A Novel Zidovudine Uptake System in Microglia
MEERA HONG,1LYANNE SCHLICHTER,2and REINA BENDAYAN1
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada
Received May 19, 2000; accepted September 19, 2000 This paper is available online at http://jpet.aspetjournals.org
In the central nervous system (CNS), brain macrophages and
microglia are the primary targets of productive human immu-
nodeficiency virus 1 (HIV-1) infection. Zidovudine (ZDV), a thy-
midine derivative, has been reported to reduce the progression
of the disease and prolong survival in patients with acquired
immunodeficiency syndrome (AIDS) and AIDS dementia com-
plex. Although a restricted ZDV distribution has been observed
in the CNS, its accumulation in brain parenchyma has not been
examined. We have investigated the uptake properties of ra-
diolabeled ZDV by a continuous rat microglia cell line (MLS-9)
grown as a monolayer on an impermeable surface. Although
the organic cations verapamil, mepiperphenidol, quinidine, ci-
metidine, and N1-methylnicotinamide moderately inhibited ZDV
uptake, the organic cation probes tetraethylammonium and
1-methyl-4-phenylpyridinium were weak inhibitors. ZDV uptake
was significantly increased when the proton gradient was out-
ward (pHi6.3 ? pHo7.4; pHi?7.1 ? pH 8.0), whereas uptake
decreased with extracellular acidification (pHi?7.1 ? pHo6.0)
or in the presence of the Na?/H?ionophore monensin. ZDV
uptake was increased under depolarized membrane conditions
(i.e., 138 mM K?in external medium) and decreased under
hyperpolarized conditions (i.e., 2 mM K?in external medium),
implying a membrane potential dependence. These results
suggest that although ZDV transport system in microglia has
some specificity features of an organic cation transporter, it
involves a carrier, distinct from other cloned organic cation
transporters, that is novel in its sensitivity to pH and membrane
potential. This system may play a significant role in the trans-
port of other weak organic cation substrates and/or metabolites
in brain parenchyma.
Human immunodeficiency virus 1 (HIV-1) infection of the
brain causes HIV-1 encephalopathy and AIDS dementia com-
plex (ADC), a syndrome characterized by cognitive, motor,
and behavioral disturbances (Navia and Price, 1998). Pa-
tients with severe ADC usually present microglial activation
and multinucleated giant cells, the hallmarks of HIV enceph-
alitis. In the central nervous system (CNS), it appears that
only macrophages and microglia harbor the virus particles
and the number of proviruses correlates with HIV encepha-
litis (Navia and Price, 1998).
Microglia are a distinct population of non-neuronal cells
that are involved in maintenance of neuronal homeostasis,
synaptic plasticity, and repair. These cells play a key role in
CNS immune and inflammatory reactions, and are suscepti-
ble to diverse insults, ranging from physical damage to in-
fectious agents (Gonzalez-Scarano and Baltuch, 1999). At the
two extremes, microglia morphology is 1) spheroid/activated,
a state that appears early in development and after brain
lesions, and 2) ramified/resting microglia, present in the nor-
mal adult brain. Microglial activation, a process that often
includes changes in morphology and surface expression of
immune-related molecules, has been described in virtually
all human neuropathologies, including neurodegenerative
diseases, multiple sclerosis, and HIV-1 infection (Gonzalez-
Scarano and Baltuch, 1999). The microglia used in our in
vitro cell system, are spheroid, proliferating, and capable of
producing nitric oxide (Hong et al., 2000; C. A. Colton and
L. C. Schlichter, unpublished data).
For the effective treatment of AIDS-ADC or AIDS enceph-
alopathy, anti-HIV drugs need to reach the CNS in signifi-
cant amounts. ZDV (3?-azido-3?-deoxythymidine, AZT, Retro-
vir), a thymidine analog, is a potent inhibitor of the in vitro
replication and cytopathic effect of HIV. Although limited CNS
This work was supported by a grant from the Ontario HIV Treatment
Network (OHTN), the Canadian Foundation for AIDS Research (CANFAR),
the Glaxo Wellcome Positive Action Fund, Ontario Ministry of Health, and the
Heart and Stroke Foundation of Ontario (no. T3726). M.H. is a recipient of an
Ontario HIV Treatment Network Studentship Award.
This work was presented in preliminary form (Hong et al., 1999). Poster
presented at the 1999 Annual Meeting of the American Association of Phar-
maceutical Scientists, New Orleans, LA, Nov. 1999.
1Current address: Department of Pharmaceutical Sciences, Faculty of
Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
2Current address: Cellular and Molecular Biology, Toronto Western Re-
search Institute, University Health Network, Toronto, Ontario M5T 2S8 and
Department of Physiology and Institute for Medical Sciences, University of
Toronto, Toronto, Ontario M5S 1A1, Canada.
ABBREVIATIONS: HIV-1, human immunodeficiency virus 1; AIDS, acquired immunodeficiency syndrome; ADC, AIDS dementia complex; CNS,
central nervous system; ZDV, zidovudine; BBB, blood-brain barrier; CSF, cerebrospinal fluid; EBSS, Earle’s balanced saline solution; MES,
2-(N-morpholino)ethanesulfonic acid; BCECF, 2?,7?-bis(carboxyethyl)-5(6?-carboxyfluorescein; BCECF-AM, 2?,7?-bis(carboxyethyl)-5(6?-carboxy-
fluorescein acetoxymethyl ester; MPP?, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium; OCT, organic cation transport; BES, N,N-bis(2-
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 296:141–149, 2001
Vol. 296, No. 1
Printed in U.S.A.
by on September 15, 2009
access has been reported for most antiretroviral drugs, after
ZDV administration, substantial improvement was noted in
ADC patients (Fischl et al., 1987). The clinical benefits include
increased survival, reduced number of opportunistic infections,
treatment is limited by its hematological toxicity (anemia, neu-
tropenia), the development of HIV resistance, and its limited
accumulation in certain tissues, including the CNS, which is an
important site for HIV replication.
ZDV can enter the CNS by passive diffusion across the
blood-brain barrier (BBB) and, more significantly, the blood-
cerebrospinal fluid (CSF) barrier (Thomas and Segal, 1997).
However, efflux via a probenecid-sensitive transport system
at the BBB contributed to the restricted distribution of ZDV
in brain tissue (Takasawa et al., 1997b). Low CSF-to-plasma
concentration ratios of ZDV after i.v. infusion have been
reported in rats (0.15) (Galinsky et al., 1990), rabbits (0.26)
(Wong et al., 1993), and humans (0.5) (Blum et al., 1988).
Thus, the low steady-state ZDV levels in the CNS depend on
both diffusive and active processes.
Because ZDV can act either as an organic anion or cation,
consistent with the resonance structures of the azido moiety
and pKavalue of 9.68 (Henry et al., 1988), in principle, its
transport could involve nucleoside transporters, organic an-
ion or organic cation transporters. Interestingly, ZDV inter-
acts with an organic anion transporter at the basolateral
membrane (Griffiths et al., 1991) and an organic cation
transporter at the brush-border membrane (Griffiths et al.,
1992) of renal cortical vesicles. Consistent with such trans-
port, an energy-dependent organic cation carrier is inhibited
by ZDV in opossum kidney cells (Chen et al., 1999). Yao et al.
(1996) provided direct evidence for Na?-dependent ZDV
transport using heterologous expression of the intestinal/
kidney nucleoside transporter rCNT1 in Xenopus oocytes.
The lipophilicity imparted by the azido group allows nonfa-
cilitated diffusion of ZDV into human erythrocytes, lympho-
cytes, macrophages, bone marrow progenitor cells, and intes-
tinal epithelial cells.
Our previous study identified a Na?-dependent nucleoside
transporter highly sensitive to ZDV in microglia cells (Hong
et al., 2000). However, it is not known which, if any, trans-
porters for ZDV exist in microglia, the primary target of HIV
infection in the CNS. The goal of this study was to determine
the in vitro disposition of ZDV by a brain parenchymal cell
line (MLS-9 microglia cells) to characterize its mechanism of
transport, and identify potential interactions with other com-
pounds at the carrier site(s).
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-cm2Falcon 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, sup-
plemented with L-glutamine, D-glucose, 5% fetal bovine serum, 5%
horse serum, and 0.5% penicillin/streptomycin suspension (all ob-
tained 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 ? 105cells/well, and only those grown to a
uniform, confluent monolayer (within 4–5 days) were used. The integ-
rity of cell monolayers was evaluated microscopically, before and after
the uptake measurements. Most importantly, the morphology and up-
take 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 mM D-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 prein-
cubated 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 quanti-
tated 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 ex-
tracellular space, determined using
mmol; NEN Life Science Products, Boston, MA), was negligible
(?0.14%); therefore, no correction was applied. The protein concen-
tration (milligrams per milliliter) in each culture plate was deter-
mined by the Bradford method, using BSA as the standard and
Bio-Rad reagent (Bio-Rad, Mississauga, Ontario, Canada). ZDV up-
take 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 ?M) or the Na?/H?
ionophore monensin (5 ?M), 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). Di-
danosine and zalcitabine were generously provided by Bristol-Myers
Squibb (Princeton, NJ) and Hoffmann-La Roche (Nutley, NJ), re-
spectively. Unless specified, other chemicals were from Sigma Chem-
ical Co. (St. Louis, MO) and were of the highest purity available.
D-[14C]mannitol (51.5 mCi/
Hong et al.
by on September 15, 2009
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 ?Ci/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-?m-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 RFvalues corre-
sponding 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 mea-
sured fluorimetrically using the pH-sensitive carboxyfluorescein de-
(Molecular Probes, Eugene, OR). Single-cell measurements were per-
formed 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. Conflu-
ent monolayers of MLS-9 cells grown in 24-well tissue culture plates
were preincubated (40 min, 37°C) in EBBS containing 20 ?M
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 flu-
orescence intensity of cells with 100 ?M BCECF acid in EBSS solu-
tion, 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 (Kmand Vmax) for ZDV trans-
port were determined by a nonlinear least-squares analysis in Sigma
Plot 4.0. To estimate the inhibitory constant (Ki) of the inhibitor
verapamil, a least-squares regression analysis was used to deter-
mine the linear correlation between V and V/[ZDV], the Eadie-Hof-
stee linear transformation. The IC50or concentration of various
organic cationic drugs causing a 50% reduction in ZDV uptake was
estimated by a sigmoidal, four-parameter inhibition model. Statisti-
cal 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 appro-
priate. A p value ?0.05 was considered significant.
Metabolism of ZDV. The intracellular metabolism of
ZDV has been examined extensively in cell lines, in retrovi-
rus-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. Be-
cause metabolism can complicate the interpretation of trans-
port 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 radio-
activity 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 frac-
tions, 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. Figure 1 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 in-
volved (see below), to approximate the apparent Kmvalue
and better describe ZDV uptake properties.
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 ?M to 2.5 mM. The total ZDV uptake
rate (V) can be described by the following equation:
V ?Vmax? ?ZDV?
Km? ?ZDV?? D ? ?ZDV?
where Vmaxis the maximum uptake rate; Kmis the affinity
constant, and D is the coefficient for nonspecific, diffusive
process. The specific ZDV uptake rate was calculated by
subtracting from the total rate, the nonspecific uptake mea-
sured with a high concentration of the most potent inhibitor
we could identify (3 mM verapamil). Figure 2 shows evidence
Fig. 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 repre-
sent the average of quadruplicate measurements.
A Novel Zidovudine Uptake System in Microglia
by on September 15, 2009
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 and V/[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 ?M, 326 ? 14 pmol/mg/min, and
0.2 ? 0.1 pmol/mg/min/?M for the Km, Vmax, and D, respec-
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 sev-
eral 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, mepiper-
phenidol, quinidine, cimetidine, and N1-methylnicotinamide,
known inhibitors of organic cation transport systems, were
the most effective inhibitors (70–79% inhibition). Thiamine,
1-methyl-4-phenylpyridinium (MPP?), guanidine, tetraeth-
ylammonium (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
Further inhibitory studies were undertaken and IC50val-
ues for the moderate inhibitors were determined by fitting
the data to a sigmoidal equation:
V ? V0?
where V and Vcrepresent ZDV uptake in the presence and
absence (control) of the inhibitor, respectively. Both V and Vc
values were corrected for the nonspecific uptake as measured
in the presence of 3 mM verapamil. Vois the zero time
uptake, C is the inhibitor concentration in the reaction mix-
ture, 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 IC50
values of 156 ? 10 (Fig. 4A), 165 ? 11, 168 ? 10, 171 ? 10,
and 200 ? 10 ?M for verapamil, mepiperphenidol, quinidine,
cimetidine, and N1-methylnicotinamide, respectively.
We next explored the type of inhibition exerted by the most
potent inhibitor we identified. Verapamil inhibited ZDV up-
take in a competitive manner with an estimated inhibitory
constant (Ki) of 167 ? 21 ?M. The Kivalue was obtained from
the competitive inhibition equation:
where [I] is the inhibitor concentration; K?m(apparent Km)
and Kmare the affinities of the carrier in the presence and
absence of the inhibitor, respectively. A least-squares regres-
sion was fitted to determine the Eadie-Hofstee linear corre-
lations (Fig. 4B). Competitive inhibition is indicated by the
Fig. 2. Kinetics of ZDV uptake. ZDV uptake was measured at various
ZDV concentrations (20 ?M 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 (Km, Vmax)
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 subtract-
ing nonspecific uptake.
Fig. 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, gua-
nidine, 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).
Hong et al.
by on September 15, 2009
common y-intercept of the regression lines, which shows the
same value for Vmax.
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. Or-
ganic 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
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 inhibi-
tory effect of nucleosides (thymidine, cytidine, guanosine,
and adenosine), nucleoside analogs (lamivudine, abacavir,
didanosine, and zalcitabine), and standard nucleoside trans-
port inhibitors [dilazep, dipyridamole, and 6-(4-nitrobenzyl)-
thio-9-?-D-ribofuranosylpurine] on ZDV uptake, in the pres-
ence and absence of Na?(Na?replaced with N-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 cor-
roborate 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 intes-
tine, 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?anti-
porter 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 con-
firmed 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), fol-
lowed 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 en-
hanced for the first 5 min, and then decreased to about
control values by 10 min, presumably due to the dissipation
Fig. 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 ?M–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 up-
take. ZDV (100, 250, and 500 ?M, 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 regres-
sion analysis was used to determine the Eadie-Hofstee linear correla-
tions. Results are expressed as mean ? S.D. of two separate experiments,
with quadruplicate measurements. (Some error bars are smaller than the
Fig. 5. Intracellular pH changes. MLS-9 cells were preincubated for 40
min at 37°C in EBSS containing 20 ?M 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 repre-
sent the combined data from four separate experiments.
A Novel Zidovudine Uptake System in Microglia
by on September 15, 2009
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 (pHi? 7.1 ? pHo8.0), and de-
creased by extracellular acidification (pHi? 7.1 ? pH06.0).
Thus, ZDV uptake appears to be increased by an outwardly
directed proton gradient and decreased by an inwardly di-
rected one. The requirement for an outwardly directed proton
gradient was further supported by the decrease in uptake (by
35%) after exposure to 5 ?M 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.
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 electro-
genic, membrane potential was changed by varying extracel-
lular 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 ?M 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.
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 incorpo-
rated into the viral DNA by HIV reverse transcriptase, re-
sulting in the termination of DNA elongation (Furman et al.,
Fig. 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 (pHi? pHo). The gradient was generated by exposing the cells to
30 mM NH4Cl, washing, and placing in uptake buffer. Results are ex-
pressed as mean ? S.D. of three different experiments, with quadrupli-
cate measurements. ??, values that are significantly different from con-
trol value (p ? 0.01).
Fig. 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 F) or EBSS
medium containing Tris (pH 8.0 ?) or MES (pH 6.0 ?). Each experimen-
tal 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).
Fig. 8. Monensin decreases specific ZDV uptake. MLS-9 cells were incu-
bated over 30 min at 37°C in standard EBSS medium (control) or in the
presence of 5 ?M monensin, an Na?/H?ionophore. Results are expressed
as mean ? S.D. of three different experiments, with quadruplicate mea-
surements. Values that are significantly different from controls are indi-
cated (*p ? 0.05; **p ? 0.01).
Hong et al.
by on September 15, 2009
1986). At 1 to 5 ?M 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 deter-
mined, 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 micro-
glia, the major target of HIV infection in the brain.
Kinetic analysis of ZDV uptake by microglia shows a sat-
urable, low-affinity system (Km? 1 mM). Similarly, weak
interactions have been reported for ZDV in rat renal mem-
brane vesicles, where ZDV inhibited transport of the organic
cation N1-methylnicotinamide at the brush-border mem-
brane and transport of the organic anion p-aminohippuric
acid at the basolateral site, with IC50values of 2.5 mM and
225 ?M, 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 pro-
benecid-sensitive organic anion transporter, and an apical
cimetidine-sensitive organic cation transporter (Aiba et al.,
1995). Interestingly, a high Km(0.5 mM) has been reported in
the transport of ZDV by a recombinant Na?-dependent nu-
cleoside transporter, functionally expressed in Xenopus oo-
cytes (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 trans-
porter (Wu et al., 1998b). Recently, two other cloned, electro-
neutral, H?-driven organic cation transporters, OCTN1 and
OCTN2, were shown to be expressed widely in human tis-
sues, including the brain (Tamai et al., 1997; Wu et al.,
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. How-
ever, the inhibition is modest (IC50range ? 156–200 ?M)
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 trans-
porter 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?anti-
porter and the H?-ATPase (Bendayan et al., 1994). To eluci-
date the energetics in ZDV transport by microglia, we inves-
tigated 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 estab-
lished 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 diffu-
sion because the zwitterionic nature of the molecule is not
Fig. 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?(F), 138 mM K?(f), 138 mM K?? 1 ?M valinomycin (?), and
2 mM K?? 1 ?M valinomycin (?), a potassium ionophore. Results are
expressed as mean ? S.D. of two separate experiments; each experimen-
tal point represents quadruplicate measurements. Values significantly
different from control values are indicated (**p ? 0.01; *p ? 0.05).
A Novel Zidovudine Uptake System in Microglia
by on September 15, 2009
modified within the pH range studied (6.3–8.0). Further-
more, 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?/
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 depolariza-
tion 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 pos-
itive 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.,
In our cell system, the prototypical organic anion p-amin-
ohippuric 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 accumu-
lation by MLS-9 cells was insensitive to a number of nucleo-
sides, 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. Fur-
thermore, ZDV, 2?,3?-dideoxyadenosine, and 2?,3?-dideoxycy-
tidine (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 trans-
porter 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 mul-
tiple drug regimens. Moreover, because interspecies differ-
ences 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 trans-
port system for ZDV), basic metabolites, and toxins. It re-
mains to be established whether other physiological roles can
be attributed to this transport system.
We thank Dr. Peter Pennefather for very helpful comments in the
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Send reprint requests to: Dr. Reina Bendayan, Department of Pharmaceu-
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Toronto, Ontario M5S 2S2, Canada. E-mail: email@example.com
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