Extract
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Results
CBZ uptake over time The accumulation of CBZ is
time-dependent and a plateau of accumulation was observed
between 15 min and 60 min. Accordingly, uptake at 30 min was
used for experiments evaluating the effects of other drugs
on the transport characteristics of CBZ (Figure 1).
Uptake of CBZ at different
concentrations After cells were incubated with a medium containing 5, 10, 20, or 40
mg/L CBZ at 37 °C for 30 min, the amounts of CBZ taken up by
cells were measured (Figure 2). We found that the uptake of
CBZ by rBMEC was concentration-dependent, but that the
ratio of the concentration in cells to that in the medium was
similar for all concentrations, indicating that the uptake of
CBZ is linear between concentrations of 5 mg/L and 40 mg/L.
Effects of other drugs on the steady-state uptake of CBZ
CBZ uptake (UT) by cells incubated with a medium
containing 10 mg/L CBZ and various other agents was measured at
37 °C for 30 min. The percentage change (D%) compared
with the control (UC) was calculated according to the
following equation:
D% =(UT/UC_1)×100%, (1)and the calculated results are shown in Figure 3.
The agents tested can be divided into 3 groups based on
the percentage change in the steady-state uptake of CBZ.
Group 1 agents, cyclosporin A, dinitrophenol, erythromycin,
clarithromycin, acyclovir and diclofenac, increased the
uptake of CBZ. The percentage uptake increased by more than
20% for all group 1 agents. Dinitrophenol, a metabolic
inhibitor, increased CBZ uptake significantly, which
indicated that some energy-dependent efflux transporters may
be involved in the transport of CBZ. Group 2 agents, OLZ,
jasminoidin and fluoxetine, decreased the uptake of CBZ by
more than 20%. Group 3 agents, levodopa and ibuprofen,
had little effect on the steady-state uptake of CBZ.
In order to investigate whether the changes in CBZ
uptake by cells in presence of the tested agents resulted from
cell damage caused by the agents used, cells were incubated
at 37 °C for 30 min with 10 mg/L CBZ, and 50
mmol/L CsA, 50 mmol/L novobiocin, 100 mmol/L aciclovir, 100
mmol/L diclofenac or 100 mmol/L OLZ. The cell activity was
measured using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H
-tetrazolium bromide (MTT) assay. No differences were found
relative to the control. Thus, we believed that the agents
used did not damage cells at the concentrations used, and
that changes in the uptake of CBZ did not result from cell
damage.
Two typical agents, CsA (which increased the uptake of
CBZ) and OLZ (which decreased the uptake of CBZ) were
selected for further study.
Effect of CsA on the uptake of CBZ by
rBMEC The uptake of CBZ by rBMEC in the presence of 50
mmol/L CsA, at both 37 °C and 4 °C, is shown in Figure 4. CsA
significantly (P<0.01) increased the uptake of CBZ at 37 °C, but
not at 4 ºC.
Effect of OLZ on the uptake of CBZ The effects of
different concentrations of OLZ on the uptake of CBZ by cells
were measured after incubation for 30 min at both 37 °C and
4 °C (Figure 5).
OLZ decreased the uptake of CBZ in a
concentration-dependent manner from 1 to 100 µmol/L at 37 °C. However,
OLZ did not influence the uptake of CBZ at 4 °C.
Effect of CsA and OLZ on the efflux of CBZ from rBMEC
The results described earlier showed that CsA increased the
uptake of CBZ, whereas OLZ decreased the uptake of CBZ.
In order to investigate whether CsA and OLZ influenced the
uptake of CBZ via the efflux of CBZ from rBMEC, the efflux
of CBZ from rBMEC after 30 min and 60 min in the presence
of the tested agents was studied, and results are shown in
Figure 6. CsA significantly (P<0.01) decreased the efflux of
CBZ from rBMEC. The BCRP (breast cancer resistance
protein, BCRP) inhibitor, novobiocin, also slightly decreased
the efflux of CBZ. OLZ did not influence the efflux of CBZ.
Discussion
The present study showed that the uptake of CBZ by
rBMEC was time-dependent, concentration-dependent,
temperature-dependent, and energy-dependent. Many
trans-porters, including ABC efflux transport systems, may be
involved in the uptake of CBZ by rBMEC. Furthermore, we
found that drugs influenced the uptake of CBZ in different
ways, and that the drugs we tested could be divided into 3
groups.
Most of the agents in group 1 may be involved in one or
more ABC transporters. For example, CsA, a common P-gp
inhibitor, also inhibits BCRP
transporters[11]. Erythromycin, clarithromycin, and verapamil are inhibitors of
P-gp[7], and erythromycin also affects
OAT(organic anion
transporter)transporters[12]. Aciclovir may affect
MRP(multidrug resistance-associated protein , MRP
) transporters[13]. Gemfibrozil can be transported by P-gp and MRP
transporters[14], and novobiocin is an inhibitor of BCRP
transporters[15]. CsA increased the uptake of CBZ by rBMEC in a
concentration-dependent and temperature-dependent manner, which may
occur via inhibiting the efflux of CBZ from rBMEC.
Diltiazem, nimodipine and berberine in group 3 did not
increase the uptake of CBZ by rBMEC, although they are all
substrates of P-gp. In contrast, these 3 agents tended to
decrease the uptake of CBZ. Andrew et al reported that CBZ
was not a substrate of P-gp in a Coca-2 model, or in
mdr1a/1b(-/-) mice[5]. A similar result was found by Maines
et al using bovine retinal endothelial
cells[16]. These results suggest that P-gp is not likely to modulate CBZ transport in
rBMEC, and that other ABC transporters are involved in the
efflux of CBZ from rBMEC.
OLZ, jasminoidin and fluoxetine significantly inhibited
the uptake of CBZ by rBMEC. OLZ inhibited the uptake of
CBZ by rBMEC in a concentration-dependent and
temperature-dependent manner. Further study showed that OLZ
did not affect the efflux of CBZ from rBMEC. These results
indicate that some transporters may be involved in the influx
of CBZ in rBMEC, and that OLZ may decrease uptake in CBZ
via inhibiting these transporters.
Together, these observations indicate that the transport
of CBZ at the BBB is mediated by many transporters. Some
specific ABC efflux transporters may be involved in the
transport of CBZ. Various drugs influence the transport of CBZ at
the BBB in different ways.
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