FIGURE 3.

Michaelis-Menten kinetics of zebrafish Oatp1d1 mediated uptake of E3S and fluorescent dye LY. Transport measurements were performed 24 h after transient transfection in HEK293 cells. Oatp1d1-overexpressing HEK293 cells were preincubated in the transport medium (145 mm NaCl, 3 mm KCl, 1 mm CaCl₂, 0.5 mm MgCl₂, 5 mm d-glucose, and 5 mm HEPES, pH 7.4) for 10 min at 37 °C. To assess transport, medium was removed, and the same medium containing a substrate was added. A, time response of Oatp1d1 [³H]E3S-mediated uptake expressed as a change in disintegration parts per minute (dpm) over the incubation time; B, dose response of Oatp1d1 [³H]E3S uptake expressed as transport rate of [³H]E3S (nmol/mg protein/min) over [³H]E3S concentration (μm) after a 5-min incubation with [³H]E3S (50 Ci/mmol). C, time response of LY uptake expressed as an increase in fluorescence (fluorescence units (f.u.)) over time (min). D, concentration dependence of LY uptake expressed as the transport rate (nmol/mg protein/min) over LY concentration (μm) after a 15-min incubation with LY. The uptake into vector-transfected HEK293 cells (mock cells) was subtracted to obtain transporter-specific uptake, and data were fitted in GraphPad Prism version 5. Each value represents the mean ± S.E. (error bars) from triplicate determinations.