Materials and Methods

MLS-9 Culture.

Cultures of MLS-9 microglia cells were prepared as previously described (Zhou et al., 1998). Microglia were originally enriched from neopallia of 2- or 3-day-old Wistar rats (Schlichter et al., 1996) to >98% purity, as judged by staining with isolectin B4, OX-42, and ED-1 antibody (Sigma Chemical Co., St. Louis, MO). Colony-stimulating factor 1 was used to induce proliferation and the continuous microglia cell line MLS-9 was established from one of the colonies that arose after several weeks of culturing. These cells also stained with isolectin B4 (100%), OX-42 antibody (98%), and ED-1 antibody (99%), but were negative for the astrocyte marker glial fibrillary acidic protein, and the fibroblast protein fibronectin (Zhou et al., 1998).

Monolayers of MLS-9 cells (passages 25–38) were maintained in either 75-cm² Falcon plastic tissue culture flasks or 24-well plates (Becton Dickinson, Lincoln Park, NJ) at 37°C in 95% air, 5% CO₂. The cells were cultured in minimal essential medium, pH 7.2, supplemented with l-glutamine,d-glucose, 5% fetal bovine serum, 5% horse serum, and 0.5% penicillin/streptomycin suspension (all obtained from Life Technologies, Grand Island, NY), with a change of medium every 2 days. When confluent, cells were passaged using a sodium citrate solution containing 130 mM NaCl, 15 mM sodium citrate, 10 mM glucose, and 10 mM HEPES, pH 7.4. As previously described (Hong et al., 2000), the morphology of MLS-9 cells in confluent monolayers was spheroid, with large cell body structures and very short uropod-like processes.

Uptake of ZDV.

For uptake experiments, MLS-9 cells were seeded on 2-cm², 24-well plates at 5 × 10⁵ cells/well, and only those grown to a uniform, confluent monolayer (within 4–5 days) were used. The integrity of cell monolayers was evaluated microscopically, before and after the uptake measurements. Most importantly, the morphology and uptake values were the same among the cell passages used.

The uptake of [³H]ZDV (15 Ci/mmol; Moravek Biochemicals, Brea, CA) was measured as previously described (Bendayan et al., 1994). Briefly, MLS-9 cells were rinsed and preincubated for 30 min with 0.5 ml of Earle's balanced saline solution (EBSS), containing: 1.8 mM CaCl₂, 5.4 mM KCl, 0.8 mM MgSO₄, 138 mM NaCl, 1.0 mM Na₂HPO₄, 5.5 mMd-glucose, and 20 mM HEPES, with Trizma base added to bring the pH to 7.4. Then, 0.5 ml of this medium containing [³H]ZDV and nonradioactive ZDV was added to the cells at 37°C for fixed time intervals. For uptake specificity studies, cells were preincubated with an inhibitor before adding ZDV. Uptake of radiolabeled probe was terminated by adding 2 ml of ice-cold “stop” solution (0.16 M NaCl). The cells were then solubilized in 1 ml of 1 N NaOH for 30 min, and the lysate transferred to scintillation vials containing 0.5 ml of 2 N HCl. Intracellular accumulation of [³H]ZDV was quantitated using a Beckman liquid scintillation counter (model LS 7000). The sample counts were corrected for variable quench, “zero time” uptake, and background radioactivity in each experiment. The extracellular space, determined using d-[¹⁴C]mannitol (51.5 mCi/mmol; NEN Life Science Products, Boston, MA), was negligible (<0.14%); therefore, no correction was applied. The protein concentration (milligrams per milliliter) in each culture plate was determined by the Bradford method, using BSA as the standard and Bio-Rad reagent (Bio-Rad, Mississauga, Ontario, Canada). ZDV uptake values were expressed in nanomoles per milligram of protein per milliliter. For uptake studies at different external pH values, EBSS was buffered with either 0.1 mM Tris, pH 8.0, or 0.1 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. To change the intracellular pH, the cells were preincubated for 15 min with 30 mM NH₄Cl, before performing the uptake measurements in standard EBSS (pH 7.4). Spectrofluorometry, using a pH indicator dye (see below), showed that exposure to NH₄Cl transiently alkalinized the cells (from pH 7.1 to 7.5) and that washing out the external NH₄Cl rapidly acidified them (from pH 7.1 to 6.3). This standard procedure for acidifying intracellular pH involves dissociating trapped NH₄⁺ into NH₃, which diffuses out, and protons, which are sequestered inside the cells. For all experiments, [³H]ZDV uptake in the presence of a high concentration (3 mM) of verapamil was used to estimate nonspecific uptake. Whenever ethanol was used as the solvent, i.e., with the potassium ionophore valinomycin (1 μM) or the Na⁺/H⁺ ionophore monensin (5 μM), solutions contained the same ethanol concentration. There was no significant difference between [³H]ZDV uptake values in the presence (<0.01%) or absence of ethanol; thus, the cells were not adversely affected.

The nucleoside analog drugs ZDV, lamivudine, and abacavir were a gift from Glaxo Wellcome (Research Triangle Park, NC). Didanosine and zalcitabine were generously provided by Bristol-Myers Squibb (Princeton, NJ) and Hoffmann-La Roche (Nutley, NJ), respectively. Unless specified, other chemicals were from Sigma Chemical Co. (St. Louis, MO) and were of the highest purity available.

Metabolism of ZDV.

Intracellular metabolism of [³H]ZDV during the uptake assay was monitored by thin-layer chromatography. MLS-9 cells were incubated for 1, 10, and 30 min with 0.5 mM [³H]ZDV (100 μCi/ml) at 37°C. After the uptake reaction these cell suspensions, including 1 mM solutions of the standards thymine and ZDV, were spotted and chromatographed for 4 h on 250-μm-thick silica gel-coated plates (silica gel 60; Sigma Chemical Co.) that were impregnated with a fluorescent indicator. The solvent was butan-1-ol saturated with water. When the plate was dried, the zones bearing the standards were located under UV light. The R_F values corresponding to ZDV, thymine, and ZDV nucleotides (mono-, di-, and triphosphate metabolites) were 0.83, 0.72, and 0, respectively. Each zone was scraped from the plate and analyzed for radioactivity by standard liquid scintillation counting methods.

Intracellular pH Measurements.

Intracellular pH was measured fluorimetrically using the pH-sensitive carboxyfluorescein derivative 2′,7′-bis(carboxyethyl)-5(6′-carboxyfluorescein (BCECF) (Molecular Probes, Eugene, OR). Single-cell measurements were performed with a Zeiss Axiovert 100TV microscope equipped with a Cohu (model 1412) charge-coupled device camera, which is controlled by the operational software Axon Imaging Work Bench, 2.1. The nonfluorescent, membrane-permeant acetoxymethyl ester BCECF-AM enters cells readily and is cleaved by cytosolic esterases to yield the nonpermeant form whose fluorescence is proportional to pH. Confluent monolayers of MLS-9 cells grown in 24-well tissue culture plates were preincubated (40 min, 37°C) in EBBS containing 20 μM BCECF-AM and 0.1% BSA. Cells were washed and preincubated in 30 mM NH₄Cl solution for 30 min, and then followed by its removal and replacement with either EBSS or 1 mM amiloride. Baseline measurements were recorded in EBSS. Fluorescence was detected with dual excitation at 440 and 495 nm, and emission at 535 nm. Emission ratios for 440- versus 495-nm excitation were computed by the software. Intracellular pH was calibrated by comparing the fluorescence intensity of cells with 100 μM BCECF acid in EBSS solution, in which the pH was buffered with 0.1 mM MES (pH 6.1), BES (pH 6.7 and 7.1), HEPES (pH 7.4 and 7.7), and Tris (pH 8.1 and 8.6). All buffers were from Sigma Chemical Co.

Data Analysis.

Experiments were repeated at least two times using cells from different passages. Data points in each experiment represent quadruplicate measurements. Results are presented as mean ± S.D. from a minimum of two separate experiments. The Michaelis-Menten kinetic parameters (K_m andV_max) for ZDV transport were determined by a nonlinear least-squares analysis in Sigma Plot 4.0. To estimate the inhibitory constant (K_i) of the inhibitor verapamil, a least-squares regression analysis was used to determine the linear correlation between V andV/[ZDV], the Eadie-Hofstee linear transformation. The IC₅₀ or concentration of various organic cationic drugs causing a 50% reduction in ZDV uptake was estimated by a sigmoidal, four-parameter inhibition model. Statistical significance was assessed by the Student's t test for unpaired experimental values, the test of repeated measures of ANOVA, and/or the post hoc multiple-comparison Bonferroni t test, as appropriate. A p value <0.05 was considered significant.

Results

Metabolism of ZDV.

The intracellular metabolism of ZDV has been examined extensively in cell lines, in retrovirus-infected mouse tissues, and in human peripheral blood mononuclear cells (Furman et al., 1986). To exert its antiviral activity, ZDV is converted by several intracellular kinases to its phosphorylated anabolites. That is, thymidine kinase, thymidylate kinase, and pyrimidine nucleoside diphosphate kinase consecutively convert ZDV to ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate, respectively. Because metabolism can complicate the interpretation of transport studies, we determined the extent of ZDV metabolism by microglia cells. After the uptake reaction, chromatographic analysis of the cell indicated no significant ZDV metabolism before 30 min. For instance, in cells incubated with 0.5 mM [³H]ZDV for 30 min at 37°C, 87% of the intracellular radioactivity was associated with the unmodified form ZDV, 5% cochromatographed with thymine, and 8% with the ZDV nucleotides (mono-, di-, and triphosphate metabolites). After a 10-min incubation, 94, 4, and 2% of the radioactivity was recovered in the ZDV, thymine, and ZDV nucleotide fractions, respectively. Similarly, after a 1-min incubation, 96, 3, and 1% of the radioactivity comigrated with ZDV, thymine, and ZDV nucleotides, respectively (data not shown).

Time Course of Specific ZDV Uptake.

Figure1 shows that specific ZDV (0.5 mM) uptake by MLS-9 cells at 37°C was essentially linear for the first 20 min and began to approach an equilibrium value by ∼30 min. Therefore, a 10-min incubation time was used to represent the initial rate of ZDV influx into the cells. The ZDV concentration (0.5 mM) was chosen after considering the kinetics of the system involved (see below), to approximate the apparent K_m value and better describe ZDV uptake properties.

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Figure 1

Time course of specific ZDV uptake by MLS-9 cells. Specific ZDV (0.5 mM) uptake was measured in standard EBSS medium over 90 min at 37°C. ZDV uptake in the presence of 3 mM verapamil was used to correct for nonspecific ZDV uptake. Results are expressed as mean ± S.D. of three separate experiments. Within each experiment, data points represent the average of quadruplicate measurements.

Kinetics of ZDV Uptake.

The initial rate of ZDV influx into MLS-9 cells was measured at 10 min (37°C) with ZDV concentrations from 20 μM to 2.5 mM. The total ZDV uptake rate (V) can be described by the following equation:V=Vmax×[ZDV]Km+[ZDV]+D×[ZDV] Equation 1where V_max is the maximum uptake rate; K_m is the affinity constant, andD is the coefficient for nonspecific, diffusive process. The specific ZDV uptake rate was calculated by subtracting from the total rate, the nonspecific uptake measured with a high concentration of the most potent inhibitor we could identify (3 mM verapamil). Figure2 shows evidence for a specific, saturable carrier-mediated component, which conformed to simple Michaelis-Menten kinetics. The linear correlation (Eadie-Hofstee plot,r = 0.97; data not shown) between V andV/[ZDV] of the specific ZDV uptake values further confirms the single nature of the transport system. The kinetic constants, determined by nonlinear regression analysis, were 1024 ± 102 μM, 326 ± 14 pmol/mg/min, and 0.2 ± 0.1 pmol/mg/min/μM for the K_m,V_max, and D, respectively.

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Figure 2

Kinetics of ZDV uptake. ZDV uptake was measured at various ZDV concentrations (20 μM to 2.5 mM) in standard EBSS medium at 10 min (37°C), with nonspecific uptake measured in the presence of 3 mM verapamil. The specific uptake rate was calculated by subtracting the nonspecific component from the total. The kinetic constants (K_m, V_max) were determined by a nonlinear least-squares fit of the simple Michaelis-Menten equation to the specific data. Results are expressed as mean ± S.D. of six separate experiments, with quadruplicate measurements in each. All subsequent uptake measurements were corrected by subtracting nonspecific uptake.

Specificity Studies.

ZDV has previously been shown to inhibit an organic cation transporter in rat renal brush-border membrane vesicles (Griffiths et al., 1992). Thus, to determine the selectivity of the transporter, we tested several organic cations (1 mM) for their ability to inhibit the influx of 0.5 mM [³H]ZDV, measured at 10 min and 37°C (Fig.3). Among these compounds, verapamil, mepiperphenidol, quinidine, cimetidine, andN¹-methylnicotinamide, known inhibitors of organic cation transport systems, were the most effective inhibitors (70–79% inhibition). Thiamine, 1-methyl-4-phenylpyridinium (MPP⁺), guanidine, tetraethylammonium (TEA), and trimethoprim (49–67% inhibition) were weak inhibitors. Because these results suggested the possible involvement of an organic cation transport (OCT) system for ZDV, we also explored the effect of ZDV on the uptake of radiolabeled TEA, a standard organic cation probe. As opposed to findings with all the other cloned OCT systems (i.e., OCT1, OCT2, OCT3, OCTN1, and OCTN2), which are readily known to transport TEA (Gorboulev et al., 1997; Kekuda et al., 1998; Wu et al., 1998b,c), the uptake of TEA by microglia cells was low and not sensitive to ZDV (data not shown).

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Figure 3

Organic cations inhibit specific ZDV uptake. Uptake of ZDV (0.5 mM in standard EBSS medium at 37°C) was measured at 10 min in the presence of 1 mM of an organic cation: MPP⁺, trimethoprim, TEA, guanidine, thiamine, choline, colchicine, quinine,N¹-methylnicotinamide (NMN), cimetidine, quinidine, mepiperphenidol (MEP), verapamil (VER). Results are expressed as mean ± S.D. of three separate experiments, with each experimental point done in quadruplicate. ∗∗, significantly different from the control value (p < 0.01).

Further inhibitory studies were undertaken and IC₅₀ values for the moderate inhibitors were determined by fitting the data to a sigmoidal equation:V−V0=Vc−V01+CIC50n Equation 2where V and V_crepresent ZDV uptake in the presence and absence (control) of the inhibitor, respectively. Both V andV_c values were corrected for the nonspecific uptake as measured in the presence of 3 mM verapamil.V_o is the zero time uptake,C is the inhibitor concentration in the reaction mixture, and n, the Hill coefficient, is the inverse of the slope at 50% inhibition. The organic cations significantly inhibited ZDV uptake in a concentration-dependent manner with IC₅₀values of 156 ± 10 (Fig. 4A), 165 ± 11, 168 ± 10, 171 ± 10, and 200 ± 10 μM for verapamil, mepiperphenidol, quinidine, cimetidine, andN¹-methylnicotinamide, respectively.

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Figure 4

Competitive inhibition by verapamil on ZDV uptake. A, dose dependence of verapamil (VER) in inhibiting ZDV uptake. Specific ZDV (0.5 mM) uptake was measured at 10 min, 37°C, and in the presence of increasing verapamil concentrations (20 μM–3 mM). The data were fit to a sigmoidal inhibition model (under Materials and Methods). Results are expressed as percentage of the control uptake ± S.D. for three separate experiments. B, Eadie-Hofstee plots of verapamil inhibition of ZDV uptake. ZDV (100, 250, and 500 μM, and 1 and 2 mM) uptake was measured in standard EBSS medium at 10 min, 37°C, in the absence (control) or presence of various concentrations of verapamil. Least-squares regression analysis was used to determine the Eadie-Hofstee linear correlations. Results are expressed as mean ± S.D. of two separate experiments, with quadruplicate measurements. (Some error bars are smaller than the symbols.)

We next explored the type of inhibition exerted by the most potent inhibitor we identified. Verapamil inhibited ZDV uptake in a competitive manner with an estimated inhibitory constant (K_i) of 167 ± 21 μM. TheK_i value was obtained from the competitive inhibition equation:Ki=[I]Km′Km−1 Equation 3where [I] is the inhibitor concentration;K′_m (apparentK_m) andK_m are the affinities of the carrier in the presence and absence of the inhibitor, respectively. A least-squares regression was fitted to determine the Eadie-Hofstee linear correlations (Fig. 4B). Competitive inhibition is indicated by the common y-intercept of the regression lines, which shows the same value for V_max.

Because previous studies have demonstrated that ZDV is both a substrate for and competitive inhibitor of the organic anion transporter in rat renal basolateral membrane vesicles (Griffiths et al., 1991), we explored the inhibitory effect of several organic anions on ZDV uptake by MLS-9 cells. Organic anions such as benzyl penicillin, salicylic acid, and the prototypic substrate p-aminohippuric acid had no significant effect on ZDV uptake (data not shown). Moreover, contrary to the ZDV efflux systems identified at the blood-brain barrier and blood-CSF barrier (Takasawa et al., 1997a,b), ZDV transporter in microglia was insensitive to probenecid, a known inhibitor of organic anion transporters (data not shown).

Nucleoside transporters (i.e., rCNT1) have also been shown to mediate ZDV transport (Yao et al., 1996). Thus, we explored the involvement of a nucleoside transport system (either equilibrative or concentrative) by testing the inhibitory effect of nucleosides (thymidine, cytidine, guanosine, and adenosine), nucleoside analogs (lamivudine, abacavir, didanosine, and zalcitabine), and standard nucleoside transport inhibitors [dilazep, dipyridamole, and 6-(4-nitrobenzyl)-thio-9-β-d-ribofuranosylpurine] on ZDV uptake, in the presence and absence of Na⁺(Na⁺ replaced withN-methyl-d-glucamine). No significant difference (P > 0.9) was observed between the control and any of the tested conditions (data not shown), suggesting that ZDV entry into the microglia cells does not involve a nucleoside transporter. These results corroborate our previous finding that ZDV acts only as a potent, noncompetitive inhibitor of the Na⁺-dependent nucleoside transporter (Hong et al., 2000).

pH and Membrane Potential Effects.

Several studies have identified organic cation/proton exchange mechanisms in brush-border membrane vesicles from kidney and intestine, and in the basolateral membranes of hepatocytes (Zhang et al., 1998). The driving force, a proton gradient, is known to be functionally linked to both the Na⁺/H⁺ antiporter and the H⁺-ATPase (Bendayan et al., 1994). To assess the presence of such a mechanism, we measured ZDV uptake in the presence of an outwardly directed proton gradient, generated by a standard NH₄Cl acidification procedure (Jans et al., 1987;Bendayan et al., 1994). Acidification was confirmed by monitoring the changes in intracellular pH with the fluorescent probe BCECF (Fig.5). Addition of NH₄Cl caused an initial intracellular alkalinization (to pH 7.5), followed by a dramatic acidification upon NH₄Cl removal (from pH 7.1 to 6.3). The pH spontaneously recovered to its initial value by ∼20 min. After a second NH₄Cl treatment and washout, the pH recovery was abolished by 1 mM amiloride, an inhibitor of the Na⁺/H⁺ antiporter. ZDV uptake was enhanced for the first 5 min, and then decreased to about control values by 10 min, presumably due to the dissipation of the proton gradient (Fig. 6). The converse effect of a proton gradient in the opposite direction (Fig.7) further supports a mechanism whereby protons exchange with ZDV in microglia cells. That is, the initial uptake value was increased by extracellular alkalinization (pH_i ∼ 7.1 < pH_o8.0), and decreased by extracellular acidification (pH_i ∼ 7.1 > pH₀6.0). Thus, ZDV uptake appears to be increased by an outwardly directed proton gradient and decreased by an inwardly directed one. The requirement for an outwardly directed proton gradient was further supported by the decrease in uptake (by 35%) after exposure to 5 μM monensin (Fig. 8), a Na⁺/H⁺ ionophore that collapses the pH gradient. Taken together, these results suggest that proton efflux is required to drive the entry of ZDV into MLS-9 cells.

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Figure 5

Intracellular pH changes. MLS-9 cells were preincubated for 40 min at 37°C in EBSS containing 20 μM BCECF-AM ester with 0.1% BSA, and baseline measurements were recorded in standard EBSS. The cells were initially exposed to 30 mM NH₄Cl in EBSS for 30 min, washed, and placed in EBSS for 30 min, followed by a second NH₄Cl treatment, removal, and replacement with EBSS + 1 mM amiloride. Results represent the combined data from four separate experiments.

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Figure 6

An outwardly directed proton gradient increases specific ZDV uptake. Specific ZDV (0.5 mM) uptake was measured over 10 min at 37°C, in the absence (control) or presence of an outwardly directed proton gradient (pH_i < pH_o). The gradient was generated by exposing the cells to 30 mM NH₄Cl, washing, and placing in uptake buffer. Results are expressed as mean ± S.D. of three different experiments, with quadruplicate measurements. ∗∗, values that are significantly different from control value (p < 0.01).

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Figure 7

Effect of various proton gradients on specific ZDV uptake. MLS-9 cells preincubated with standard EBSS (pH 7.4) were incubated at 37°C over 10 min in either standard EBSS medium (pH 7.4, control ●) or EBSS medium containing Tris (pH 8.0 ■) or MES (pH 6.0 ♦). Each experimental point represents the mean ± S.D. of quadruplicate measurements from two separate experiments. Significant differences from the control value are indicated (*p < 0.05; **p < 0.01).

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Figure 8

Monensin decreases specific ZDV uptake. MLS-9 cells were incubated over 30 min at 37°C in standard EBSS medium (control) or in the presence of 5 μM monensin, an Na⁺/H⁺ ionophore. Results are expressed as mean ± S.D. of three different experiments, with quadruplicate measurements. Values that are significantly different from controls are indicated (*p < 0.05; **p < 0.01).

MLS-9 cells express a K⁺ current that presumably sets the cell membrane potential (Zhou et al., 1998) in both NaCl and KCl solutions. To determine whether ZDV uptake is electrogenic, membrane potential was changed by varying extracellular K⁺ concentrations (NaCl replaced by equimolar KCl) to depolarized (138 mM K⁺), hyperpolarized (2 mM K⁺), and control (5 mM K⁺) conditions. With 138 mM K⁺ in the uptake buffer, ZDV uptake increased by 50%, when measured after 10 min (Fig.9). Because no further increase in uptake was observed when 1 μM valinomycin, a potassium ionophore, was added to the KCl solution, the cells were effectively depolarized by the high concentration of K⁺. Conversely, when the cells were hyperpolarized with valinomycin and reduced external K⁺, ZDV uptake was significantly reduced (Fig.9). These results are consistent with a transporter that is membrane potential-dependent.

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Figure 9

Effect of membrane potential on specific ZDV uptake. ZDV (0.5 mM) uptake was measured at 37°C over 10 min in the presence of 138 mM Na⁺ (●), 138 mM K⁺ (▪), 138 mM K⁺ + 1 μM valinomycin (■), and 2 mM K⁺ + 1 μM valinomycin (⋄), a potassium ionophore. Results are expressed as mean ± S.D. of two separate experiments; each experimental point represents quadruplicate measurements. Values significantly different from control values are indicated (**p < 0.01; *p < 0.05).

Discussion

ZDV, a thymidine analog, is considered one of the first-line therapeutic agents for AIDS and AIDS dementia complex (Fischl et al., 1987). To be active, intracellular ZDV must be phosphorylated to its triphosphate derivative and incorporated into the viral DNA by HIV reverse transcriptase, resulting in the termination of DNA elongation (Furman et al., 1986). At 1 to 5 μM concentrations, ZDV is an effective and selective inhibitor of viral DNA replication in vitro.

Penetration of anti-HIV drugs into the CNS is of clinical concern due to the neurological effects of HIV (Navia and Price, 1998). Results from in vivo studies examining ZDV uptake into brain, after a single pass through the cerebral circulation, have demonstrated nonsignificant net transport of ZDV across the BBB (Terasaki and Pardridge, 1988; Wu et al., 1998a). Moreover, Thomas and Segal (1997) have determined, using a ventriculocisternal perfusion technique, that ZDV influx across the blood-CSF and BBB occurs primarily by a diffusional process. Because at present no information is available on the mechanism of transport of nucleoside analog drugs within brain parenchyma, the purpose of this study was to characterize the properties of ZDV uptake by microglia, the major target of HIV infection in the brain.

Kinetic analysis of ZDV uptake by microglia shows a saturable, low-affinity system (K_m = 1 mM). Similarly, weak interactions have been reported for ZDV in rat renal membrane vesicles, where ZDV inhibited transport of the organic cationN¹-methylnicotinamide at the brush-border membrane and transport of the organic anionp-aminohippuric acid at the basolateral site, with IC₅₀ values of 2.5 mM and 225 μM, respectively (Griffiths et al., 1991, 1992). These in vitro experiments further support in vivo results that showed that in the kidney, ZDV is transported by a basolateral probenecid-sensitive organic anion transporter, and an apical cimetidine-sensitive organic cation transporter (Aiba et al., 1995). Interestingly, a highK_m (0.5 mM) has been reported in the transport of ZDV by a recombinant Na⁺-dependent nucleoside transporter, functionally expressed in Xenopusoocytes (Yao et al., 1996).

Specific transporters responsible for handling cationic compounds have been reported mainly in the kidney, liver, and intestine. Three polyspecific, potential-sensitive organic cation transporters (OCT1, rOCT2/hOCT2, OCT3) cloned originally from rat kidney, and placenta, are differentially expressed in brain (Zhang et al., 1998). Sensitive reverse transcription-polymerase chain reaction and Northern blot studies reported low brain expression of OCT1 and OCT2, although several cationic neurotoxins and neurotransmitters are accepted as substrates by both transporters (Gorboulev et al., 1997; Grundemann et al., 1997). In contrast, mRNA transcripts specific for OCT3 were detected in significant amounts by Northern analysis in brain (i.e., cerebral cortex, cerebellum, hippocampus) and several other tissues (Kekuda et al., 1998; Wu et al., 1998b). Not only is OCT3 capable of transporting various cationic neurotoxins (i.e., MPP⁺) and neurotransmitters but also it has been shown to exhibit transport properties of an extraneuronal monoamine transporter (Wu et al., 1998b). Recently, two other cloned, electroneutral, H⁺-driven organic cation transporters, OCTN1 and OCTN2, were shown to be expressed widely in human tissues, including the brain (Tamai et al., 1997; Wu et al., 1998c).

Results from our substrate specificity studies show that several endogenous and exogenous organic cations (i.e.,N¹-methylnicotinamide, quinidine, mepiperphenidol, verapamil) inhibit specific ZDV uptake in a concentration-dependent manner with verapamil being a competitive inhibitor. However, the inhibition is modest (IC₅₀ range = 156–200 μM) and the system was weakly inhibited by the OCT probes TEA and MPP⁺. Furthermore, we observed that the cell line was unable to efficiently transport TEA. This specificity pattern is not shown for other OCT members such as OCT1, OCT2, OCT3, OCTN1, and OCTN2, which are all known to readily transport TEA (Gorboulev et al., 1997; Grundemann et al., 1997; Kekuda et al., 1998; Wu et al., 1998b,c). Thus, despite some OCT specificity similarities, the characterized transporter for ZDV in microglia appears to be distinct from any of the other cloned members of the OCT family.

Several studies have characterized the involvement of a H⁺/organic cation transporter in the renal luminal transport of organic cations. The driving force, a proton gradient, is known to be functionally linked to both the Na⁺/H⁺ antiporter and the H⁺-ATPase (Bendayan et al., 1994). To elucidate the energetics in ZDV transport by microglia, we investigated the effect of an outwardly directed proton gradient generated by a standard NH₄Cl loading procedure (Jans et al., 1987;Bendayan et al., 1994; Faff et al., 1996). The 20-min recovery of internal pH to baseline 7.1 from an intracellular acidification (pH 6.3, a decrease of 0.8 units) was confirmed by microfluorimetry. Moreover, the diuretic drug amiloride, a known inhibitor of the Na⁺/H⁺ antiporter, abolished the pH recovery and maintained the cell acidification, suggesting functional activity of a Na⁺/H⁺ antiporter. It is well established that amiloride rapidly and competitively inhibits the Na⁺ site of the Na⁺/H⁺ antiporter without affecting other Na⁺-coupled transport processes, shown, for instance, by its selective inhibition of the Na⁺/H⁺ exchanger in rabbit renal microvillus membrane vesicles (Kinsella and Aronson, 1981). Our studies show that with intracellular acidification, ZDV uptake is significantly enhanced within the first 5 min. The accumulation of ZDV cannot be explained by nonionic diffusion because the zwitterionic nature of the molecule is not modified within the pH range studied (6.3–8.0). Furthermore, ZDV uptake was significantly reduced (i.e., by 35% at 30 min) in the presence of the Na⁺/H⁺ ionophore monensin, thus suggesting the involvement of a H⁺exchanger. In mouse microglia, several pH regulatory systems (i.e., Na⁺/HCO₃⁻cotransporter, Cl⁻/HCO₃⁻exchanger, H⁺/K⁺- ATPase, Na⁺/H⁺ antiporter) are functionally expressed (Faff et al., 1996; Shirihai et al., 1998).

To test for membrane potential effects, ZDV uptake was investigated in high- and low-K⁺ medium in the presence of the potassium ionophore valinomycin. Membrane depolarization enhanced ZDV uptake, whereas hyperpolarization caused a decrease in ZDV uptake. Unlike other potential-driven OCT systems (Kekuda et al., 1998; Wu et al., 1998b), when depolarized with high extracellular K⁺, an inside positive potential difference drives an increased uptake of ZDV in microglia cells. Thus, although ZDV uptake system in microglia has some specificity features of an organic cation transporter, our results suggest a system that is novel in its sensitivity to membrane potential and pH. Various normal mechanisms that depolarize microglia could enhance ZDV uptake and, ultimately, increase its pharmacological activity. Activation of a number of channels has been reported to depolarize microglia, i.e., Ca²⁺ (Colton et al., 1994), Na⁺ (Korotzer and Cotman, 1992), anion channels (Schlichter et al., 1996), or purinergic receptor-gated channels (Illes et al., 1996).

In our cell system, the prototypical organic anionp-aminohippuric acid and the inhibitor probenecid had no effect on ZDV uptake, implying the lack of involvement of an organic anion transporter in MLS-9 cells. In addition, ZDV accumulation by MLS-9 cells was insensitive to a number of nucleosides, nucleoside analog drugs, and standard nucleoside transport inhibitors, suggesting that nucleoside transporters are not involved. These findings are consistent with reports documenting that the absence of the 3′-hydroxy group of the ribose moiety greatly reduces the ability of compounds to be transported by equilibrative nucleoside transporters. Furthermore, ZDV, 2′,3′-dideoxyadenosine, and 2′,3′-dideoxycytidine (zalcitabine) did not have measurable affinity for the BBB nucleoside transporter (Terasaki and Pardridge, 1988) or the Na⁺-dependent nucleoside transporter at the choroid plexus (Wu et al., 1994). In microglia, we have previously observed that ZDV was a potent noncompetitive inhibitor of a Na⁺-nucleoside transporter (Hong et al., 2000).

In summary, a novel, electrogenic, proton-driven ZDV transporter has been characterized in microglia, the target and reservoir of HIV infection in the brain. This system may play a significant role in the transport of other weak organic cation substrates and metabolites in brain parenchyma. The low affinity of ZDV for the rat microglia transporter suggests that drug-drug interactions may readily occur at the transporter site(s) with other weak organic cations, most likely causing either a decrease in therapeutic efficacy, an increase of toxicity, or both in AIDS patients receiving routinely multiple drug regimens. Moreover, because interspecies differences in the kinetic and selectivity properties of organic cation transporter homologs from rodent, rabbit, and human have been reported (Dresser et al., 2000), it is possible that a ZDV transporter with different kinetics may be expressed in humans. This could result in substantial differences in the in vivo microglial handling of drugs (i.e., higher affinity transport system for ZDV), basic metabolites, and toxins. It remains to be established whether other physiological roles can be attributed to this transport system.