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Results Methotrexate Uptake. To evaluate the time-dependent uptake of methotrexate in S2 hOAT1, S2 hOAT3 and S2 hOAT4, S2 cell monolayers were incubated in a solution MTX Transport and Its Drug Interaction 667 containing 1 _M methotrexate (hOAT1) or 100 nM methotrexate (hOAT3 and hOAT4) for various periods at... 顯示更多 Results
Methotrexate Uptake. To evaluate the time-dependent
uptake of methotrexate in S2 hOAT1, S2 hOAT3 and S2
hOAT4, S2 cell monolayers were incubated in a solution
MTX Transport and Its Drug Interaction 667
containing 1 _M methotrexate (hOAT1) or 100 nM methotrexate
(hOAT3 and hOAT4) for various periods at 37°C. As
shown in Fig. 1, when the amount of methotrexate uptake in
mock was subtracted from those in S2 hOAT1, S2 hOAT3, and
S2 hOAT4, the specific uptake in S2 hOAT1 (Fig. 1A) and S2
hOAT4 (Fig. 1C) increased in a time-dependent manner and
reached steady state, whereas that in S2 hOAT3 (Fig. 1B)
decreased after reaching peak. The latter may be due to the
observation that nonspecific methotrexate efflux overwhelmed
hOAT3-mediated methotrexate uptake depending
on the nature of monoclonal cells into which hOAT3 cDNA
was transfected.
The kinetics of methotrexate uptake was examined to evaluate
the pharmacological characteristics of hOAT1, hOAT3,
and hOAT4 on methotrexate transport. The concentrationdependent
uptake of methotrexate was observed in S2
hOAT1, S2 hOAT3, and S2 hOAT4 after subtraction of uptake
by mock (data not shown). As shown in Fig. 2, Eadie-Hofstee
plot analysis of hOAT1-, hOAT3-, and hOAT4-mediated
methotrexate uptake gave a single straight line. The estimated
Km values of methotrexate uptake by hOAT1, hOAT3,
and hOAT4 were 553.8 _ 43.2 _M, 21.1 _ 2.8 _M, and 17.8 _
1.6 _M, respectively (from three determinations in one typical
experiment of two separate experiments). These results
suggest that hOAT1, hOAT3, and hOAT4 mediate transport
of methotrexate.
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