Fig. 2.

Bidirectional transport of MPP⁺. (A–D) EM4 cells stably overexpressing rat Oct3 or empty vector (EV), astrocytes from new born C57BL/6 mice (E–G), astrocytes from Oct3^+/+ and Oct3^−/− mice (H–J), were assessed for their transport activities of [³H]-MPP⁺. As described in the SI Materials and Methods, briefly, for the uptake studies (A, B, E, F, and H–J), cells were incubated with 10 nM [³H]-MPP⁺ and various concentrations of unlabeled MPP⁺, in the presence or absence of 5 μM D22 (a potent Oct3 inhibitor). Cell pellets were collected and radioactivity was counted using a scintillation counter. Specific uptake (in J) represents transport activity in the absence of D22 minus that of the group with D22. For the release studies (C, D, G), cells were preloaded for 20 min with 10 nM [³H]-MPP⁺ (plus 100 μM MPP⁺), washed, and then incubated at 37 °C for different time points in the assay buffer with or without 5 μM D22. Radioactivity released into the buffer and remaining in cells was counted using a scintillation counter. Oct3-mediated transport was time and concentration dependent and was blocked by D22 and Oct3 deficiency. Data represent mean ± SEM from 3–5 independent experiments with n = 4 per experiment.