Fig. 8. Effect of MRP1 inhibitors on NO-mediated 59Fe (A,B) and GSH release (C,D) from MCF7-WT and MRP1 hyper-expressing MCF7-VP cells. MRP1 inhibitors used were: MK571 (20 mM), difloxacin (20 mM), probenecid (0.5 mM), verapamil (20 mM) or vincristine (VCR; 20 mM). (A,B) Cells were labelled for 3 h at 37°C with 59Fe-Tf (0.75 mM), washed and reincubated for 30 min at 37°C with the inhibitors. A further 3-h incubation at 37°C was performed with the inhibitors in the presence or absence of SperNO (0.5 mM) and 59Fe efflux assessed. (C,D) Cells were incubated as in (A,B) and GSH release examined. The results are mean ± SD (4 determinations) from a typical experiment of 4. (Taken with permission from Ref. [89].)

The role of GSH in NO-mediated Fe release may not only be important for its transport out of the cell via the GSH transporter, MRP1. In fact, we hypothesize that it is also essential for Fe release from cellular proteins targeted by NO such as those with [Fe—S] clusters, e.g. aconitase [18,103]. Thus, we suggest that there is an intracellular equilibrium between protein-bound DNICs and low-Mr DNICs and that GSH is necessary for the conversion to the low-Mr form that is then transported out of the cell by MRP1 [89]. This idea is supported by our studies showing that incubation of cells with BSO markedly prevented both GSH and 59Fe release (Fig. 6A,D) and the potent MRP1 transport inhibitors, MK571 or probenecid, resulted in an accumulation of DNICs (Fig. 9). Our hypothesis is also consistent with the studies of Ding and colleagues that postulate that GSH and/or Cys are necessary for the release of DNICs from [Fe—S] clusters [104,105].

Considering how Fe and GSH are transported by MRP1, five potential mechanisms have been proposed to account for the interaction of the transporter with GSH and its

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