was thus avoided.
Controls Using Peptides.
Preabsorption experiments were conducted to test for specificity of the antibodies. The antibodies (working dilution) directed against -rENaC and
-rENaC were preincubated with their corresponding peptide (up to 2 mg/ml) at RT for 24 h. The staining procedure was then carried out as described above.
Immunohistochemistry in Sheet Preparations
Freshly peeled anterior epithelia (see above) had areas up to 9 x 9 mm and contained up to 90 fungiform papillae with taste buds that could easily be seen when viewed with a binocular microscope from the "inner" side. In a Petri dish with a cast silicon bottom, the sheets were stretched out with needles and covered with Bouin's fixative (prepared on the same day) for 48 hr at 4C. Sheets were cut into pieces having about 10 fungiform papillae each and were transferred to a beaker containing 200 ml of working buffer [TRIS (tris(hydroxymethyl)aminomethane) buffer, pH 7.4, containing 5 mM NaN3 and 1% fish gelatin (Sigma)] which was magnetically stirred overnight at 4C. The sheets were individually placed in 1 ml of working buffer containing primary antibody and were stored for 24 hr at 4C, then washed for 8 hr in 25 ml of working buffer at 4C. Sheets were incubated individually in 1 ml of working buffer containing secondary antibody and stored overnight at 4C, then washed for 5 hr at 4C. Primary antibodies were used at the following final dilution: anti--rENaC 1:250, anti-ß-hENaC 1:250, anti-
-rENaC 1:250, RA6.3 1:50, anti
-gustducin 1:250. Secondary antibodies were affinity-purified goat anti-rabbit and goat anti-mouse conjugated to the fluorescent dye Cy3 (Jackson ImmunoResearch; West Grove, PA). Alternatively, secondary antibodies conjugated to peroxidase (Dako) were used and staining performed as described for sections.
After staining, the whole mounts were placed between two large coverglasses with a drop of working buffer and were inspected in a fluorescence microscope. The objective was a Nikon Fluor x40 (oil immersion) of 1.3 NA. The fluorescence of individual buds was first documented from the inner side in an optical plane that intersected the long diameter of the bud roughly in the middle. The sheet was then turned around for imaging of taste pores. Each plane was also documented with transmitted light. Stacks of images were taken by changing the focal plane of the microscope in steps of 0.5 or 1 µm, using a feedback-controlled motor (RS8, HA-400; Harmonic Drive, Limburg, Germany) activated by the imaging software (T.I.L.L. Photonics; Planegg, Germany). For controls, the fixed and washed whole mounts were incubated with preimmune serum instead of primary antibody and the subsequent steps performed as described above. In addition, whole mounts were imaged with a confocal microscope (Bio-Rad; München, Germany) to confirm the intracellular location of the immunoreactivity.
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Results |
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Immunohistochemistry with Sections
A survey of immunoreactivity for -, ß-, and
-subunits of ENaC is shown in Figure 1. In the anterior tongue (upper row),
- and
-subunit-like reactivity was easily observed in the taste buds and in the epithelium of the fungiform papillae, whereas ß-subunit-like reactivity was restricted to a few taste cells. In the foliate papillae (center row),
- and ß-subunit-like reactivity was easily seen whereas
-subunit-like reactivity was weak. In the vallate papilla (bottom row)
-subunit-like reactivity was easily observed. Intensity of staining for the ß-subunit was less, provided that incubation times for the primary antibody were 24 hr. However, the intensity became more comparable to that of
-subunit when incubation times were extended to 48 hr. The reactivity for
-subunit was very low or absent in the vallate papilla, even at long incubation times (Figure 1I and Figure 2D).
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Control experiments with antibodies that had been preincubated with their antigenic peptide for 24 hr (see Materials and Methods) showed increasingly weaker immunostaining when the peptide concentration was increased. At 2 mg peptide/ml, reactivity was minimal (Figure 2B and Figure 2E). Controls omitting primary antibody are shown in Figure 2C and Figure 2F. Furthermore, positive controls were performed with sections from rat kidney and rat submandibular gland. The expected targets (-subunit immunoreactivity localized in the brush border (data not shown). However, we do not consider this target unexpected because of the recent demonstration of ENaC mRNA in the straight proximal segment (
According to electrophysiological evidence (- or ß-subunit emphasized cellular borders (Figure 1B, Figure 1E, and Figure 1G), suggesting that the signal was at or near the basolateral membrane of the cells. Whether apical reactivity was present in addition was difficult to ascertain because the sections seldomly passed through the taste pore. However, when sections were cut tangentially through the anterior lingual epithelium, taste pores were sometimes revealed. They contained immunoreactivity for ENaC subunits, as shown in Figure 3. (An alternative procedure, which used epithelial sheet preparations, is described below.)
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The monoclonal antibody RA 6.3, directed against amiloride binding sites, labeled a subset of elongated taste cells. The label was mostly intracellular, as was the pattern obtained with the anti -subunit antibody. This suggested that the intracellularly located subunits of ENaC are able to bind amiloride. To check for the presence of alternative binding sites of amiloride, we used a polyclonal antibody directed against epithelial histaminase (
To further probe the nature of the ENaC-subunit immunoreactivity, double staining with anti -subunit antibody and RA 6.3 was carried out as described in Materials and Methods. This was especially interesting to do with the vallate papilla, in which the functional role of ENaC is unknown. At the cellular level, there was good coincidence between the reactivity of
-rENaC and of amiloride binding sites (Figure 4). Furthermore, Figure 4 shows rather clearly that the reactivity was limited to a subset of elongated cells within a bud and that at least some of these cells had slim extensions pointing towards the base of the bud. These cells may therefore be of Type I, a subtype of elongated cells that maintain contact with the basement membrane (reviewed in
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In addition, double staining of vallate papillae was carried out with antibodies directed against -ENaC and against
-gustducin, a G-protein specific for taste tissue (
-Gustducin was previously localized to a subset of Type II elongated taste cells (
Immunohistochemistry with Sheet Preparations
On the anterior tongue, fungiform papillae are distributed randomly, most of them containing only one taste bud. In consequence, the buds were difficult to find, especially in paraffin-embedded material, and only seldom did a section contain a taste pore. As an alternative, therefore, we prepared sheets (whole-mounts) of the anterior tongue epithelium (see Materials and Methods). With this approach, it was easy to subject many anterior taste buds to immunostaining and subsequently to image their taste buds and taste pores. Immunoreactivity of anterior taste buds was found for -, ß-, and
-ENaC in the sheet preparations, as shown in Figure 5. The labeling was further checked by confocal microscopy. These experiments confirmed the mainly intracellular location of the ENaC-like reactivity. When in control experiments preimmune serum was used instead of primary antibody, staining became insignificant (Figure 5C).
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Taste pores were searched for possible fluorescent labeling of the apical microvilli of taste cells. It turned out that such labeling was not seen at the level of the outer opening of a taste pore but at 0.51 µm downward where, indeed, the apical poles of taste receptor cells are to be expected. Figure 6 depicts apical immunoreactivity for -, ß-, and
-ENaC. Labeling of apical cellular poles close to the taste pores was generally evident, even though it was not possible to distinguish the fluorescence of microvilli from that of other less superficial structures.
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PCR of Isolated Buds
Although the anti-ENaC antibodies were raised against peptides with no significant homology to other known proteins, the possibility exists that they reacted nonspecifically with non-ENaC proteins. However, if ENaC proteins exist in a cell, then the corresponding RNA must also be present. Therefore, we used RT-PCR to corroborate expression of the three subunits in taste cells. To minimize epithelial contamination, taste buds were physically isolated from the anterior tongue and the vallate papilla and were pooled into groups of 3050 buds. Total RNA was extracted, reverse-transcribed, and subjected to the PCR procedure. The quality of the RNA was extensively checked, as described in Materials and Methods.
Occurrence of ENaC mRNA in Posterior Tongue and in Anterior Tongue
A single VPt preparation (i.e., vallate tissue enriched in taste buds but containing some epithelium) yielded about 900 ng of total mRNA (-Gustducin (
Subsequently, pools of 30 isolated buds were analyzed (Figure 7). Vallate buds (labeled V) were strongly positive for -gustducin and for
-ENaC mRNA. Signals for ß- and
-ENaC mRNA were weaker and were only demonstrable after hybridization with specific probes. Because the efficiency of the three oligonucleotide pairings used was similar (see Materials and Methods) and because the material was quantified during the exponential phase of the PCR reaction (although not detectable by staining with ethidium bromide), the relative abundance of the RNAs encoding
-ENaC, ß-ENaC, and
-ENaC was directly deduced from data like those shown in Figure 7 and Figure 8, yielding
-ENaC >> ß-ENaC >
-ENaC for taste buds from the vallate papilla.
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The lingual epithelium (pLE) posterior to the vallate papilla was also subjected to RT-PCR, as described in Materials and Methods. The amount of each PCR product obtained from vallate papilla isolated buds and from pLE was quantified by scanning on a phosphor imager and was normalized to 1 ng of total RNA. Ratios were then computed for the abundance of ENaC RNA found in the taste buds over ENaC RNA found in the posterior epithelium in the same assay. The ratios for -, ß-, and
-ENaC were 3.3, 2.7, and 0.7, respectively. This result clearly shows the differential expression of subunits when taste buds and lingual epithelium of the posterior tongue are compared.
In pooled anterior tongue taste buds, a high abundance was noted for all three species of ENaC mRNA (Figure 7). The differential occurrence of the three subunits in anterior vs vallate papilla was confirmed by the semiquantitative approach described below.
Semiquantitative Estimation of RNA Abundance
The specific signal in isolated fungiform buds and VPt was measured for each subunit and for gustducin after 25, 30, 35, and 40 cycles of amplification (Figure 8). In the anterior tongue taste buds, -, ß-, and
-ENaC were detectable by staining with ethidium bromide after only 30 cycles of PCR, and the relative amounts of the three amplicons did not vary with the number of cycles used. In contrast, in the vallate papilla, despite some contamination by epithelium included in the VPt-preparation, the signals for ß-ENaC and
-ENaC were much lower than the signal for
-ENaC. At 30 cycles of amplification, only
-ENaC was detected, whereas ß-ENaC and
-ENaC were absent. Detection of ß-ENaC required 35 cycles and
-ENaC 40 cycles. This comparison between vallate papilla and the fungiform papilla shows that abundance of
-ENaC RNA was similar, whereas ß-ENaC and
-ENaC RNA were more abundant in anterior taste buds than in vallate papilla taste buds. The abundance of
-gustducin RNA was consistently stronger in the vallate papilla than in the anterior tongue.
Splice Variants of ENaC
Using the RT-PCR with material from rat VP and rat lingual epithelium, Li and co-workers (1995) have identified two alternatively spliced transcripts of -rENaC. These splice variants,
-rENaC-a and
-rENaC-b, have deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxy terminus. The presence of the splice variants in taste buds of the vallate papilla and the anterior tongue was investigated using oligonucleotide AR1327S, located in exon 7, and oligonucleotide AR1592asns, located in exon 10 (see Materials and Methods). In
-hENaC, ß-hENaC, and
-hENaC, identical sizes of 118 BP and 79 BP have been described for the eighth and ninth exons, respectively. A greater phylogenetic distance exists between
-ENaC and ß-ENaC or
-ENaC than between
-rENaC and
-hENaC. It is therefore quite likely that the same genomic organization exists also for the three rat genes. RT-PCR with oligonucleotides AR1327S and AR1592asns can theoretically, in that case, amplify several additional bands. In addition to amplification of a 265-BP band, which corresponds to the normal form of the transcript, a deletion of the eighth exon would provide an additional band at 147 BP. A deletion of the ninth exon would provide an additional band at 186 BP (observed by Li et al). A deletion of both exons would provide an additional band at 68 BP. According to
With our set of specific oligonucleotides, only a single unique band of 265 BP was amplified. It corresponded to the normal form of the transcript (data not shown). This suggests that the two splice variants were not present in our material, or at least that their levels of expression were below the threshold of detection of our assay. In any case, it appears unlikely that these spliced forms, expressed at a low level, can affect the function of the normal proteins and modulate sodium perception. A more trivial explanation of the results obtained by Li et al. is that their use of degenerate oligonucleotides has caused the amplification of artifactual products. A similar problem was encountered during the cloning of ß-hENaC (
Occurrence of MDEGs and ASICs
The presence of further members of the ENaCdegenerinFaNaCh gene superfamily was investigated using the two pairs of oligonucleotides specified under Materials and Methods. The pairing ASIC1-7S and ASIC1-10AS amplified specifically a 500-BP fragment of ASIC, which encodes an H+-gated cation channel (
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Discussion |
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Sensory cells contained in taste buds of the anterior tongue respond to increases in mucosal Na+ concentration with inward current and firing of action potentials. This is especially true in rodents, in which the responses are blockable by mucosal amiloride in low micromolar concentrations, suggesting the involvement of the epithelial Na+ channel ENaC in the Na+- (and Li+)-specific component of salt taste (
In contrast to the anterior tongue, the salt taste signals arising from the posterior tongue (vallate papilla) of rats in sodium balance are not Na+- and Li+-specific and are not blockable by amiloride (
In this study we physically isolated taste buds from the vallate papilla and used purification of mRNA followed by RT-PCR to investigate the presence of ENaC-type message in taste buds isolated from the anterior and posterior tongue. In this way we hoped to reduce or overcome the contamination with ENaC stemming from the lingual epithelium. In addition, we used antibodies against peptides derived from the three subunits of ENaC (
ENaC-RNA in Taste Buds from Anterior and Posterior Tongue
RT-PCR revealed message for ENaC subunits in isolated buds derived from the vallate papilla and in buds from the anterior tongue. These results provide the first molecular evidence for the presence of ENaC subunits in taste buds that had reduced, presumably almost negligible, epithelial contamination. The relative abundance of subunit message varied with lingual area. In the anterior buds, -, ß-, and
-ENaC RNA was readily detected. The same was true for the anterior and posterior lingual epithelium. In posterior buds, however, only
-ENaC RNA appeared to be abundant, whereas ß- and especially
-ENaC RNA was less easily detected.
It appears likely that the functional channel contains all three subunit proteins, for example, in the proportion , ß,
,
(
ß
in the bronchiolar epithelium, whereas in the nasal and tracheal epithelium the relationship is
>
>> ß (
, ß,
,
. For example, only one of the RNA species may be under hormonal control and may therefore determine the rate of overall channel synthesis. In the cortical collecting duct, aldosterone controls
-subunit mRNA, whereas in the distal colon aldosterone controls ß- and
-mRNA (
- and/or ß-mRNA does not allow the assembly of the multimeric complex. Interestingly, a recent study performed parallel to ours describes that treatment of Na+-deprived rats with aldosterone enhanced immunoreactivity to ENaC subunits in the rat vallate papilla (
Other Channels of the ENaC Family
ENaC is homologous to a large number of proteins which are expressed in epithelial as well as excitable cells (reviewed by
ENaC Immunoreactivity in Taste Buds from Anterior and Posterior Tongue
- and ß-ENaC-like reactivity was found in taste cells of the vallate papilla, whereas
-like reactivity was scarce or absent. In buds of the foliate papilla,
-like reactivity was weak even though not absent. In contrast,
-, ß-, and
-reactivity was clearly present in taste cells of the anterior tongue. In taste cells of all lingual areas, some of the immunoreactivity was at the apical cellular pole, either at microvilli or at apical and basolateral membranes close to the tight junction. However, the major part of the immunoreactivity was not apical but intracellular. The same result was obtained with the RA6.3 antibody directed against amiloride binding sites. Double staining showed that those taste cells that had ENaC-like reactivity also had RA6.3 reactivity.
Localization of the Immunoreactivity
Most of the ENaC-like reactivity was located intracellularly. Although this may be surprising for a channel expected to reside in the apical membrane, it is entirely compatible with reports by others. Using an antibody against the native (at the time, putative) channel protein (-, ß-, and
-subunit of ENaC, all indicated a predominantly intracellular location of the amiloride-blockable Na+ channel in taste cells.
Similar observations made with other membrane proteins are on record. For example, the -subunit of the taste-specific G-protein gustducin, expected to be located close to receptor proteins at the apical membrane, was found in addition intracellularly, associated with intermediate filaments (
We can only speculate about the significance of ENaC immunoreactivity at intracellular locations. One attractive explanation is provided by recent insights into the protein editing performed by the endoplasmic reticulum. Incorrectly folded proteins and heteromeric complexes that remained incomplete were "edited out" in the ER (pre-Golgi degradation) and in the lysosomal pathway of the Golgi system (
In addition, in the fungiform papilla, part of the intracellular immunoreactivity may represent ENaC units in transit to the apical membrane (-subunit associates with a new ENaC subunit, as yet unknown, that confers a low amiloride affinity to the complex. An ENaC-like channel located on the lateral membrane would cause a strong depolarization of the cell unless the channels had a low open probability, i.e., were practically closed. The interesting possibility that basolateral ENaC is closed at rest but opens in response to cellular or extracellular signals will have to be considered in future experimentation.
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft (SFB 246-C1 and Li 126/7-1), by the Centre National de la Recherche Scientifique, and by the Association Française de Lutte contre la Mucoviscidose.
Thanks are due to Weihong Lin, Thomas E. Finger, Bernhard C. Rossier, and Sue C. Kinnamon for discussing their results freely with us before publication (-gustducin was generously provided by Robert F. Margolskee (Mount Sinai School of Medicine, New York, NY). The expert technical assistance of Valérie Friend (Pharmacologie Moléculaire, Valbonne) is particularly acknowledged.
Although our results, based on RT-PCR, did not reveal the presence of MDEG or ASIC in the taste tissues, MDEG1 was recently found in the rat vallate papilla by expression cloning (Ugawa S, Minami Y, Guo W, Saishin Y, Takatsuji K, Yamamoto T, Tohyama M, Shimada S (1998) Receptor that leaves a sour taste in the mouth. Nature 395:555556)
Received for publication May 20, 1998; accepted September 22, 1998.
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Literature Cited |
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