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. 2002 Nov 29;546(Pt 2):375–385. doi: 10.1113/jphysiol.2002.033175

Multidrug resistance transporters in the olfactory receptor neurons of Xenopus laevis tadpoles

Ivan Manzini 1, Detlev Schild 1
PMCID: PMC2342511  PMID: 12527725

Abstract

Olfactory receptor neurons (ORNs) are the only class of neurons that is directly exposed to the environment. Therefore, they need to deal with xenobiotic and potentially cytotoxic substances. Here we show for the first time that ORNs possess transporter systems that expel xenobiotics across the plasma membrane. Using calcein and calcium-indicator dyes as xenobiotics, we demonstrate that ORNs appear to express the multidrug resistance P-glycoprotein (MDR1) and multidrug resistance-associated proteins (MRP). This endows ORNs with the ability to transport a large number of substrates including calcium-indicator dyes and calcein across their plasma membranes. Conversely, blocking P-glycoprotein and MRP increases the net uptake of these dyes.

Olfactory receptor neurons (ORNs) sample the environment for odorants. Situated in the olfactory epithelium, they are the only neurons that are in direct contact with the animal's environment (for reviews, see Getchell et al. 1984; Schild & Restrepo, 1998). Due to this exposure, some odorants and other xenobiotic compounds constitute a potential cytotoxic threat to ORNs. Removal of xenobiotics from the olfactory epithelium is thus essential. Previous studies have shown that odorants can be degraded by cytochrome P450 and UDP glucuronosyl transferase (UGT) in the vertebrate olfactory epithelium (Lazard et al. 1991) and by degrading enzymes in the sensilla lymph of insects (Vogt et al. 1985; Maida et al. 1995).

The possibility of xenobiotic compounds being expelled across the plasma membrane of ORNs has not yet been investigated. In other cells and tissues, such as the liver, kidney, gut, trachea and tumour cells, multidrug resistance P-glycoprotein (MDR1) and multidrug-resistance-associated proteins (MRP) are known to extrude xenobiotics (Galietta et al. 1996; König et al. 1999; Inui et al. 2000; Mottino et al. 2000; Renes et al. 2000; Tang et al. 2000; Abrahamse & Rechkemmer, 2001; Barnes, 2001; Doi et al. 2001). Multidrug resistance transporters are also expressed in taste sensory cells, where they are reported to extrude fluorescent dyes (Jakob et al. 1998).

The starting point for this investigation was our finding that the cells in the olfactory epithelium of Xenopus laevis tadpoles cannot be loaded with the acetoxymethyl (AM) ester derivates of calcium-indicator dyes. We investigated the mechanisms underlying this phenomenon and demonstrate that ORNs express two transport systems with a wide substrate spectrum in their plasma membrane.

Methods

Slice preparation

Tadpoles of Xenopus laevis (stage 51-54; Nieuwkoop & Faber, 1956) were chilled in a mixture of ice and water and decapitated, as approved by the Göttingen University Committee for Ethics in Animal Experimentation. A block of tissue containing the olfactory mucosa (OM), the olfactory nerves and the anterior two-thirds of the brain were cut out and kept in bath solution of the following composition (mm): 98 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 5 glucose, 5 sodium pyruvate, 10 Hepes; 230 mosmol, pH 7.8. The tissue block was glued onto the stage of a vibroslicer (VT 1000S, Leica, Bensheim, Germany) and cut horizontally into 120-125 μm-thick slices (Peters et al. 2000).

Incubation and fluorescence measurements

The slices were placed between two grids in a recording chamber to allow diffusion from both sides. The AM esters of the fluorescent dyes and the transport inhibitors were applied to the bath solution at the required concentrations. We used fura-red, fura-2, Calcium Green and calcein as fluorescent dyes and potential transporter substrates, and probenecid, sulphinpyrazone, MK571, verapamil and PSC 833 as transport inhibitors. The dyes were dissolved in DMSO and Pluronic F-127. The inhibitors used were dissolved according to the instructions provided by the suppliers. The fluorescent dyes used and Pluronic F-127 were purchased from Molecular Probes (Leiden, The Netherlands). PSC 833 was a generous gift from Novartis Pharma (Basel, Switzerland) and MK571 was purchased from Alexis Biochemicals (Grünberg, Germany). All other chemicals were from Sigma (Deisenhofen, Germany).

The recording chamber was placed on the microscope stage and recording was started approximately 2 min after the beginning of incubation. To observe the time course of dye loading we used either an Axiovert 100M microscope (Zeiss, Jena, Germany) to which a laser-scanning unit (LSM 510, Zeiss, Jena, Germany) was attached, or an Axioskop 2 microscope (Zeiss, Göttingen, Germany) with a CCD camera attached (Micromax, Visitron, München, Germany). The fluorescence intensities of all dyes except fura-2 were measured using the LSM 510 and 488 nm as the excitation wavelength. fura-2 was excited at 380 nm and its loading was followed using the CCD camera.

The progressive increase in fluorescence in the slice was measured by taking an image every 3 min (in the case of fura-red or Calcium Green) or every 1 min (in the case of calcein). The fluorescence images were evaluated by calculating intensity histograms and average intensities, leaving out all pixels not covered by the slice. Thus, the intensities shown in this paper are averages over all cells in a slice.

Transporter model

To describe the uptake and removal of fluorescent dyes into and from cells, we started out with the model used by Jakob et al. (1998). The tissue under investigation is incubated in an AM ester of a fluorescent dye with extracellular concentration co. The molecules diffuse through the plasma membrane (rate constant, kD; flux, kD (co – cAM)) into the cytosol, and intracellular dye/AM-molecules with concentration cAM are hydrolysed with rate ke or extruded through P-glycoprotein across the plasma membrane (rate kp). Assuming the hydrolysis of the AM ester as the rate-limiting step (Goodfellow et al. 1996; Jakob et al. 1998), cAM approaches a quasi-stationary concentration, which follows from the condition dcAM/dt = 0. In other words:

graphic file with name tjp0546-0375-mu1.jpg

or

graphic file with name tjp0546-0375-m1.jpg (1)

While cAM cannot be observed directly, the fluorescence of the corresponding salt is a measure of its concentration cs, which increases with ongoing hydrolysis.

With

graphic file with name tjp0546-0375-m2.jpg (2)

and eqn (1) we have:

graphic file with name tjp0546-0375-m3.jpg (3)

Thus, the concentration cs increases linearly, and the slope of the increase is limited by the pump rate kp. In case the pump is completely blocked or not expressing (kp = 0), the increase in cs is maximum. Otherwise the pump activity reduces the increase of cs, and thus the increase in fluorescence, with time.

Fluorescence intensities are assumed to be proportional to cs, so that a constant rise of cs is reflected by a constant increase in fluorescence F, i.e. ΔF/Δt = constant.

Immunocytochemistry

Tadpoles of Xenopus laevis (stages 52-54) were chilled and killed as described above. A block of tissue containing the OM, the olfactory nerves and the anterior part of the brain were cut out and immediately transferred in 4 % paraformaldehyde (PFA) in phosphate buffer, pH 7.4, for at least 2 h at room temperature or overnight at 4 °C. For further processing, the tissue blocks were washed in phosphate buffered saline (PBS), embedded in 5 % low-melting-point agarose (Sigma), and sectioned at 70 μm on a vibroslicer (VT 1000S, Leica, Bensheim, Germany). Sections were then washed in PBS containing 0.2 % Triton X-100 (PBST), and non-specific binding was blocked with 2 % normal goat serum (NGS; ICN, Aurora, Ohio, USA) in PBST for 1 h at room temperature. The tissue was then incubated overnight at 4 °C in primary monoclonal antibody P-glycoprotein C219 (1:20 dilution; Alexis Biochemicals) diluted in 2 % NGS/PBST or in natural mouse serum (NMS, 1:20; Sigma) as a negative control. Primary antibody or NMS was washed off with PBS, and Alexa 488-conjugated goat anti-mouse secondary antibodies (Molecular Probes) were applied at a dilution of 1:250 in 1 % NGS/PBS for 2 h at room temperature. The secondary antibody was washed off in several changes of PBS and then the sections were incubated for 15 min in 25 μg ml−1 propidium iodide (Molecular Probes) in PBS for cell nuclei staining. Sections were washed in at least five changes of PBS, transferred into 60 % glycerol/PBS for at least 1 h and then mounted on slides in 80 % glycerol/PBS. Preparations were viewed and imaged using a laser-scanning confocal microscope (Zeiss LSM 510/Axiovert 100, Jena, Germany).

Results

The starting point for this study was the surprising observation shown in Fig. 1. When a tissue slice containing the OM and the olfactory bulb (OB) (Fig. 1A) was incubated for 60 min in fura-red/AM, the cells of the OB were clearly loaded with the dye (Fig. 1B), while the OM exhibited almost no staining (Fig. 1C).

Figure 1. Effect of probenecid on the net uptake of calcium-indicator dyes in the olfactory mucosa (OM) and the olfactory bulb (OB).

Figure 1

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A, low-magnification view of the OM, the olfactory nerve (ON) and the OB. B and C, laser scanning images of the OB (B) and the OM (C) of a slice incubated for 60 min in bath solution containing 50 μm fura-red/AM. D and E, OB (D) and OM (E) of a slice incubated for 60 min in bath solution containing 50 μm fura-red/AM and probenecid 2.5 mm. F and G, slice of an OM exposed to 50 μm fura-red/AM for 30 min without probenecid (F), and then for 30 min following addition of 2.5 mm probenecid to the bath (G). H and I, CCD images of a slice of an OM incubated for 30 min in 50 μm fura-2/AM without probenecid (H) and with probenecid (2.5 mm) (I). For clarity, the borders of the epithelium are drawn in H and I. Scale bars, 500 μm in A and 50 μm in F and I. Bar scales from low (black) to high (white) fluorescence intensities (12 bit, B-G).

Assuming transporter-mediated destaining during incubation, we repeated the dye-loading experiment with probenecid (2.5 mm) added to the incubation solution (all other parameters kept constant). Probenecid is a known inhibitor of organic anion transport (Pritchard & Miller, 1993; Burckhardt & Pritchard, 2000), which also inhibits MRP-mediated calcein efflux from cells (Feller et al. 1995; Versantvoort et al. 1995). With probenecid added to the bath, the dye uptake by the OM was markedly increased (Fig. 1E) and became similar to the uptake by the OB (Fig. 1D). The OB itself, especially the neuropil, was also stained more intensely (Fig. 1D).

Poor staining of the OM occurred not only with fura-red/AM incubation (observed in 10 slices), but likewise after incubation with Calcium Green/AM (three slices), fura-2/AM (eight slices) and calcein/AM (four slices). Adding probenecid (2.5 mm) to the bath solution increased the staining with fura-red, Calcium Green, fura-2 and calcein (n = 6, 3, 5 and 4 slices, respectively). To see the probenecid-induced increase of staining in one slice at a time, we incubated six slices for 30 min in fura-red/AM without probenecid (Fig. 1F) and then for another 30 min with probenecid (Fig. 1G). Clearly, the staining during the second 30 min was more effective. The intensity histograms (see Methods) underwent a marked right-shift during incubation (Fig. 2).

Figure 2. Intensity histograms of confocal images of the olfactory epithelium.

Figure 2

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A, intensity histogram of the image shown in Fig. 1F (control). B, intensity histogram corresponding to Fig. 1G (probenecid in the incubation solution). Pixels that were not covered by the slice were not considered for calculating the histograms.

These experiments were carried out using a laser-scanning microscope. To measure the net uptake of fura-2/AM we used CCD-imaging. The resulting images, although blurred and devoid of confocal contrast, show very little fluorescence after 30 min incubation in fura-2/AM in the absence of probenecid (Fig. 1H), but marked fura-2 fluorescence after another 30 min incubation in fura-2/AM and probenecid (2.5 mm; Fig. 1I). Similar results were observed in seven other slices.

We investigated the time course of staining by taking images at constant time intervals while incubating the slice and representing the intensities I averaged over the slice as a function of time (t, I(t)).

The average intensities I(t) of fura-red increased very slowly if no transport inhibitor was present (Fig. 3A, from t = 0 to 40 min). In the example shown, the fluorescence increase was 0.12 min−1 without probenecid and 0.69 min−1 with probenecid (2.5 mm). The net uptake ratio was thus 5.8. In 11 identically performed fura-red/AM incubation experiments we found an average net uptake ratio of 5.78 ± 1.41.

Figure 3. Time courses of fura-red accumulation in mucosal slices in the absence and presence of probenecid, MK571 and of cyclic AMP.

Figure 3

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Mucosal slices were incubated in 50 μm fura red/AM (in bath solution). The bars under the traces show the time over which the indicated drug was added to the incubation solution. The concentration of fura red/AM was kept constant over the whole incubation. Effects of 2.5 mm probenecid (A) and 50 μm MK571 (B). C, fura-red accumulation in six individual olfactory receptor neurons before and after application of 50 μm MK571. D, no effect of 5 mm cAMP upon the uptake of fura-red.

MK571, a specific inhibitor of MRP (Gekeler et al. 1995; Abrahamse & Rechkemmer, 2001), affected the staining with fura-red/AM in a way that was comparable to probenecid (Fig. 3B). The average net uptake ratio in nine identically performed experiments, with MK571 added at a concentration of 50 μm, was 6.73 ± 1.41. In individual ORNs randomly selected from a slice of the OM, MK571 (50 μm) had the same effect on the uptake of fura-red, producing net uptake ratios in the range between 5.91 and 11.32 (n = 6 ORNs, Fig. 3C).

One of the MRP isoforms, MRP-5, is known to transport cyclic nucleotides such as cAMP (Jedlitschky et al. 2000). As cAMP is the second messenger in many ORNs (Schild & Restrepo, 1998), it was intriguing to see whether or not cAMP increased the net uptake of fura-red (i.e. whether cAMP would be extruded across the plasma membrane). In six out of six slices this was not the case (Fig. 3D).

Fluorophore bleaching did not affect our experiments. Even a 60-fold increased laser power exposure did not lead to a noticeable amount of bleaching. In this test experiment we loaded slices with calcein/AM in presence of MK571 (50 μm) for 1 h. After the loading phase we applied a bath solution containing MK571 (50 μm) but no dye. We then measured the fluorescence at a laser power P0, as used in all of our experiments. Even at a high frame acquisition rate of 1 frame s−1 (i.e. 60 or 180 times faster than in our experiments with calcein or calcium dyes, respectively), no bleaching was observed (Fig. 4). However, when taking pairs of images, one pair per 2 s, the first at 10 times the laser power used before (10P0) and the second at P0, a fast bleaching of fluorescence occurred (Fig. 4). As the average exposure in our experiments was at least 60 times less than in the first part of Fig. 4, we conclude that bleaching did not affect our measurements.

Figure 4. Fluorophore bleaching does not interfere with dye accumulation measurements.

Figure 4

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A mucosal slice loaded with calcein and placed in a bath solution containing MK571 (50 μm) was exposed, once every second, to a laser beam power P0. In all experiments reported in this manuscript (except for this figure), the power P0 was applied either once every minute (calcein) or once every 3 min (calcium dyes). For the experiments shown in this figure, frames were taken once every second. The exposure to P0, even at a frame rate of 1 s−1, did not induce fluorophore bleaching (grey bar, laser power P0). In a second phase of this experiment (black bar, laser power 10P0), we applied, once every 2 s, twin pulses, the first being ten times stronger (10P0) than the second (P0). This resulted in a substantial fluorophore bleaching.

Interestingly, verapamil, a known inhibitor of P-glycoprotein (Ford & Hait, 1990; Fujita et al. 1997; Jakob et al. 1998; Abrahamse & Rechkemmer, 2001; Laupeze et al. 2001), improved the net uptake of fura-red, although with a lower net uptake ratio (Fig. 5A). The inhibitory effects on the extrusion system(s) of verapamil and MK571 were clearly saturated at 125 and 50 μm, respectively (Fig. 5A and B). Still, verapamil together with MK571, both at saturating concentrations, were more efficient in blocking the extrusion of fura-red than was MK571 alone (Fig. 5C). This suggests that fura-red was extruded by both MRP and P-glycoprotein.

Figure 5. Time courses of fura-red accumulation in mucosal slices in the absence and presence of inhibitors of multidrug resistance P-glycoprotein (MDR1) and of multidrug resistance-associated proteins (MRP).

Figure 5

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Mucosal slices were incubated in bath solution containing 50 μm fura-red/AM. A, effects of 125 μm (dashed line) and 500 μm verapamil (continuous line) upon dye uptake are similar. B, MK571 at 50 μm (dashed line) had virtually the same effect as the same drug at 200 μm (continuous line). The average net uptake ratios in A for verapamil concentrations 125 and 500 μm were 2.73 ± 0.78 (n = 4) and 2.55 ± 0.52 (n = 5), respectively. With MK571 50 and 200 μm, the average net uptake ratios were 6.73 ± 1.41 (n = 9) and 6.36 ± 0.66 (n = 6), respectively. C, a mucosal slice incubated in bath solution with 50 μm fura-red/AM was exposed, first, for 40 min to MK571 (50 μm) and then for another 40 min to both MK571 (50 μm) and verapamil (125 μm). The accumulation effects are clearly additive. The average net uptake ratio was 1.69 ± 0.12 (six slices). The laser power in C was half the power used in A and B.

The presence of P-glycoprotein in Xenopus laevis tadpole ORNs was confirmed by incubation and staining with a monoclonal antibody (see Methods), showing a spotty antibody staining predominantly in the basolateral compartments of cells in the epithelium (Fig. 6).

Figure 6. Immunostaining of the olfactory epithelium with the MDR1-specific monoclonal antibody C219.

Figure 6

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A, immunostaining of a mucosal slice of a Xenopus laevis tadpole by C219 antibody (1:20, green fluorescence). B, higher magnification of A. The slice was counterstained with propidium iodide to show cell nuclei (red fluorescence). Note that the antibody stains the basolateral part (cell bodies and dendrites) of the cells. No detectable staining was observed in knobs and cilia. C and D, negative control: mucosal slice incubated in natural mouse serum (1:20) instead of primary antibody. Scale bars, 50 μm in A and C, and 10 μm in B and D.

The linear time course of the net uptake curves of fura-red is consistent with its salt not being removed from the cells (see Discussion, eqn (4), kM,s = 0). However, if Calcium Green/AM was used as a calcium-indicator dye and probenecid (2.5 mm) as a transport inhibitor, the intensity time course, I(t), deviated markedly from linearity (Fig. 7A). I(t) instead saturated and appeared to be proportional to (1 – e−t/τ) suggesting a concentration-dependent extrusion of the salt of Calcium Green (see Discussion, eqn (4)).

Figure 7. Non-linear time course of Calcium Green accumulation in mucosal slices without and with probenecid and of calcein accumulation without and with probenecid, sulphinpyrazone and MK571.

Figure 7

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Mucosal slices were incubated in bath solution containing 50 μm Calcium Green/AM (A) or 250 nm calcein/AM (B, C and D). Effects of the addition of 2.5 mm probenecid (A and B), 1 mm sulphinpyrazone (C) and 50 μm MK571 (D) are shown. Virtually identical results were obtained from 5, 12, 6 and 5 slices treated the same way as shown in A, B, C and D, respectively.

Calcein, the fluorescence of which is calcium independent and which is structurally related to Calcium Green rather than to fura-red, had net uptake kinetics similar to that of Calcium Green (Fig. 7B). Virtually identical results were obtained when using sulphinpyrazone (1 mm), another known inhibitor of organic anion transport (Decléves et al. 2000; Abrahamse & Rechkemmer, 2001; Fig. 7C) or MK571 (50 μm, Fig. 7D) as transport blockers.

While Fig. 7 shows that calcein is extruded by MDR, its extrusion was also reduced by verapamil (250 μm, Fig. 8A) and PSC 833 (10 μm, Fig. 8B), two specific blockers of P-glycoprotein (Decléves et al. 2000; Miller et al. 2000; Thévenod et al. 2000). Thus, both MDR and P-glycoprotein are expressed in Xenopus laevis tadpole ORNs.

Figure 8. Verapamil and PSC 833 increase calcein accumulation.

Figure 8

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Mucosal slices were incubated in a bath solution containing 250 nm calcein/AM. Effects of the addition of 250 μm verapamil (A) and 10 μm PSC 833 (B) are shown. Virtually identical results were obtained from five and six slices treated the same way as shown in A and B, respectively.

We have thus shown the increased efficiency of dye uptake by blocking MDR and P-glycoprotein while the slices were incubated in a solution containing AM esters of fluorescent dyes. As a last part of our study we analysed the effect of MK571 after the dye had been taken up and was washed out of the bath. Figure 9 shows an example of blocked calcein extrusion. After removal of calcein/AM from the bath, the fluorescence decreased slowly as long as MK571 (50 μm) was applied, while without MK571 in the bath, the decrease of fluorescence was markedly faster (Fig. 9A). On the other hand, in standard bath solution (i.e. without calcein/AM and without MK571), the fast extrusion of calcein was almost completely stopped by adding MK571 (50 μm) to the bath (Fig. 9B).

Figure 9. MK571 blocks calcein extrusion.

Figure 9

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A, fluorescence of a mucosal slice loaded with calcein and placed in a bath solution containing MK571 (50 μm). After 40 min MK571 was removed leading to a fast decay of fluorescence. B, the complementary case where a mucosal slice loaded with calcein was first placed in standard bath solution without blocker leading to a fast decay of fluorescence. After 40 min addition of MK571 (50 μm) virtually stopped the fluorescence decay. Identical results were obtained from three and four slices treated the same way as shown in A and B, respectively.

Discussion

Membrane transport systems for neutral compounds as well as for organic cations and organic anions have been studied primarily in the liver, gut and kidney (Koepsell, 1998; Szakacs et al. 1998; Borst et al. 2000; Burckhardt & Pritchard, 2000; Burckhardt & Wolff, 2000; Renes et al. 2000; Barnes, 2001; Leslie et al. 2001), and calcium-indicator dyes are known substrates of several of these transport systems (Neyfakh, 1988; Di Virgilio et al. 1989; Homolya et al. 1993). Taste sensory cells, which are in direct contact with the environment, have recently been shown to express the multidrug resistance P-glycoprotein, MDR1 (Jakob et al. 1998).

That Xenopus laevis tadpole ORNs could not be loaded with calcium/AM-dyes was surprising because calcium was successfully imaged in ORNs in a number of species (Lischka & Schild, 1993; Jung et al. 1994; Leinders-Zufall et al. 2000; Ma & Shepherd, 2000). Calcium-indicator dye-removal mechanisms thus appear to differ in different species and even at different stages in one particular species (e.g. Xenopus laevis). When calcium-indicator dyes are dialysed into the cytosol through a patch pipette, removal is of minor importance, since removal is compensated for by dye molecules entering the cell through the pipette. On the other hand, in cases of loading with AM esters of dyes, it has to be borne in mind that some dye removal can easily be overlooked. In many studies where fluorescence intensities are sufficiently high for imaging purposes, one usually does not consider the possibility of indicator dye removal. Figure 1B and D exemplifies such a case.

Loading calcium dyes or calcein into tadpole ORNs could be modulated by probenecid, sulphinpyrazone, MK571, verapamil and PSC 833 (see Figs 1, 3, 5, 7 and 8). The dynamics of fluorescence increase was linear if ORNs were incubated in fura-red/AM and nonlinear in the case of incubation with the AM esters of calcein or Calcium Green. In other species and other cell systems, the AM esters of calcium dyes and of calcein are reported to be transported by P-glycoprotein (Homolya et al. 1993; Brezden et al. 1994; Essodaigui et al. 1998) and MRP (Essodaigui et al. 1998; Olson et al. 2001). In addition, MRP has been reported to transport the salts of calcein, fluo-3,2′-7′bis(2-carboxyethyl)-5(6)-carboxyfluorescein and fluorescein (Feller et al. 1995; Draper et al. 1997a, b; Essodaigui et al. 1998; Nies et al. 1998; Vernhet et al. 2000; Abrahamse & Rechkemmer, 2001; Sun et al. 2001). Verapamil is known to block the P-glycoprotein (Ford & Hait, 1990; Homolya et al. 1993), while it is believed to have no specific effect on MRP. PSC 833 is a specific inhibitor of P-glycoprotein that has no effect on MRP (Decléves et al. 2000; Miller et al. 2000; Thévenod et al. 2000). On the other hand, probenecid, which is a known blocker of many anion transport systems (Pritchard & Miller, 1993; Burckhardt & Pritchard, 2000), also blocks MRP but not P-glycoprotein (Feller et al. 1995; Versantvoort et al. 1995; Evers et al. 1996; Gollapudi et al. 1997; Jakob et al. 1998). MK571 and sulphinpyrazone are believed to specifically block MRP, with no effect upon P-glycoprotein (Gekeler et al. 1995; Evers et al. 1996). Taking into account these properties, our data are consistent with the model shown diagrammatically in Fig. 10. In this model, the AM esters of all dyes used are extruded through both MRP and P-glycoprotein. In addition, the salts of Calcium Green and calcein, but not of fura-red are extruded through MRP. Let the extrusion rates of MRP for the AM and the salt species be kM, AM and kM, s, respectively, then eqn (3) (see Methods) takes the form:

graphic file with name tjp0546-0375-m4.jpg (4)

The solution of this first-order differential equation is an exponential function, proportional to 1 – e−t/τ. Thus, the fluorescence increases in a non-linear, saturating way if the salt of a calcium-indicator dye is extruded from the cell, and in a linear way if this is not the case.

Figure 10. Model of fluorescent dye accumulation in olfactory receptor neurons.

Figure 10

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co and cAM, concentrations of AM ester of fluorescent dyes outside and inside the cell; cs, concentration of the hydrolysed dye inside the cell. AM-dye molecules enter the cytosol of the cell by diffusion through the plasma membrane (rate constant kD). The AM-dye molecules can be extruded from the cytosol of the cell either by the P-glycoprotein (P-gp, rate constant kp) or by an MRP (rate constant kM, AM). In addition, it is converted to the corresponding salt of the dye by cellular esterases (rate constant ke). The MRP also transports the salt form of some fluorescent dyes (rate constant kM,s).

This description is a simplification in that possible interactions between AM dye and salt transport as well as the ATP-dependence of MRP are neglected. However, the model appears to be adequate in that it reflects the linear fluorescence increase of fura-red (for which kM, s = 0) and the saturating behaviour of calcein and Calcium Green (for which kM,s =/ 0). There are, however, too many free and unknown parameters to fit, in an unambiguous way, the model to our data.

Our results regarding the drug modulation and the time course of fluorescent dye net uptake differ in two respects from the removal mechanism found in taste sensory cells. First, dye removal can be blocked not only by P-glycoprotein inhibitors (verapamil and PSC 833), as in taste cells, indicating removal through P-glycoprotein, but also by probenecid, MK751, and sulphinpyrazone, indicating strongly an additional removal route through MRP. The presence of two different transporter systems is further supported by the facts that MK571 had a larger inhibitory effect upon dye removal than verapamil or PSC 833 and that the effect of verapamil is added to that of MK571. Second, the kinetics of removal is linear (as in taste cells) for fura-red, but non-linear for Calcium Green and calcein, indicating the concentration-dependent removal of the salts of the latter two. MRP-mediated removal of the calcein salt was confirmed by the fact that destaining of slices loaded with calcein was blocked by MK571. As the calcein salt is known to be extruded by MRP (Essodaigui et al. 1998; Vernhet et al. 2000), this confirms the involvement of MRP.

Antibodies against P-glycoprotein stained the non-apical parts of ORNs and sustentacular cells. Although this qualitative evidence does not rule out a minor expression of P-glycoprotein on cilia, the major transport route appears to be across the basolateral membranes. Our attempts to stain MRP in the Xenopus laevis tadpole mucosa using antibodies against rat, mouse or human MRP were unsucccessful. Considering that dye extrusion was observed not only in ORNs but also in sustentacular cells, it may thus be hypothesized that sustentacular cells are involved in degrading xenobiotics.

Acknowledgments

We are grateful to Professor Dr G. Burckhardt for valuable discussions.

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