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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
Received March 29, 2005; accepted June 13, 2005.
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Abstract |
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; Ghersi-Egea and Strazielle, 2002; Kusuhara and Sugiyama, 2004). Transport from CSF to blood involves three steps: uptake from CSF at the apical plasma membrane, transit across the epithelial cell, and efflux at the basolateral membrane into the subepithelial space and fenestrated capillaries. Recently, all of these steps have been visualized in living rat and mouse choroid plexus using confocal imaging of fluorescent xenobiotics (Breen et al., 2002, 2004; Sweet et al., 2002; Sykes et al., 2004).
Choroid plexus morphology is complex, with the apical plasma membrane of the epithelium exposed to CSF and the basolateral membrane facing blood vessels within the tissue. When isolated from the brain, choroid plexus from the lateral ventricles presents an epithelium-covered surface in which the apical plasma membrane is in direct contact with the medium. Confocal imaging studies of isolated, intact rat choroid plexus show that fluorescein, a fluorescent organic anion and Oat3 substrate, enters the cells well before the vascular space (Breen et al., 2002), indicating that immediate access is to the apical (ventricular) surface of the tissue. Thus, in vitro measurements of solute uptake over short incubation times provide a means to characterize uptake at the apical membrane, the first step in transport from CSF to blood (Miller, 2004).
Although the ability of the choroid plexus to transport organic anions has been known for some time (Pappenheimer et al., 1961), it has only been recently that the molecular basis of transport has been explored. Reverse transcriptase-polymerase chain reaction studies show that mRNA for 10 multispecific organic anion transporters from the Oat, Oatp, and Mrp families are expressed in the tissue (Choudhuri et al., 2003). Of these, six organic anion transport proteins have been localized to one side of the tissue or the other (Fig. 1), and recent attempts to functionally map transport of specific organic anions in intact tissue suggest important roles for Oat3 and Oatp3 at the apical membrane and Oatp2 and Mrp1 at the basolateral membrane (Sweet et al., 2002; Breen et al., 2004; Sykes et al., 2004). Such studies have been particularly successful in the mouse because of the availability of transgenic animals that fail to express specific transport proteins, e.g., Mrp1-, Mrp4-, and Oat3-null mice (Wijnholds et al., 2000; Sweet et al., 2002; Leggas et al., 2004; Sykes et al., 2004). However, it is also becoming increasingly clear that not all transport processes identified in intact tissue match transporters that have both been characterized in expression systems and shown to be expressed in the tissue (Breen et al., 2004; Sykes et al., 2004).
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; Kim and O'Tuama, 1981; Pritchard et al., 1999; Villalobos et al., 2002). Using isolated apical (brush border) membrane vesicles from cow choroid plexus and intact choroid plexus from cow and rat, Pritchard et al. (1999) showed that 2,4-D uptake is indirectly coupled to sodium. Although these authors initially concluded that Oat1 was responsible for sodium-dependent 2,4-D uptake in choroid plexus, it now appears that Oat3, not Oat1, is the primary sodium-dependent organic anion transporter at the apical membrane (Nagata et al., 2002; Sweet et al., 2002, 2003; Sykes et al., 2004). Consistent with this, Nagata et al. (2004) recently found that 2,4-D was a moderately high-affinity substrate in Oat3-transfected LL-CPK1 cells (apparent Km, 20 µM) and that the uptake kinetics and inhibitor profile in intact rat choroid plexus matched that of the transfected cells. These authors also concluded that the kinetics of 2,4-D uptake by rat choroid plexus could be described by a single, Oat3-mediated component plus diffusion. In the present study, we used inhibitor profile, sodium dependence, and organic anion exchange to confirm Oat3 as the transporter responsible for high-affinity 2,4-D uptake. However, we also present evidence for a second, lower affinity, sodium-independent component of 2,4-D uptake. |
Materials and Methods |
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Animals. Lateral choroid plexuses were isolated from adult, male Harlan Sprague-Dawley rats (250–400 g; Taconic Farms, German-town, NY) using Dumont no. 5 forceps inserted into each hemisphere of the brain. Tissue was immediately transferred to ice-cold aCSF (103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM glucose, and 1 mM sodium pyruvate at pH 7.4), previously gassed with 95% O2/5% CO2. Tissue was preincubated in gassed aCSF at 37°C for 15 min before transport experiments. Animal housing was in accordance with institutional guidelines and the National Institutes of Health Guide for the Use and Care of Laboratory Animals.
[3H]2,4-D Transport. For uptake measurements, each lateral choroid plexus (about 1 mg) was incubated with shaking at 37°C in 1 ml of continuously gassed (95% O2/5% CO2) aCSF containing [3H]2,4-D, unlabeled 2,4-D, and the indicated additions. After incubation, tissue was removed, rinsed briefly, weighed, and processed for liquid scintillation counting. Tissue accumulation of 2,4-D was calculated from disintegrations per minute per milligram of tissue wet weight and medium-specific activity. In some experiments, tissue was incubated in sodium-free aCSF, with NaCl and NaHCO3 replaced with cholineCl and cholineHCO3, respectively. Initial experiments indicated no loss of viability (preserved morphology and ability to concentrate organic anions) when tissue was incubated in gassed aCSF for periods up to 90 min.
For efflux measurements, tissue was loaded by incubation for 30 min at 37°C in continuously gassed (95% O2/5% CO2) aCSF containing 20 µM [3H]2,4-D. Each plexus was then briefly washed and transferred to 1.5 ml of gassed (95% O2/5% CO2) aCSF without (control) or with unlabeled compounds. Vials were shaken and continuously gassed. At preset intervals, 50 µl of efflux medium was taken for liquid scintillation counting. After 60 min, a final medium sample was taken, and the tissue and all medium samples were removed and processed for liquid scintillation counting. Total tissue disintegrations per minute at the beginning of the efflux period were calculated from final tissue disintegrations per minute and final medium disintegrations per minute plus disintegrations per minute removed for the timed samples. At each sampling time, the fraction of total label remaining in the tissue was calculated from initial total tissue disintegrations per minute and medium disintegrations per minute at the sampling time (corrected for disintegrations per minute removed in previous samples).
Statistics. Data are presented as mean ± S.E. Means were compared using Student's t test or one-way analysis of variance. Means were deemed to be significantly different when P < 0.05. Linear and nonlinear regression analyses were carried out using GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA).
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Results |
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). For each species, Oat1 and Oat3 could be differentiated based on substrate and inhibitor specificity: p-aminohippurate (PAH) was a substrate and inhibitor of both, but estrone sulfate (ES) interacted with Oat3, not Oat1. Transport data from kidney and choroid plexus of Oat3-null mice confirmed this dichotomy (Sweet et al., 2002; Sykes et al., 2004). Thus, for sodium-dependent organic anion transport, differential sensitivity to PAH and ES would functionally map pathways of sodium-dependent organic anion transport. This is one approach we have used in the present study.
The uptake of 20 µM 2,4-D into choroid plexus tissue was initially rapid but then fell off and reached a plateau after about 10 min (Fig. 2). At steady state, the uncorrected tissue/medium ratio (picomoles per milligram of tissue divided by picomoles per microliter of medium) was about 7. Since this value substantially exceeded unity, it suggests concentrative transport. A closer examination of the early time course of 2,4-D uptake revealed that the data could be fitted with a line passing through the origin (Fig. 2, inset). The zero intercept indicates the absence of detectable binding to the tissue, a finding in agreement with the data of Nagata et al. (2004) for uptake of 0.05 µM 2,4-D. The linearity of uptake indicates that measurements taken over the first 2 min approximate an initial rate. To focus on 2,4-D uptake at the apical membrane, all subsequent measurements were made under initial rate conditions.
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). Figure 3A shows that the uptake of 20 µM 2,4-D was sensitive to inhibition by PAH. Uptake was reduced in a concentration-dependent manner by PAH concentrations below 2.5 mM but from 2.5 to 10 mM inhibition was constant at about 50%. We interpret these data to indicate that 2.5 mM PAH blocked all common pathways of 2,4-D uptake but that additional pathways remained that were not sensitive to PAH.
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We characterized further the PAH-insensitive component of 20 µM 2,4-D uptake by determining whether organic anions in combination with 10 mM PAH could reduce uptake to a greater extent than 10 mM PAH alone. As shown in Fig. 5, both DHEAS and taurocholate significantly reduced the PAH-insensitive component of 2,4-D uptake. In contrast, the anionic, neurotransmitter metabolite 5-hydroxyindole acetic acid (HIAA) was without effect. Since HIAA alone partially reduced 2,4-D uptake in the absence of PAH (Fig. 5), these data indicate HIAA only affected the PAH-sensitive component of uptake.
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Note that for Oat3 to drive organic anion uptake in an sodium-dependent manner, anion exchange must be energetically coupled to sodium-dicarboxylate cotransport. In this regard, using apical membrane vesicles from pig choroid plexus, Pritchard et al. (1999) demonstrated both 2,4-D/glutarate exchange and sodium/glutarate cotransport. They also found glutarate stimulation of 2,4-D uptake by intact rat choroid plexus. To determine whether sodium-dependent dicarboxylate uptake also occurs in intact rat choroid plexus, we measured the time course of 10 µM glutarate uptake in the presence and absence of sodium. Figure 7 shows rapid initial uptake of glutarate that reached a plateau within 30 min. At steady state, the uncorrected tissue/medium ratio was nearly 20, indicating concentrative uptake. This concentrative uptake was abolished in sodium-free medium (Fig. 7).
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, 2003). Based on inhibitor specificity and sodium dependence, the PAH-sensitive component of 2,4-D uptake defined above matches this profile. If Oat3 was indeed responsible for a substantial component of 2,4-D uptake, we should also be able to demonstrate exchange of 2,4-D for other monovalent organic anions that are Oat3 substrates as well as for certain dicarboxylates. To demonstrate organic anion exchange, we preloaded choroid plexus tissue to steady state in medium containing [3H]2,4-D, transferred the tissue to 2,4-D-free medium, and measured efflux of label over 60 min. Figure 8A shows a representative experiment in which [3H]2,4-D efflux into control medium and medium containing 1 mM unlabeled 2,4-D is plotted as a function of time. In controls, efflux was rapid and essentially complete within 60 min. It could be described by a single exponential (Fig. 8A, inset). Adding unlabeled 2,4-D to the medium accelerated efflux by a factor of about 8. Moreover, all Oat3 substrates tested significantly increased the rate of [3H]2,4-D efflux (Fig. 8B). Compounds that stimulated efflux included monocarboxylates (PAH, ES, and benzylpenicillin), dicarboxylates (glutarate and 
-ketoglutarate), and one organic cation (cimetidine), although none of these compounds were nearly as effective as 2,4-D itself.
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To determine whether 2,4-D could influence the transport of an Oat3 substrate, we measured the effects of the herbicide on the uptake and efflux of PAH. Figure 9A shows that 1 to 100 µM 2,4-D caused concentration-dependent reductions in the initial rate of uptake of 10 µM PAH. Consistent with these findings, 1 mM 2,4-D as well as several Oat3 substrates significantly increased the rate of [3H]PAH efflux from choroid plexus (Fig. 9B).
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Kinetics of 2,4-D Uptake. To further characterize transport, we measured the kinetics of 2,4-D uptake. Figure 10 shows the initial rate of uptake as a function of substrate concentration over the range 0.5 to 500 µM. Clearly, the rate was not a linear function of concentration but fell off as concentration was increased. To define the nonmediated component of uptake (diffusive entry plus nonspecific accumulation), we measured uptake in the presence of a high concentration of unlabeled 2,4-D. Over a substrate concentration range of 1 to 100 µM, uptake in the presence of 5 mM 2,4-D was a linear function of substrate concentration; the regression line passed through the origin of the plot and had a slope of 1.04 ± 0.04 (Fig. 10, inset). Consistent with this, uptake at high 2,4-D concentrations (3–5 mM) was found to be linear; the slope of the regression line connecting these data points was 1.05 ± 0.05 (not shown).
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 | (1) |
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We used the analytical function that describes the concentration dependence of 2,4-D uptake shown in eq. 1 to calculate the contribution of each of the three components to total uptake (Fig. 12). As expected, at 20 µM and below, the high-affinity component accounted for well over 50% of total uptake. At about 80 µM, each of the three components contributed equally and at higher concentrations, nonmediated uptake dominated. At 20 µM 2,4-D, the model predicted 19% of total uptake to be nonmediated, 58% to be on the highaffinity component, and 23% to be on the low-affinity component. Using the data for uptake of 20 µM 2,4-D from Figs. 3 and 4, we calculated total uptake to be partitioned as follows: nonmediated uptake, 13 ± 3% (based on inhibition by 5 mM unlabeled 2,4-D); and Oat3, 52 ± 12% (based on inhibition by 10 mM PAH) or 51 ± 11% (based on sodium dependence). Thus, it appears that the sodium-dependent, PAH-sensitive component of uptake that we have attributed to Oat3 matches well with the high-affinity component disclosed by kinetic analysis.
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Discussion |
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With 20 µM 2,4-D, the major component of uptake, which was both ES- and PAH-sensitive, corresponded to the sodium-dependent component of uptake. Thus, by the criteria established previously (Sweet et al., 2003), Oat3 mediated this component of uptake. It is important to note that we could not detect any component of 2,4-D uptake that was sensitive to PAH but not to ES (present study). Such a component would be indicative of transport mediated by Oat1. This was also the case when we examined the mechanisms driving PAH uptake in choroid plexus from wild-type and Oat3-null mice (Sykes et al., 2004). All mediated PAH uptake in tissue from wild-type mice was ES-sensitive, and mediated uptake was abolished in tissue from Oat3-null mice. Apparently, in tissue from mouse and rat, Oat1-mediated organic anion uptake is undetectable.
Note that in the inhibition studies, millimolar PAH concentrations were needed to block uptake shared by 2,4-D and PAH. Although these concentrations may appear to be high, they reflect the low apparent Km for transport of 2,4-D on Oat3 [5 µM in present study and 10 µM from Nagata et al. (2004)] and the substantially higher apparent Ki for PAH [Ki against 398 µM benzylpenicillin (Nagata et al., 2004), but Km for uptake, 67 µM (Kusuhara et al., 1999)]. Assuming Michaelis-Menten kinetics, competitive inhibition, a Km for 2,4-D of 7 µM and a Ki for PAH of 250 µM (Nagata et al., 2002), one calculates that 50% inhibition of uptake mediated by Oat3 will be obtained with 960 µM PAH and 90% inhibition with nearly 8 mM PAH. Although these calculated values appear higher than those suggested by Fig. 3A, they emphasize the need for high PAH concentrations in the present Oat3 blocking studies.
The second approach was based on the kinetics of 2,4-D efflux from preloaded tissue. Consistent with 2,4-D transport on Oat3, efflux from choroid plexus was accelerated by several Oat3 substrates, including monovalent organic anions, dicarboxylates, and cimetidine. Conversely, 2,4-D accelerated the efflux of the Oat3 substrate PAH. It is noteworthy that with one exception all of the Oat3 substrates tested at millimolar concentrations at most doubled the rate of 2,4-D efflux. That exception was 2,4-D itself, which increased the rate nearly 8-fold. This finding suggests a second component of 2,4-D efflux likely on another transporter that supports organic anion exchange.
The final approach was based on analysis of the kinetics of 2,4-D uptake. As in the inhibitor studies, we found multiple components of uptake: one nonmediated and at least two mediated. Nonlinear regression indicated apparent Km values of 5.4 and 312 µM. Because of the limitations of the kinetic approach, we cannot, by kinetic criteria alone, know whether either of the mediated components represents multiple processes with similar Km values. Of the two discernible mediated components, the high-affinity one matched the PAH-sensitive and sodium-dependent component defined in inhibitor studies. That is, we found very good agreement when we compared the calculated contribution of the high-affinity component with total uptake with the measured contribution of the PAH-sensitive, sodium-dependent component. Thus, the high-affinity component appeared to represent transport mediated by Oat3. The transporter(s) responsible for the low-affinity, sodium-independent component of 2,4-D uptake remains to be identified. At this point, we know that 2,4-D uptake by this component is inhibited by ES, taurocholate, and DHEAS. Certainly, Oatp3, which has been localized to the apical membrane of choroid plexus and transports those organic anions (Abe et al., 1998; Kusuhara et al., 2003; Ohtsuki et al., 2003), is one candidate. However, at present, it is not known whether 2,4-D is a substrate for Oatp3.
Until recently, there was considerable uncertainty as to the transporter(s) responsible for 2,4-D uptake in choroid plexus. Uptake of 2,4-D was known to be indirectly coupled to sodium (Pritchard et al., 1999), and Oat3 was shown to be both capable of sodium-dependent transport (Sweet et al., 2003) and the major sodium-dependent organic anion transporter at the apical side of choroid plexus epithelial cells (Nagata et al., 2002; Sweet et al., 2002; Sykes et al., 2004). However, initial experiments using a cell line that overexpressed rat Oat3 showed that 2,4-D was not transported (Hasegawa et al., 2003). More recently, Nagata et al. (2004) showed that 2,4-D was indeed a substrate for transport in the same Oat3-expressing cell line and that in rat choroid plexus, the inhibitor profile and kinetics of uptake matched those of the cloned transporter. These authors also concluded that Oat3 was the only transporter responsible for 2,4-D uptake.
The present results, showing multiple mediated components of 2,4-D uptake in rat choroid plexus, are not necessarily incompatible with the data of Nagata et al. (2004). First, Nagata et al. studied the effects of inhibitors using 0.05 µM 2,4-D. At that substrate concentration, our model indicates that 87% of total uptake would be on Oat3 and only 7% on the low-affinity component. The latter could easily have been too small for Nagata et al. (2004) to detect given experimental error in the inhibitor studies. Second, one can use nonlinear regression to produce a highly significant fit for our data to a function with a single Michaelis-Menten term. If we had not first found from inhibitor studies that multiple mediated components contributed to 2,4-D uptake and determined that a single Michaelis-Menten term poorly fit the data for mediated transport (nonlinear Lineweaver-Burke and Eadie-Hofstee plots), we, like Nagata et al. (2004), might have concluded that a second mediated component was not needed. Clearly, experiments designed to reconcile transport in intact, native tissue with data obtained from systems expressing single, cloned transporters require full evaluation of all aspects of transport.
Finally, as an Oat3 substrate, 2,4-D has the potential to interfere with the removal from CSF of a number of potentially toxic neurotransmitter metabolites, e.g., HIAA and indoxyl sulfate, which are themselves Oat3 substrates (Ohtsuki et al., 2002). Since the herbicide is also transported by a second organic anion transporter, albeit with substantially lower affinity, it may also alter the removal of additional compounds that are handled by the second transporter. Molecular identification of the sodium-independent transporter involved is likely to expand the list of possible competitive interactions.
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Footnotes |
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ABBREVIATIONS: CSF, cerebrospinal fluid; aCSF, artificial CSF; Oat, organic anion transporter subtype; Oatp, organic anion transporting polypeptide subtype; Mrp, multidrug resistance-associated protein subtype; 2,4-D, 2,4-dichlorophenoxyacetic acid; PAH, p-aminohippurate; ES, estrone sulfate; DHEAS, dehydroepiandrosterone sulfate; HIAA, 5-hydroxyindole acetic acid.
Address correspondence to: Dr. David S Miller, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: miller{at}niehs.nih.gov
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Breen CM, Sykes DB, Baehr C, Fricker G, and Miller DS (2004) Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy. Am J Physiol 287: F562–F569.
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0.1 mM and ES concentrations 
