Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Christopher M. Breen, Destiny B. Sykes, Carsten Baehr, Gert Fricker, and David S. Miller作者单位：1 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and 2 Institute of Pharmacy and Molecular Biotechnology, INF 36 D-69120 Heidelberg, Germany


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      【摘要】
   One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 µM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1. 1 o6 m& l8 x; L1 D( \  D
      【关键词】 image analysis xenobiotic transport' p6 d- w) c& a, x% w9 w" j
   THE CELLS OF THE central nervous system (CNS) are particularly sensitive to chemical injury and thus require a highly regulated extracellular environment. In this regard, the choroid plexus is a potent excretory tissue that transports xenobiotics and metabolic wastes from cerebrospinal fluid (CSF) to blood for eventual elimination in urine and bile. Like the renal proximal tubule and liver, choroid plexus possesses one or more excretory transport systems for organic anions. A number of compounds are substrates for transport; these include small organic anions, neurotransmitter metabolites, drugs, environmental pollutants ( 6, 8, 18 ), and larger organic anions, e.g., methotrexate (MTX; see Refs. 1 and 17 ). Molecular studies indicate that members of the organic anion transporter (Oat), organic anion transport polypeptide (Oatp), and multidrug resistance-associated protein (Mrp) subfamillies of transporters are expressed in the tissue ( Fig. 1 ). Our understanding of the mechanisms by which choroid plexus handles organic anions is hampered by the small size of the tissue and its inaccessible location and morphological complexity. To date, information about transport function in intact tissue has come from studies of solute movements in vivo and from tracer uptake experiments in vitro. These approaches treat the tissue as a "black box," providing some understanding of the mechanisms that drive xenobiotic uptake at the apical membrane but little information on subsequent steps.9 C5 L. D, l: B! [3 Q$ y! }
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Fig. 1. Distribution of transporters for anionic xenobiotics in rat choroid plexus epithelial cells. Only shown are those transporters that have been immunolocalized to a specific side of the cell ( 7, 11, 12, 13, 15, 24 ). In addition, high to moderate levels of mRNA for organic anion transporter (Oat) 2, multidrug resistance-associated protein (Mrp) 4, and Mrp5 were detected in the tissue; very low levels of mRNA for Oat1, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K1/2, Mrp 2, and Mrp3 were also detected ( 5 ). At present, it is not clear to what extent any of these transport proteins are expressed in the tissue. CSF, cerebrospinal fluid.5 r9 Y! V$ b* f3 N1 E
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One approach to understanding transport function in intact tissue is to image the movement of fluorescent substrates using microscopy. Twenty-five years ago, Bresler and co-workers ( 3 ) used conventional fluorescence microscopy to demonstrate that the fluorescent small organic anion fluorescein (FL) accumulated within choroid plexus tissue. More recently, we used confocal microscopy and quantitative image analysis to visualize and measure FL distribution within several compartments of the tissue and within the epithelial cells responsible for vectorial transport ( 2 ). The images were consistent with a two-step mechanism of transepithelial transport: Na-dependent FL uptake at the apical (CNS side) plasma membrane and electrical potential (PD)-driven efflux from the cells at the basolateral membrane.

In the present study, we used confocal microscopy to investigate the mechanisms responsible for the transport in rat choroid plexus of a larger organic anion, a fluorescent derivative of MTX (FL-MTX; 900 Da). It is known that this tissue transports the chemotherapeutic, MTX, from CSF to blood and that such transport is uphill and mediated ( 1, 17 ). Our findings for the fluorescent MTX analog also show concentrative and Na-dependent transport from CSF to blood. FL-MTX uptake at the apical membrane and efflux at the basolateral membrane were carrier mediated. However, it is likely that FL-MTX transport across the tissue is mediated by transporters that are different from those that handle FL.
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MATERIALS AND METHODS

Chemicals. FL-MTX and FL were obtained from Molecular Probes. All other chemicals were of reagent grade or better and were obtained from commercial suppliers. [ 3 H]MTX (sp act 20 Ci/mmol) was obtained from American Radiolabeled Chemicals.

Animals. Lateral choroid plexuses were isolated from adult, male Harlan Sprague-Dawley rats (250-400 g, obtained from Taconic Farms) using Dumont no. 5 forceps inserted in each hemisphere of the brain. The experiments were carried out under protocols approved by the National Institute of Environmental Health Sciences Animal Use Committee. Tissue was immediately transferred to ice-cold, artificial cerebrospinal fluid (aCSF: 103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2, 1.2 mM KH 2 PO 4, 1.2 mM MgSO 4, 25 mM NaHCO 3, 10 mM glucose, and 1 mM sodium pyruvate, at pH 7.4), previously gassed with 95% O 2 -5% CO 2.4 m% @1 f) c. G6 }2 P1 G7 t

Confocal imaging. Each plexus was divided into two pieces, and pieces were transferred to covered Teflon microscope imaging chambers ( 2 ), each containing 1.0 ml pregassed aCSF with 1 µM FL or 2 µM FL-MTX and added effectors. In some experiments, medium ionic composition was altered as follows: 1 ) for low-Na aCSF (Na 29 mM), NaCl was replaced by N -methylglucamine, 2 ) for Na-free aCSF, NaCl was replaced by choline chloride and NaHCO 3 by choline bicarbonate, and 3 ) for high-K aCSF, the KCl concentration was increased to 47 mM (isoionic replacement of NaCl). All FL-MTX transport experiments were conducted at room temperature (incubation and microscopy at 18-20°C), and all chambers with tissue were maintained in Ziploc plastic bags containing 95% O 2 -5% CO 2 under slight positive pressure until removed for confocal imaging. While on the microscope, tissue was gassed with 95% O 2 -5% CO 2 through a port in the chamber cover. Fluorescent compounds and inhibitors were added to the incubation medium as stock solutions in aCSF or DMSO. Preliminary experiments showed that the final concentrations of DMSO used ( 0.5%) had no significant effects on the uptake and distribution of FL or FL-MTX or the uptake of [ 3 H]MTX.
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To acquire images, the chamber containing tissue was mounted on the stage of a Zeiss inverted confocal laser scanning microscope (model 510 or 410) and viewed through a x 40 water immersion objective (numeric aperture = 1.2). A 488-nm laser line (Ar ion laser), a 505-nm dichroic filter, and a 515-nm long-pass emission filter were used. Low laser intensity was used to avoid photobleaching. With the photomultiplier gain set to give a subepithelial space fluorescence intensity of 150-200 (full scale, 255) in control tissue loaded with 2 µM FL-MTX, autofluorescence of unloaded tissue was undetectable. From each piece of tissue under investigation, 10-15 areas of epithelium and adjacent vasculature were selected for measurement. Image acquisition and analysis have been described previously in detail ( 2 ).

[ 3 H]MTX uptake. Each lateral choroid plexus ( 1 mg) was incubated with shaking at 37°C in continuously gassed (95% O 2 -5% CO 2 ) aCSF containing 1 µM [ 3 H]MTX and the indicated additions. After incubation, tissue was removed, rinsed briefly, weighed, and processed for liquid scintillation counting. Tissue accumulation of MTX was calculated from disintegrations per minute per milligram tissue wet weight and medium specific activity.

Statistics. Figures 1 - 10 show data from single experiments using tissue from six to eight animals; these are representative of two to four replicate experiments. Data are presented as means ± SE. Means were compared using Student's t -test. Values were deemed to be significantly different at P ; r9 q9 w! U6 e9 j/ D3 _# E
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Fig. 10. Inhibition of steady-state MTX accumulation by organic anions and digoxin. Tissue was incubated for 15 min in medium with 1 µM [ 3 H]MTX without (control) and with the indicated additions. Shown are mean values for 4-6 intact choroid plexuses; variability is given by SE bars. *Significantly different from control, P 6 r- ]* I6 ?  o4 `! |. h: i' ]
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RESULTS
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FL-MTX transport. Figure 2 shows transmitted light and confocal micrographs of choroid plexus tissue after 45 min incubation in aCSF containing 1 µM FL or 2 µM FL-MTX. In the transmitted light images, the apical brush-border membrane appears as a refractile element between cells and medium ( Fig. 2, A and C ). At high magnification, the epithelial cells are seen to contain a large central nucleus and smaller vesicular organelles ( 2 ). Beneath the epithelium is a distinct subepithelial space and blood vessels containing erythrocytes. Unlike most other capillaries in the brain, those in choroid plexus are fenestrated and thus the endothelium is no barrier to exchange of solutes between the vasculature and the interstitium. As shown previously ( 2 ), FL accumulated in both the epithelial cells and the subepithelial/vascular spaces, with fluorescence intensities in vascular/subepithelial medium ( Fig. 2 B ). The pattern of FL-MTX fluorescence was clearly different, with levels in the cells slightly lower than the medium and levels in vascular/subepithelial spaces substantially higher than the medium ( Fig. 2 D ). The only cellular element exhibiting FL-MTX fluorescence higher than the medium was the apical plasma membrane. In Fig. 2, B and D, erythrocytes can be seen within the blood vessels as areas of very low fluorescence, indicating exclusion of FL and FL-MTX from these cells or quenching of fluorescence by Hb. In spite of the low fluorescence within erythrocytes, spaces between and around these cells are highly fluorescent, with the fluorescence intensity being roughly equivalent to that seen in the subepithelial space., z# d4 F! u: w0 C. w
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Fig. 2. Transmitted light and confocal images of choroid plexus tissue after 45-min incubation (steady state) in medium with 1 µM fluorescein (FL; A and B ) or 2 µM FL-methotrexate (FL-MTX; C and D ). Red arrows point to the lumens of erythrocyte-filled blood vessels. White arrows point to epithelial cells. The white bar indicates 50 µm.+ Z. q5 s7 q& `( i* M7 {/ s
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Figure 3 shows the time course of FL-MTX accumulation in cells and vascular/subepithelial spaces. FL-MTX accumulated rapidly in the vascular/subepithelial compartment, with steady-state levels attained within 30 min. Although initial accumulation in the vascular/subepithelial compartment was rapid, a line drawn through the first two points intersects the time axis at 3 min, suggesting an initial delay in filling this compartment that may reflect the time required for the compound to cross the epithelium. The time course of cellular accumulation roughly paralleled vascular/subepithelial accumulation, but at a considerably lower level ( Fig. 3 ). For comparison, medium fluorescence in this experiment averaged 25 units. At steady state, fluorescence in the vascular/subepithelial compartment was fivefold higher than the medium, and cellular fluorescence was only 40% of medium fluorescence. Thus average fluorescence in the vascular/subepithelial compartment was at least an order of magnitude higher than in the cells, indicating potent basolateral efflux.' x& d9 u3 |+ W4 e# p9 _/ {" x) H

Fig. 3. Time course of FL-MTX accumulation in epithelial (cells) and vascular/subepithelial (vessels) compartments of choroid plexus. Tissue was incubated in medium with 2 µM FL-MTX, and at each indicated time confocal images were acquired. Mean fluorescence intensity in each compartment was measured as described in MATERIALS AND METHODS. The broken line indicates the fluorescence intensity of the medium. Each point represents the mean value for 8-10 measurements; variability is shown as SE bars.6 j& f% d9 {# b

FL-MTX transport from bath to vascular/subepithelial spaces was dependent on metabolism and sensitive to replacement of medium Na. Inhibiting metabolism with NaCN reduced steady-state FL-MTX accumulation in the vascular/subepithelial spaces by 80% ( Fig. 4 ); cellular accumulation was not affected (data not shown). Decreasing medium Na (replacement with N -methylglucamine or choline) caused a progressive decrease in FL-MTX accumulation in the vascular/subepithelial spaces ( Fig. 4 ). In Na-free medium, transepithelial transport was reduced by 80%. Consistent with this, 1 mM ouabain substantially reduced FL-MTX accumulation. However, increasing medium K from 4.7 to 47 mM had no effect ( Fig. 4 ). It was previously demonstrated that increasing medium K by an order of magnitude both depolarized choroid plexus cells by 40 mV, decreased organic cation uptake ( 23 ), and blocked FL exit from the cells at the basolateral membrane ( 2 ). Together, the present data indicate that transepithelial FL-MTX transport across the epithelium was metabolism driven and Na dependent, but not PD sensitive.
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Fig. 4. Effects of metabolic inhibition and ionic disruption on steady-state FL-MTX accumulation by choroid plexus. Tissue was incubated in medium with 2 µM FL-MTX without (control) and with the indicated modifications. After 45 min, confocal images were acquired. Shown are mean values for 12-15 measurements of fluorescence intensity in vascular/subepithelial (vessels) compartments; variability is given by SE bars. **Significantly lower than controls, P

Steady-state FL-MTX accumulation in both the cellular and vascular/subepithelial compartments saturated with increasing medium substrate concentration ( Fig. 5 ). For both compartments, FL-MTX accumulation was a function of substrate concentration at low concentrations, but clearly 5 µM. Note that parallel experiments at lower photomultiplier gain settings confirmed saturation of vascular/subepithelial space FL-MTX accumulation at 5-10 µM substrate concentrations (data not shown).7 n# l$ N; L, ?1 W; n) k: ~4 r

Fig. 5. Effect of increasing medium FL-MTX concentration on steady-state accumulation in choroid plexus. Tissue was incubated in medium with 1-10 µM FL-MTX, and, after 45 min, confocal images were acquired. Shown are mean values for 10-15 measurements of fluorescence intensity in cellular and vascular/subepithelial (vessels) compartments; variability is given by SE bars. Inset : cellular fluorescence values plotted on an expanded scale.
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To begin to identify transporters responsible for transepithelial transport, we measured the effects of several compounds on steady-state FL-MTX accumulation in choroid plexus. Compounds were chosen based on their abilities to interact with Oats currently known to be expressed in the tissue, i.e., Oat1, Oat3, Oatp3, Mrp1, and Oatp2 ( 11, 12, 16, 19, 20 ). Note that because of the wide specificity limits of these Oats, it is difficult to identify compounds that interact specifically with only one transporter. Possible exceptions to this generalization are digoxin for Oatp2 and MK-571 (low affinity) for Mrp1 ( 16, 18 ). Figure 6 shows the effects of several organic anions on steady-state accumulation of FL-MTX in the vascular/subepithelial spaces. At 10 and 50 µM, MTX significantly reduced transepithelial transport. Probenecid, which affects transport on Oats, Oatps, and Mrps ( 16 ), inhibited at 100 µM. p -Aminohippurate (PAH), which interacts with Oat1 and Oat3, but not with Oatps and only weakly with Mrps ( 16, 19 ), was a poor inhibitor of transport. It was ineffective at 100 µM and only inhibited by 30% at 1 mM.
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Fig. 6. Inhibition of steady-state vascular/subepithelial accumulation of FL-MTX by organic anions. Tissue was incubated in medium with 2 µM FL-MTX without (control) and with the indicated additions. After 45 min, confocal images were acquired. Shown are mean values for 13-20 measurements of fluorescence intensity in the vascular/subepithelial compartment; variability is given by SE bars. *Significantly lower than controls, P
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Dose-response data for four additional inhibitors are shown in Fig. 7. Two patterns of inhibition are evident. Estrone sulfate, a compound transported by Oat3, Oatps, and Mrps ( 9, 11, 16, 19 ), and leukotriene C 4 (LTC 4 ), a high-affinity substrate for Mrps ( 16 ) and Oatps ( 10 ), decreased vascular/subepithelial accumulation of FL-MTX in a concentration-dependent fashion but did not affect cellular accumulation ( Fig. 7, A and B ). Digoxin, a high-affinity substrate for Oatp2 ( 16 ), and MK-571, a selective inhibitor of Mrps ( 16 ), also decreased vascular/subepithelial accumulation of FL-MTX in a concentration-dependent fashion ( Fig. 7, C and D ). However, both compounds significantly increased cellular accumulation. For digoxin, this increase was seen with concentrations as low as 0.1 µM. With MK-571, significant increases were found with 50 and 100 µM. With 300 µM MK-571, both vascular/subepithelial accumulation and cellular accumulation were reduced substantially. Apparently, digoxin and MK-571 (unlike the other organic anions tested in Figs. 6 and 7, A and B ) were more potent inhibitors of basolateral FL-MTX efflux than of apical uptake, and, when efflux was partially blocked, concentrative cellular accumulation became evident.

Fig. 7. Alteration of steady-state FL-MTX accumulation by estrone sulfate ( A ), leukotriene C 4 (LTC 4; B ), digoxin ( C ), and MK-571 ( D ). Tissue was incubated in medium with 2 µM FL-MTX without (control) and with the indicated additions. After 45 min, confocal images were acquired. Shown are mean values for 10-15 measurements of fluorescence intensity in cellular and vascular/subepithelial (vessels) compartments; variability is given by SE bars. **Significantly different from control vessels, P   W2 f0 P+ U& c

Using 10 µM digoxin and 100 µM MK-571 as tools to partially block efflux and thus enhance cellular accumulation, we could then begin to characterize the mechanism of apical uptake. In the presence of 10 µM digoxin, cellular accumulation of FL-MTX was nearly abolished by 200 µM probenecid. Replacement of medium Na had a similar inhibitory effect ( Fig. 8 A ). In the presence of 100 µM MK-571, cellular accumulation of FL-MTX was reduced by 90% by 200 µM probenecid, 50 µM estrone sulfate, and 1 µM LTC 4 ( Fig. 8 B ). Consistent with a two-step transport mechanism, all of these treatments further reduced accumulation of FL-MTX in the vascular/subepithelial compartment. Thus FL-MTX accumulation at the apical membrane was indeed saturable, concentrative, specific, and Na dependent. In the absence of digoxin or MK-571, these characteristics of apical transport appeared to be masked by a particularly avid efflux mechanism at the basolateral membrane. Even at steady state, basolateral efflux maintained a very low cellular level of FL-MTX while concentrating the compound in the vascular/subepithelial space.  `* K& b5 p2 O4 f2 l

Fig. 8. Effects Na replacement and organic anions on steady-state FL-MTX accumulation by digoxin-treated ( A ) or MK-571-treated ( B ) choroid plexus. Tissue was incubated in medium with 2 µM FL-MTX without (control) and with the indicated additions. After 45 min, confocal images were acquired for later analysis. Shown are mean values for 12-17 measurements of fluorescence intensity in cellular and vascular/subepithelial (vessels) compartments; variability is given by SE bars. **Vessels significantly different from 10 µM digoxin or 100 µM MK-571 alone, P

Although organic anions in combination with MK-571 reduced FL-MTX accumulation in both tissue compartments, a different result was found when digoxin was used in combination with MK-571. Figure 8 B shows that incubating tissue in medium containing MK-571 plus digoxin reduced vascular/subepithelial accumulation of FL-MTX and increased cellular accumulation compared with tissue exposed to MK-571 alone. Because MK-571 and digoxin likely interact with different basolateral transporters, this result suggests that FL-MTX efflux is mediated by both Oatp2 and Mrp1.- o1 K, k# Y4 {0 o) ~0 A$ ]
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MTX transport. It is of interest to compare the present findings using confocal imaging with those obtained using radiotracer methodology. Figure 9 shows the time course of uptake of 1 µM [ 3 H]MTX by rat choroid plexus. Initially, uptake was rapid and linear with 5 min, uptake was nonlinear, suggesting an approach to steady state. Even at times as short as 5 min, accumulation of MTX by the tissue was concentrative, with calculated tissue-to-medium ratios at steady state approaching 20 (for 1 µM MTX, 1 pmol/mg tissue is equivalent to an uncorrected tissue-to-medium ratio of unity). Incubating tissue in low-Na medium (26 mM Na rather than 128 mM Na) caused a significant reduction in MTX uptake at 10, 30, and 60 min ( Fig. 9 ). Uptake of MTX was also sensitive to inhibition by other organic anions. PAH, FL-MTX, and probenecid reduced uptake significantly and, as with FL-MTX (above), estrone sulfate was a particularly good inhibitor ( Fig. 10 ). Interestingly, 1 µM LTC 4 did not inhibit, 1.0 µM digoxin was without effect, but 10 µM digoxin significantly reduced MTX uptake. Based on inhibition by 50 µM estrone sulfate, at least 85% of total MTX uptake at 15 min was mediated. Raising medium K from 4.7 to 47 mM did not significantly alter MTX uptake. Thus, in agreement with present experiments using FL-MTX, MTX uptake was concentrative, specific, Na-dependent, and mediated but not potential dependent. However, transport did not appear to be affected by LTC 4 or by 1 µM digoxin.
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Fig. 9. Time course of MTX accumulation in choroid plexus. Tissue was incubated in control and low-Na medium with 1 µM [ 3 H]MTX. Shown are mean values for 4-6 intact plexuses; variability is given by SE bars. **Significantly different from controls, P

DISCUSSION* V; x( Y1 i; U* ?6 ~

A crucial obstacle to understanding xenobiotic transport in epithelia, such as renal tubule and choroid plexus, is correlating findings on transporter expression obtained from molecular/immunological studies with data on transport of individual compounds in the intact tissue or in vivo. This is particularly difficult for solutes that are handled by multiple transporters in tissues that are morphologically complex. For these solutes and tissues, tools are needed to define and characterize the separate components of transport and identify the molecular basis for each component. We recently utilized the power of quantitative, confocal microscopy-based techniques to probe in intact, living choroid plexus organic anion transport mechanisms at the cellular and subcellular levels ( 2 ). We found that transport of FL across the epithelium involved the following two concentrative steps: 1 ) Na-dependent uptake at the apical (CNS side) plasma membrane and 2 ) mediated, PD-driven efflux from the cells at the basolateral membrane. Based on findings with an Oat3 knockout mice ( 20, 21 ) and a recent reassessment of Oat3 energetics in Xenopus oocytes and rat kidney ( 19 ) and choroid plexus (Miller DS and Lowes S, unpublished observations), the Na-dependent, apical transporter for FL in choroid plexus appears to be Oat3. The PD-driven basolateral transporter remains to be identified.% y+ W% d; {: ]' k2 T+ C
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In the present study, we used a similar approach to characterize the transport of a large, fluorescent organic anion, FL-MTX (900 Da), across rat choroid plexus and found a more complicated picture. As with FL, transepithelial transport of FL-MTX was specific (reduced by other organic anions), concentrative, metabolism driven (inhibited by NaCN), and Na dependent. Unlike basolateral efflux of FL, which is blocked by high K, raising medium K by an order of magnitude had no effect on FL-MTX transport. Uptake of [ 3 H]MTX measured by scintillation counting was also specific, concentrative, and Na dependent, but it was not clear where in the tissue nonfluorescent MTX accumulated. MTX did inhibit FL-MTX transport, and FL-MTX did inhibit MTX transport, indicating that they shared at least one transporter. However, there were some disparities in inhibitor profiles (compare, e.g., the effects of digoxin and LTC 4 in Figs. 7 and 10 ), suggesting differences in the ways that the tissue handled MTX and FL-MTX.
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The steady-state tissue distribution of FL-MTX was clearly different from that found for FL, implying different mechanisms of transport. For FL, confocal micrographs showed the steady-state distribution medium ( 2 ). In contrast, the distribution of FL-MTX was vascular/subepithelial cells (present study). Thus, based on tissue distribution of the substrates, it was clear that two concentrative steps in series drove transepithelial FL transport, but it was not clear whether FL-MTX transport involved mediated steps at both membranes or just at the basolateral membrane.0 c- h3 q$ y8 U) B0 A
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Experiments that tested the metabolism and ion dependence of FL-MTX transport provided no evidence for a two-step mechanism. That is, although exposure to NaCN and Na replacement greatly reduced vascular/subepithelial accumulation, these treatments did not alter cellular accumulation. This was also true for most of the organic anions that inhibited transepithelial transport. The first evidence that apical FL-MTX uptake might be mediated came from the observation that steady-state cellular accumulation appeared to saturate with medium substrate concentration. This could only have happened if apical uptake were mediated. However, it was when we tested digoxin and MK-571 as inhibitors that convincing evidence was found for a two-step transport mechanism. Both drugs increased cellular FL-MTX accumulation and decreased vascular/subepithelial accumulation. With 10 µM digoxin and 100 µM MK-571, mean cellular and vascular/subepithelial FL-MTX accumulation were roughly equivalent to 50% of control vascular/subepithelial accumulation but exceeded medium FL-MTX by a factor of three to four. These results are consistent with digoxin and MK-571 blocking FL-MTX efflux from the cells. Because increased cellular accumulation was accompanied by decreased vascular/subepithelial accumulation, it is likely that these drugs blocked basolateral efflux.5 W) ~5 R3 E1 j( e: Z

With vascular/subepithelial accumulation reduced by digoxin or MK-571, concentrative uptake at the apical membrane was revealed and could be characterized. When tissue was incubated in medium containing digoxin plus probenecid, cellular and vascular/subepithelial accumulation of FL-MTX were substantially reduced compared with tissue exposed to digoxin alone, indicating mediated uptake at the apical membrane. Moreover, in the presence of digoxin, removal of medium Na greatly reduced cellular and vascular/subepithelial accumulation of FL-MTX, showing that apical uptake was Na dependent. Similarly, when tissue was incubated in medium containing MK-571 plus probenecid, estrone sulfate, or LTC 4, cellular and vascular/subepithelial accumulation of FL-MTX were substantially reduced compared with tissue exposed to MK-571 alone. Thus, in the absence of digoxin or MK-571, concentrative, mediated, and Na-dependent uptake of FL-MTX at the apical membrane was hidden by avid pumping of the compound from the cells in the vascular/subepithelial space.
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Which transporters are responsible for transepithelial FL-MTX transport in choroid plexus? Although several Oats are expressed in the tissue, these possess greatly overlapping substrate specificities, and potent inhibitors of one, e.g., estrone sulfate and LTC 4, often also inhibit others ( 16 ). At present, we know that Oat1, Oat3, and Oatp3 are localized to the apical membrane and Mrp1 and Oatp2 are localized to the basolateral membrane ( Fig. 1 ). However, Choudhuri et al. ( 5 ) recently detected mRNA coding for several additional Oats in rat choroid plexus. These include high to moderate levels of mRNA for Oat2, Mrp4, and Mrp5 and very low levels of mRNA for Oatp1, Oatp4, Oatp5, Oatp12, Oat-K1/2, Mrp2, and Mrp3. Neither the level of transporter protein expression nor the cellular location of any of these is known in choroid plexus." t6 n. P6 U9 y; l- r/ n) T0 }" ^

Of all the Oats expressed at the apical membrane, only those of the Oat subfamily can be energetically coupled to the Na gradient (indirect coupling; see Ref. 16 ). Although mRNA for Oat1 has been detected in rat choroid plexus by RT-PCR ( 14 ), Oat3 appears to be the major Oat family member at the apical membrane of choroid plexus ( 11, 21 ), and the Na-dependent component of organic anion uptake originally attributed to transport on Oat1 appears to be a result of transport on Oat3 (Refs. 19 and 21 and Miller et al., unpublished data). MTX is clearly an Oat3 substrate ( 4, 9 ), and inhibition of FL-MTX and MTX transport by estrone sulfate and PAH is consistent with transport on Oat3. However, recent experiments with an Oat3-null mouse showed no reduction in FL-MTX transport in choroid plexus even though transport of FL was substantially reduced ( 20 ). Experiments with choroid plexus from wild-type mice indicate that transepithelial FL-MTX transport was indeed Na dependent and that the tissue distribution pattern was identical to that shown here for the rat (Ref. 20 and Miller et al., unpublished data). If orthologous transporters mediate Na-dependent FL-MTX uptake in mouse and rat choroid plexus, an Oat other than Oat3 must be involved.

The present data provide some guidance in further narrowing down the list of possibilities. Note that digoxin and MK-571 proved to be particularly useful tools in the present study because they were more potent inhibitors of basolateral efflux than of apical uptake. Other compounds tested have not shown this property, but other organic anions clearly inhibited FL-MTX transport. This means that each of the other inhibitors had a greater effect on uptake than on efflux. Assuming that inhibitor potency reflects specificity of the apical FL-MTX process, we are clearly looking for transporters that are driven directly or indirectly by Na, PD insensitive, and sensitive to inhibition by MTX, estrone sulfate, PAH, and LTC 4. Because most Oats are remarkable in their ability to handle a wide range of anionic compounds, identification of the apical transporter(s) for FL-MTX will not be possible using specificity data alone. Available evidence points to Oatp2 and Mrp1 on the basolateral membrane as being the transporters responsible for basolateral FL-MTX efflux. Two findings indicate that FL-MTX efflux from the cells at the basolateral membrane is mediated. First, depolarizing the cells with high K had no effect on transport. If efflux of the organic anion was a result of simple diffusion (or even facilitated diffusion), it should have been reduced by depolarization. This was not the case. Second, digoxin and MK-571 reduced vascular/subepithelial accumulation, while at the same time increasing cellular accumulation of FL-MTX, consistent with inhibition of efflux at the basolateral membrane mediated by Oatp2 and Mrp1. Digoxin is a potent inhibitor of transport on Oatp2 ( 12 ), which can operate as an organic anion exchanger and as a uniporter ( 10 ). For Oatp2 to mediate FL-MTX efflux in choroid plexus, transport must be electrically silent, e.g., organic anion exchange. Such exchange would require a counter ion with a concentration gradient that is appropriately directed to drive FL-MTX efflux. It is not clear at present what the physiological counter ion would be.& ?, \. }$ [" s7 u
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MK-571 is a selective inhibitor of Mrps, which are ATP-driven efflux pumps and thus not responsive to changes in PD. Although MTX is not apparently a substrate for transport on Oatp2 ( 12 ), it is transported by a number of Mrp isoforms ( 16 ), and FL-MTX is clearly a potent inhibitor and substrate for Mrp2 ( 22 ). Because Mrp1 and Mrp2 do not differ radically in specificity, it is likely that, in rat choroid plexus, Mrp1 (along with Oatp2) mediates FL-MTX efflux at the basolateral membrane.! f6 w* j# G: h5 q
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In summary, confocal imaging demonstrated that FL-MTX accumulated to high levels in the vascular/subepithelial space of rat choroid plexus, but not in the epithelial cells. Nevertheless, transepithelial transport involved two mediated steps: Na-dependent uptake at the apical membrane and potent, electroneutral efflux at the basolateral membrane. The transporters responsible for uptake remain to be identified, but efflux is most likely mediated by Oatp2 and Mrp1.; _% z& I' }, @; ^* c
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