1 Department of Physiology, Multidrug resistance P-glycoprotein (MDR1) is a membrane protein
of 150-170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells. Expressed in many
epithelial tissues and in the endothelia of the blood-brain barrier,
the MDR1 protein provides major routes of detoxification. We found that
taste cells of the rat vallate papilla (VP; posterior tongue) had only
a slow increase in fluorescence due to uptake of the hydrophobic dye
calcein acetoxymethyl ester. However, the development of fluorescence
was accelerated two- to threefold by substrates and/or
inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A,
and by addition of the transport-blocking antibody to MDR1, UIC2.
Western blots of vallate tissue rich in taste buds with
the MDR1-specific monoclonal antibodies C219 and C494 revealed an
immunoreactive protein at ~170 kDa. In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies. Furthermore, using the antibodies C494 and UIC2 with tissue
sections, MDR1-like immunoreactivity was found in taste cells. These
results show that MDR1 is present and functional in vallate taste cells
of the rat. MDR1-related transport may achieve active elimination of
xenobiotics from the sensory cells and thereby protect the peripheral
taste organs from potentially harmful molecules contained in an
animal's food.
fura 2-acetoxymethyl ester; calcein-acetoxymethyl ester; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl
ester; C219; C494; UIC2
THE RECEPTOR CELLS of oral taste buds must necessarily
come into contact with a large variety of environmental molecules, many
of which may interfere with the processes of sensory function or may be
generally harmful for the cells. Therefore, have the taste cells
acquired mechanisms of protection? It is known from many other
epithelia and tissues that there is active elimination of xenobiotics
by means of multidrug resistance P-glycoproteins (MDR1). These membrane
proteins convey the property of multiple drug resistance by virtue of
an ATP-driven outward transport of a large variety of hydrophobic
"drugs" (11).
We previously noticed that taste buds are especially difficult to load
with hydrophobic dyes like fura 2-acetoxymethyl ester (AM)
(2). In the present study we found that the uptake of calcein-AM and of
other hydrophobic dyes into taste cells, as indicated by the
development of cellular fluorescence, can be accelerated by the
presence of inhibitors and/or substrates of the P-glycoprotein
isoform MDR1 and by the inhibitory antibody to MDR1, UIC2.
In addition, MDR1-specific antibodies used in Western blots indicated
the presence of MDR1 protein in vallate papilla (VP) rich in taste
buds, whereas much less MDR1 protein was found in the surrounding
lingual epithelium with this method. Furthermore, immunohistochemistry
showed the presence of MDR1-like molecules in taste cells, suggesting
that MDR1 is one of the routes by which xenobiotics are eliminated from
taste buds.
Lingual slice preparation. Adult Sprague-Dawley rats
of both sexes (weighing 180-300 g) that had been fed
with standard pellets were used. In addition, rats 8 or 15 days old were used occasionally, but no differences in dye
transport properties were observed. Animals were briefly
anesthetized with chloroform or
CO2 and were decapitated. Tongues
were removed, stored in ice-cold Tyrode solution containing (in mM)
140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), adjusted to pH 7.4 with NaOH, for at least 10 min, and then were treated as described below. The VP or posterior lingual
epithelium (PLE) surrounding the VP was cut out of the tongue and
separated from connective tissue, resulting in small tissue blocks of 3 × 5 × 3 mm and 3 × 8 × 3 mm, respectively. With
the use of a cyanoacrylate glue (Uhu; Bühl), the blocks were
glued into a space carved into a rectangular block of carrot, with the
epithelial surface oriented downward. The blocks of carrot were glued
onto a pedestal of a sliding tray filled with ice-cold saline. The
preparations of VP were sectioned parallel and the PLE perpendicular to
the tongue surface using a vibrating blade microslicer (Campden
Instruments, Lancaster, UK), yielding two to three slices of ~100
µm thickness that contained taste buds or lingual epithelium. By
means of a scalpel with a curved blade, the VP sections were
partitioned into small pieces, each containing a row of 10-20
taste buds. These small VP sections and small strips made from the
epithelial sections were placed into a saline-filled recording dish
and, with a piece of Parafilm, were gently pressed against the bottom,
which was a coverglass coated with a layer of the cell and tissue
adhesive Cell-Tak (Labor-Schubert, Munich, Germany). The slices would
then remain in place while superfused with the various media described
below.
Fluorescence measurements. Chambers
containing a slice were placed on an inverted microscope (IMT2-F;
Olympus, Hamburg, Germany) and were imaged with a ×20, 0.85 (numerical aperture) or ×40, 0.85 objective (Fluor
20 or 40; Nikon, Düsseldorf, Germany). A variable monochromatic
light source (Till Photonics, Munich, Germany) was coupled to the
microscope with a light guide. Images were recorded with an integrating
slow scan camera (Theta System, Munich, Germany) and were digitized to
8 bits/pixel (19). Camera and scanner were controlled by a computer
program written in the laboratory (2). The program was also used to
analyze the stored images. For evaluation, subregions of
interest were defined on an image (Fig.
1), and the fluorescence
intensities were expressed as the sum of pixel values per 100-ms
exposure time per area. Excitation wavelengths used for measurements of
fluorescence were calcein-AM, 495 nm; fura 2-AM, 340 and 380 nm; and
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM, 450 and 480 nm. Emitted light was collected with the appropriate dichroic mirrors.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
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Fig. 1.
Transmission light and fluorescence micrographs
(A-D) and effect of verapamil on
fura 2-acetoxymethyl ester (AM) loading
(E and
F) in lingual slices of vallate
papilla (VP). A and
C: horizontal sections of lingual
slices in transmission light. B and
D: corresponding fluorescence images
after incubation with fluorescence probes.
A: taste buds are seen as a dense
layer between mucosal space (m) and subjacent connective tissue (c).
B: fluorescence of same section is
shown after 120 min incubation with 10 µM fura 2-AM.
C: for clarity 2 taste buds are
outlined with apical (a) and basolateral (b) parts and subjacent
connective tissue (c) labeled. D:
fluorescence of same slice is shown after 30-min incubation with 1 µM
calcein-AM. E: transmission micrograph
of horizontal sections of VP and corresponding fluorescence images in
F. F,
left: the slice was exposed to 10 µM fura 2-AM for 60 min; right: same slice 15 min after
addition of fura 2-AM and 200 µM verapamil. Fluorescence
images are in pseudocolors. Color bar scales from low (blue)
to red (high) fluorescence intensities. Scale bars, 50 µm.
Calcein accumulation in viable tissue slices was measured by replacing the normal saline in the recording chamber by saline containing 1 µM calcein-AM. The gradual increase in fluorescence intensity in the slice was recorded by taking an image every 3 min. After 20-30 min, the section was superfused by replacing the chamber content with a solution of the previous concentration of calcein-AM containing, in addition, one of the nonfluorescent MDR1 substrates and/or inhibitors, the MDR1-specific antibody UIC2, or mouse immunoglobulin G (IgG) used as a control. Results were obtained from 42 lingual sections of the VP and from 7 sections of epithelium surrounding the VP (16 rats). Images were stored on disk for further analysis.
To measure the activity of intracellular esterases, VP tissue was homogenized by sonication in a buffer containing (in mM) 280 mannitol, 5 EDTA, 0.1 MgSO4, 0.2 Pefabloc, and 10 HEPES, adjusted to pH 7.0 with tris(hydroxymethyl)aminomethane (Tris) and kept on ice until use. Calcein-AM (1.25 µM) was added to 2 ml of a constantly stirred solution containing (in mM) 130 KCl, 5 NaCl, 2 MgCl2, 1.2 KH2PO4, 0.1 CaCl2, 20 HEPES, and 10 glucose, as well as 0.1 mg/ml trypsin inhibitor (from hen egg white) and 2 mg/ml bovine serum albumin (pH adjusted to pH 7.0 with KOH), and the increase in calcein fluorescence was measured after addition of VP homogenate (200 µg protein) with a FluoroMax-2 (Jobin Yvon-Spex Instruments, Edison, NJ) fluorometer (excitation wavelength 493 nm; emission wavelength 515 nm; bandwidth 5 nm). When the increase in fluorescence was linear, inhibitors and/or substrates of MDR1 were added to the cuvette. In some experiments, bis-(p-nitrophenyl) phosphate (BNPP), a nonspecific esterase inhibitor, was added to the cuvette before VP homogenate.
All experiments were carried out at room temperature and were repeated at least three times with different tissue batches.
Immunohistochemistry. Tissue blocks containing VP were fixed in Bouin's solution (prepared freshly) for 3 days at 4°C. For dehydration, isopropanol of 80, 96, and 99% was used, followed by three washes in methyl benzoate. Tissues were embedded in paraffin (histo-comp; Vogel, Giessen, Germany) at 60°C. Sections of ~10-µm thickness were cut perpendicular to the epithelial surface and were affixed to glass slides with egg albumin. Paraffin sections were deparaffinized and rehydrated by consecutive submersions in xylene, 100% ethanol, 96% ethanol, and distilled water. Immunohistochemical staining of MDR1 was carried out according to Toth et al. (27, 28) with minor modifications. Endogenous peroxidase activity was quenched for 15 min with 1% H2O2 in methanol (vol/vol) at room temperature. After the slides were washed in phosphate-buffered saline (PBS), they were placed in a buffer solution containing 1.8 mM citric acid and 8.2 mM sodium citrate and were heated in a microwave oven at 600 W for 4 min and at 250 W for 10 min. To reduce unspecific binding, slides were incubated for 30 min with 4% human serum albumin in PBS. Sections were then exposed sequentially to the primary antibody followed by two secondary antibodies labeled with horseradish peroxidase (HRP) and peroxidase (POD) to amplify the immunoperoxidase reaction. Tissue sections were incubated for 60 min at room temperature with mouse IgG as a negative control or with the following monoclonal antibodies against MDR1: UIC2 (1:50) (18), C494 (1:50) (14), and C219 (1:10) (14). The first secondary antibody, an HRP-conjugated rabbit anti-mouse antibody, was applied for 30 min at room temperature at a dilution of 1:50. The second secondary antibody, a goat anti-rabbit POD-conjugated antibody, was also applied at a dilution of 1:100 for 30 min at room temperature. After the slides were washed, they were incubated with the chromogenic substrate 3-amino-9-ethylcarbazole (AEC) until staining developed (maximally 5 min) at room temperature. Slides were washed once more, mounted in glycerol-gelatin, and viewed with a noninverted microscope using objectives of ×10, ×20, and ×40. Images were recorded with charge-coupled device cameras connected to a frame grabber that was mounted in an IBM-compatible personal computer.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sheet preparations of the VP and the PLE, which directly surrounded the VP, were dissected from one rat tongue, as described earlier (24). Briefly, a syringe fitted with a small needle and containing an enzymatic cocktail [0.5 mg/ml collagenase A, 2.5 U/ml dispase grade I (both Boehringer, Mannheim, Germany), and 1.0 mg/ml trypsin inhibitor I-S (Sigma, Deisenhofen, Germany)] was used to inject between the muscle layer and the lingual epithelium of the back part of the tongue. After an incubation time of ~30 min, the epithelial layer was peeled off and the VP was carefully trimmed from epithelial tissue. The protein content of a VP was determined by the method of Bradford (3) and amounted to 8-14 µg. The protein content of the PLE sample was about twice that of the VP sample. Both samples were solubilized for 30 min at 37°C in 40 µl of three times concentrated sodium dodecyl sulfate (SDS) sample buffer and were sonicated for 30 s. After centrifugation at 12,000 g for 5 min, the supernatants were loaded onto the gel. Electrophoresis and blotting procedures were performed essentially as described earlier (25). Proteins were separated by SDS-polyacrylamide gel electrophoresis on 7.5% acrylamide Laemmli (15) minigels and transferred onto polyvinylidene difluoride (PVDF) membranes overnight. The efficiency of protein transfer was monitored with prestained protein standards. Blots were blocked with 3% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 6 h and were incubated with primary antibodies against MDR1 (5 µg/ml C219 or C494 monoclonal antibodies) overnight. After incubation with HRP-conjugated secondary antibody (1:6,000 dilution) for 60 min, blots were developed in enhanced chemiluminescence reagents and signals were visualized on X-ray films. X-ray films were scanned with a single-pass flatbed scanner (Linotype-Hell, Eschborn, Germany) and were processed for documentation using JASC Paint Shop Pro 4.1 software (Jameln, Germany). The contrast of the Western blot signals was enhanced by 100% with Corel Photo-Paint 5.0 software (Corel, Rüsselsheim, Germany).
Chemicals and reagents. Stock solutions of fura 2-AM, BCECF-AM, calcein-AM (Molecular Probes, Eugene, OR), BNPP, and tamoxifen (Sigma) were made by solubilization in dimethyl sulfoxide. Cyclosporin A (Sigma) and verapamil (Aldrich Chemie, Steinheim, Germany) were dissolved in ethanol. Probenecid (Sigma) was dissolved in diluted solutions of NaOH buffered to pH 7.4. Cell-Tak was from Labor-Schubert (Munich, Germany). C494 monoclonal antibody, HRP-conjugated rabbit anti-mouse antibody, goat anti-rabbit POD-conjugated antibody, and the chromogenic substrate AEC were from DAKO Diagnostika (Hamburg, Germany). UIC2 monoclonal antibody was from Coulter-Immunotech Diagnostics (Hamburg, Germany). HRP-conjugated sheep anti-mouse IgG and enhanced chemiluminescence reagents were purchased from Amersham-Buchler (Braunschweig, Germany). Nonfat dry milk and prestained protein standards were from Bio-Rad (Munich, Germany). C219 monoclonal antibody (for the detection of P-glycoprotein) was from Alexis Deutschland (Grünberg, Germany). PVDF membranes were from NEN-Dupont (Bad Homburg, Germany). All other substances were from commercial sources and of analytical grade.
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RESULTS |
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Distribution of accumulated dyes. When viable slices of the VP were incubated in a medium containing the fluorescent indicator dye fura 2-AM or BCECF-AM, staining remained weak in the course of 60 min. Figure 1A shows a horizontal VP slice in transmitted light. The taste buds were seen between the mucosal space (m) and the subjacent connective tissue (c). When slices (Fig. 1A) were loaded with fura 2-AM, fluorescence distribution in the VP taste buds was always nonuniform and patchy (Fig. 1B). Incubation with the fluochrome BCECF-AM showed the same distribution pattern as with fura 2-AM (data not shown). Longer incubation times up to 150 min did not significantly enhance dye uptake in the unstained parts.
Because of the known action of MDR1 to actively expel a large variety of fluorescent dyes (21), another fluorescent dye was used, calcein-AM, a viability and esterase activity probe (17). Figure 1D illustrates the fluorescence distribution after incubation with calcein-AM for a period of 30 min. The corresponding micrograph in Fig. 1C shows in its center a row of taste buds. Single taste buds (two are outlined for clarity in Fig. 1, C and D) are visible by their bulb-shaped or oval profiles lined up side by side. The layer subjacent to the taste bud region represents connective tissue and nerve fibers (c), and the mucosal space is seen as the topmost, unstructured part of this image. The dye distribution was nonhomogeneous, with the strongest calcein fluorescence found in the basal parts of the taste buds (b). Several of the taste buds showed even stronger dye exclusion. Staining of connective tissue was also weak and always patchy.
Epithelial slices incubated with calcein-AM showed in some cases almost homogeneously distributed dye and in other cases a predominant fluorescence in basal parts of the epithelial cross section (data not shown).
The Ca2+ channel blocker verapamil is one of several pharmacological agents known to inhibit MDR1-mediated efflux (8), thereby enhancing dye accumulation. Figure 1F illustrates the increase in fura 2 fluorescence in a lingual slice by application of verapamil. The row of taste buds (seen as a darker layer between "c" and "m" in Fig. 1E) showed a faint fluorescence of fura 2 (Fig. 1F, left) after 60 min of incubation. Incubation with 200 µM verapamil for an additional 15 min enhanced dye accumulation in taste buds and connective tissue (Fig. 1F, right). Although fluorescence intensity increased, the general pattern of distribution remained the same, and a uniform distribution was not achieved. Similar results were obtained with another multidrug resistance-reversing agent, cyclosporin A (5 µM), which also increased fura 2 fluorescence intensity (data not shown).
Functional assay for P-glycoprotein. The involvement of a putative MDR1 transporter in dye extrusion was further assessed by measurement of the time course of intracellular accumulation of calcein in the absence or presence of three known inhibitors and/or substrates of MDR1. The dye calcein has the advantage that its AM form is nonfluorescent, whereas the intracellular hydrolysis product calcein is fluorescent and practically insensitive to changes in pH, Ca2+, and Mg2+. Calcein-AM, but not calcein, is a known substrate of MDR1 (12, 13). By means of fluorescence cell imaging we measured the effect of the three inhibitors of MDR1 on the intracellular accumulation of calcein.
Since fluorescence images like those in Fig. 1 indicated local differences of dye distribution, we selected subregions, the basal and apical regions of taste buds and the region of connective tissue subjacent to the layer of taste buds (labeled a, b, and c in Fig. 1C) to study uptake of calcein as a function of time. In the presence of calcein-AM (1 µM) the increase in fluorescence was linear or only slightly curved over 60 min in all three subregions. Solution change (arrow in Fig. 2A) with readdition of 1 µM calcein-AM had little effect on the slope of the fluorescence time course (Fig. 2A). Addition of verapamil (200 µM, arrow in Fig. 2B) increased the rate of increase in fluorescence in all subregions in four of five experiments. Verapamil was, however, not effective at lower concentrations. Addition of 5 µM cyclosporin A, an immunosuppressive cyclic peptide, also accelerated the development of fluorescence in four of five instances (Fig. 2C). Cyclosporin A already had an effect at a concentration of 2 µM in two of three experiments (data not shown). Tamoxifen (50 µM), a synthetic steroid analog and antiestrogen, also accelerated the development of calcein fluorescence in four of five experiments and was also effective at the lower concentration of 25 µM in two of three experiments. This suggests that the inhibitors of MDR1 (verapamil, cyclosporin A, and tamoxifen) prevented dye extrusion by inhibiting the transport function of MDR1 and thereby accelerated the net uptake of the fluorescent calcein.
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The intracellular hydrolysis products of the fluorescent dyes used in this study (calcein, fura 2, and BCECF) are polyanions. These may be eliminated from the cell by a variety of membrane proteins, including the multidrug resistance-associated protein (MRP), which is related to MDR1 (4). However, probenecid (2 mM), a known specific inhibitor of organic anion transport (22), which also prevents MRP-mediated calcein efflux from cells (7, 29), did not affect the calcein uptake rate in four of four experiments (data not shown).
A dye accumulation ratio was calculated by dividing the rate of net calcein accumulation in the presence of the inhibitor by the rate observed before addition of inhibitor. Table 1 shows the accumulation ratios obtained with the three inhibitors and/or substrates tested. The rates of calcein uptake varied considerably from slice to slice, but the quantitative differences of calcein uptake observed within the three subregions of one slice were consistent in all slices tested. Each of the drugs tested accelerated the net uptake of calcein 1.5- to 4-fold in the apical area of the taste buds (Table 1). Cyclosporin A (5 µM) was the most effective drug, producing up to a fourfold increase in rates. With tamoxifen and verapamil, a 50- to 200-fold higher concentration, respectively, of the drugs was necessary to produce similar effects. Table 1 also shows that, despite the differences in fluorescence distribution observed in the three subregions in individual experiments (see Fig. 1), the dye accumulation ratio, calculated in the presence of drugs, was similar in all three subregions selected for measurements.
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To ascertain that the increases in fluorescence accumulation seen with verapamil, cyclosporin A, and tamoxifen had not been caused by stimulation of intracellular esterase activities as opposed to inhibition of the MDR1 exit pathway, experiments were carried out with VP tissue homogenate, in which the exit pathway is no longer involved in the increase in calcein fluorescence. As shown in Fig. 3A, after addition of 1.25 µM calcein-AM to a cuvette containing 100 µg/ml VP homogenate suspended in a K+-rich buffer, calcein fluorescence increased steadily. Verapamil (0.2 mM), cyclosporin A (5 µM), or tamoxifen (20 µM) had no effect on calcein fluorescence in the cell-free esterase system. These results confirm the corresponding observations by Homolya et al. (13) with MDR1-transfected cell lines. The increase of calcein fluorescence observed with VP homogenate was caused by cellular esterases, since the nonspecific esterase inhibitor BNPP prevented fluorescence development in a concentration-dependent manner (Fig. 3B). At 30 mM BNPP, calcein fluorescence was abolished to the level observed that was measured in the absence of tissue homogenate.
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Further evidence that MDR1 is modulating the net uptake of calcein-AM was obtained with the blocking antibody UIC2, which is directed against extracellular epitopes of MDR1 (18). This monoclonal antibody has previously been shown to inhibit multidrug resistance (18) and to increase calcein fluorescence (12) in MDR1-expressing cell lines. When UIC2 (40 µg/ml) was added to slices of rat VP incubated in the presence of 1 µM calcein-AM (arrow in Fig. 4A), a significant increase of the intracellular fluorescence accumulation of calcein occurred in all selected areas of the VP slice. This effect was reproducible in slices obtained from three different animals, although the dye accumulation ratio (see above) varied between experiments. With a lower concentration of the antibody (20 µg/ml), an increase in fluorescence accumulation was seen in one of three different experiments only (data not shown). The effect of the MDR1 antibody UIC2 was specific, since addition of mouse IgG (40 µg/ml) did not cause any increase in calcein fluorescence (Fig. 4B).
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Immunohistochemistry. When paraffin sections of rat tongue lingual slices were immunostained with MDR1-specific antibodies, the pattern of immunoreactivity differed depending on the antibodies used. With the monoclonal antibody C494 (1 µg/ml), which detects a cytosolic epitope next to the COOH-terminal ATP-binding domain in the MDR1 isoform of P-glycoprotein only (9), immunoreactivity on the surface of taste bud cells was clearly demonstrated (Figs. 5, A and C). In addition, the basal cells of the lingual epithelium germinative stratum were labeled (Figs. 5, A and B). Here labeling appeared restricted to the lateral and apical cellular surfaces, whereas the membranes facing the basal lamina were not labeled. Diffuse cellular staining was shown by serous salivary glands as well as secretion by products accumulated in the luminal cleft of the VP (Fig. 5A). Furthermore, thin fiberlike structures in the interstitial space close to the VP, possibly nerve fibers, showed MDR1-like immunoreactivity with the C494 antibody. Controls carried out in the absence of primary antibody or with mouse IgG instead of C494 antibody were negative (data not shown).
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The monoclonal antibody C219 binds to two homologous sequences in the neighborhood of the NH2- and COOH-terminal ATP-binding domains present in all P-glycoprotein isoforms (9). Surprisingly, taste buds and lingual epithelium did not stain when C219 was used at 10 µg/ml (Fig. 5D), but serous portions of the lingual salivary glands and secretions accumulated in the luminal cavity of the VP were stained by the antibody C219 (Fig. 5D).
The monoclonal antibody UIC2 (1 µg/ml) had a staining pattern similar to that seen with C494, even though a higher background was present (data not shown).
Western blot analysis. The presence of MDR1 was further ascertained by immunoblotting with the MDR1-specific monoclonal antibodies C219 and C494. Single or dual VP and samples (30 mm2) from the PLE surrounding the VP were homogenized and processed for Western blot analysis with the monoclonal antibody C219. As shown in the first lane of Fig. 6, 5 µg/ml of the C219 antibody labeled a band of ~170 kDa, as expected for full size MDR1. In contrast, the Western blot of the sample of nonsensory epithelium (PLE, see Fig. 6, second lane) did not show labeling at ~170 kDa. In another Western blot, in which PLE from four rats was loaded onto the gel, a weak band with an apparent molecular mass of ~170 kDa was detected with the C219 monoclonal antibody (third lane), suggesting that MDR1 is expressed in rat tongue VP tissue and to a lesser degree in the surrounding nonsensory epithelium. Similarly, the monoclonal antibody C494 (5 µg/ml) labeled an ~170-kDa double band in VP preparations (fourth lane) but not in the PLE (fifth lane). The monoclonal antibodies C219 and C494 also cross-reacted with additional, lower-molecular-mass protein bands, which were detected both in VP and PLE (see Fig. 6).
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DISCUSSION |
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In the present study we provide evidence that MDR1 is expressed in taste buds of rat circumvallate papilla, where it counteracts the decreased accumulation of fluorescent dyes, such as calcein, in the cells.
MDR1 (6, 13, 21), MRP (7, 29), and as-yet-unidentified other ATP-dependent transport systems (1) have been implicated in the extrusion of fluorescent dyes from cells. Although fluorescent dyes may be extruded from taste cells by other transporters as well, several lines of evidence indicate that the ester forms of dyes are extruded from taste bud cells by MDR1, i.e., whereas substrates of MRP are most likely organic anionic compounds (7, 16, 29), MDR1 transports a large variety of hydrophobic, amphiphilic substrates as well as organic cations (11). Thus BCECF-AM, fura 2-AM, and calcein-AM are hydrophobic, electroneutral fluorescent dyes, which are substrates of MDR1. They are metabolized by intracellular esterases to organic anions, which are not transported by MDR1. Stimulation of calcein and fura 2 accumulation in taste cells displayed a distinct pharmacological profile, indicative of MDR1-mediated fluorescent dye extrusion, i.e., the substrates and/or inhibitors of MDR1 [cyclosporin A, verapamil, and tamoxifen (8, 11)] increased the accumulation of calcein or fura 2 into taste buds (Figs. 1 and 2, and Table 1). This effect was not caused by stimulation of intracellular esterases, as shown in Fig. 3. In contrast, probenecid, an inhibitor of organic anion transport that also inhibits MRP-mediated efflux of the anionic hydrolysis products of the fluorescent dyes from the cytosol into the extracellular space (7, 29), had no effect on calcein accumulation into taste cells. Finally, UIC2, a monoclonal antibody to extracellular domains of MDR1, which inhibits multidrug resistance in MDR1-expressing cell lines only (18), increased calcein accumulation in rat VP (see Fig. 4). This strongly indicates that MDR1 but not MRP is the main route of extrusion that interferes with the accumulation of fluorescent dyes in taste cells.
Additional evidence for the presence of MDR1 in taste bud cells was obtained by immunostaining of taste tissues with MDR1-specific antibodies (Fig. 5). Both monoclonal antibodies C494 (Fig. 5, A and C) and UIC2 (data not shown) stained the surface of taste bud cells. However, with the monoclonal antibody C219, no staining of taste bud cells was found (Fig. 5D). This is in agreement with a previous report that showed lack of P-glycoprotein detection in paraffin-embedded sections with this antibody (5), even though Western blots of the same tissues were positive (11). In Western blots of tissue from an isolated circumvallate papilla, the C219 and C494 antibodies detected an immunoreactive band of ~170 kDa (Fig. 6), as expected for full-length P-glycoprotein.
Cells in the germinative layer of the lingual epithelium were also labeled by the C494 antibody, suggesting expression of MDR1 in stem cells of the lingual epithelium. The presence of MDR1 in the lingual epithelium was confirmed in Western blots of epithelial tissue with the C219 antibody (Fig. 6, third lane). Expression of MDR1 in the lingual epithelium, however, must be low, since much higher amounts of protein had to be loaded onto the gel for immunodetection of the 170-kDa protein band. Furthermore, in epithelial slices free of taste buds, cyclosporin A and tamoxifen occasionally increased calcein accumulation (data not shown). An interesting report has demonstrated the expression of MDR1 in squamous cell carcinomas of the tongue base by immunohistochemistry with the C494 monoclonal antibody (23). Unfortunately, expression of MDR1 in normal tissue of the tongue base was not included in this study.
The distribution of fluorescent dyes within the taste bud cells showed a preferential accumulation of dye over the contraluminal poles of the cells (Fig. 1D), suggesting that extrusion of the dye mainly occurred at the luminal membrane. However, immunohistochemistry showed no polar distribution of immunolabeling in taste cells. One possible explanation is that, due to spatial restrictions by the tissue surrounding the basolateral area of taste bud cells, differences in the concentration gradients of the fluorescent dye generated across the apical or basolateral plasma membrane of taste cells favor luminal extrusion of the dye into the oral cavity. To test this hypothesis, dye extrusion studies of calcein across cell membranes should be carried out (see Ref. 20).
Surprisingly, the dye accumulation ratios calculated in the presence of drugs in all three subregions selected for measurements were similar (Table 1), irrespective of the differences of the absolute values of the accumulation rates found in the different subregions of taste bud cells (Fig. 2). Kinetic considerations described below (APPENDIX) suggest, however, that the accumulation ratios will be constant and independent of the rate of accumulation if the membrane density of MDR1 molecules is the same in the subregions selected within the taste bud cells. Indeed, immunohistochemistry suggests an even distribution of MDR1 molecules on the surface of taste bud cells (see Fig. 5C).
MDR1 is expressed in the luminal membrane of cells lining internal surfaces, such as kidney proximal tubule, small intestine, bile canaliculi, and capillary endothelia of the brain (26). Based on this tissue distribution, it has been proposed that MDR1 plays a role in the protection of the organism against toxic xenobiotics (11), i.e., by actively excreting these toxic molecules into bile, urine, or the intestinal lumen, MDR1 would prevent toxic injury of the tissues. Receptor cells of the taste buds line the oral cavity and are therefore permanently exposed to a large variety of environmental molecules, which may interfere with the sensory function and be harmful to the cells. The presence of MDR1 in the plasma membrane of taste cells, therefore, appears to be a useful cytoprotective mechanism developed by these cells.
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APPENDIX |
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Consider a dye molecule (e.g., calcein-AM) that is present in the
extracellular space as a nonfluorescent AM in concentration c1. The molecule will enter a cell
by diffusion through the cell membrane (rate constant
km) and will be
found in the cytosol in concentration
c2 (Fig.
7). The net uptake by diffusion will be km(c1 c2). From the cytosol
the dye ester can be moved outward by MDR1-mediated, ATP-driven
transport (rate constant
kp) or be converted to a fluorescent molecule by cellular esterase (rate constant
ke). Because of
this esterase step, the charged fluorescent dye will accumulate in the
cytosol.
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Because dye accumulation was generally slow, we will assume that the
esterase step was rate limiting for the generation of c3, i.e., that
ke km,
kp. This means
that a partial equilibrium is established with respect to
c2, as also assumed by Goodfellow et al. (10). We obtain by the law of mass action
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(1) |
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(2) |
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(2a) |
In a given lingual slice the rate of accumulation of fluorescent dye, although varying with position in the slice (Fig. 1, C and D), was enhanced everywhere by inhibitors of MDR1-mediated transport (Fig. 2). However, when the rate of accumulation observed with the inhibitor was divided by the control rate, only values between 2 and 3 were found (the "accumulation ratios" of Table 1), irrespective of the absolute values of accumulation rates. We are now asking under which conditions this might occur. For simplicity, the discussion is restricted to the case of a linear increase of fluorescence with time (kc = 0).
In the presence of an MDR1 inhibitor, kp will be decreased to k'p. Equation 2 then becomes
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(2b) |
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(3) |
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Riess (Institute for Pathology, City Hospital of Nuremberg, Germany) and B. Hausknecht (Department of Internal Medicine IV, University of Erlangen/Nuremberg, Germany) for expert advice in setting up the immunohistochemistry protocol. Valuable suggestions for the immunohistochemistry protocol were also made by Dr. O. Kretz (Department of Anatomy, Saar University, Homburg, Germany).
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246-C1 to B. Lindemann and 246-C6 to F. Thévenod).
Address for reprint requests: F. Thévenod, Physiologisches Institut, Universität des Saarlandes, Medizinische Fakultät, D-66421 Homburg/Saar, Germany.
Received 9 June 1997; accepted in final form 29 September 1997.
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