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Fig. 9. Electron paramagnetic resonance (EPR) spectroscopy demonstrates that SperNO results in dinitrosyl Fe complex (DNIC) formation and incubation with MRP1 inhibitors increases intracellular DNICs. Low-temperature EPR spectra (77 K) of MCF7-VP cells (1010 cells) preincubated with media alone (control) or the GSH synthesis inhibitor, BSO (0.1 mM), for 20 h at 37°C. The cells were then washed and incubated for 3 h at 37°C in the presence or absence of SperNO (0.5 mM). Alternatively, cells were incubated for 30 min with medium alone (control) or the MRP1 inhibitors, probenecid (0.5 mM) or MK571 (20 ^M), and then SperNO (0.5 mM) added and the incubation continued for 3 h at 37°C. Results are typical spectra from 6 experiments. (Taken with permission from Ref. [89].)

Fig. 9. Electron paramagnetic resonance (EPR) spectroscopy demonstrates that SperNO results in dinitrosyl Fe complex (DNIC) formation and incubation with MRP1 inhibitors increases intracellular DNICs. Low-temperature EPR spectra (77 K) of MCF7-VP cells (1010 cells) preincubated with media alone (control) or the GSH synthesis inhibitor, BSO (0.1 mM), for 20 h at 37°C. The cells were then washed and incubated for 3 h at 37°C in the presence or absence of SperNO (0.5 mM). Alternatively, cells were incubated for 30 min with medium alone (control) or the MRP1 inhibitors, probenecid (0.5 mM) or MK571 (20 ^M), and then SperNO (0.5 mM) added and the incubation continued for 3 h at 37°C. Results are typical spectra from 6 experiments. (Taken with permission from Ref. [89].)

substrates [84]. First, GSH itself can directly act as a MRP1 substrate and be transported out of the cell. Second, GSH can form a complex or conjugate with a metal or compound that is then transported by MRP1, e.g. leukotriene C4 [106] or the As(GS)3 complex [107]. Third, GSH can be co-transported with some MRP1 substrates, e.g. vincristine. Fourth, GSH can stimulate the transport of certain compounds (e.g. estrone-3-sulfate) by MRP1 without itself being transported out of the cell, and fifth, GSH transport by MRP1 can be enhanced by interaction with certain compounds (e.g. verapamil) that are not translocated themselves across the membrane [84,108,109].

We showed that the GSH synthesis inhibitor, BSO, markedly inhibited both NO-mediated 59Fe and GSH efflux suggesting a dependence on GSH for transport [65] (Fig. 6). Considering this, it can be suggested that either a complex containing both 59Fe and GSH are effluxed together or 59Fe and GSH are separately transported across the membrane by MRP1. We favor a hypothesis necessitating the formation of a complex intracellularly. This is because we can detect intracellular DNICs directly by EPR and the fact that these accumulate after incubation with the MRP1 inhibitors MK571 and probenecid (Fig. 9) [89]. Relevant to the molecular mechanism of GSH and Fe transport by MRP1, it is of interest that verapamil and difloxacin markedly prevent NO-mediated 59Fe efflux from MCF7-VP and MCF7-WT cells (Fig. 8A,B), while both of these MRP1 inhibitors stimulate GSH release (Fig. 8C,D).

Fig. 10. Schematic illustration of hypotheses proposing the consequences of MRPl-mediated DNIC efflux from cells. (A) The efficient efflux of DNICs by an active transport mechanism could be crucial where NO is produced in small quantities as a messenger molecule, e.g. in blood vessels where endothelial NOS (eNOS) generates NO. The ability of cells to actively transport and traffic NO overcomes the random process of diffusion that would be inefficient and non-targeted. The small quantities of GS-Fe—NO complexes released from endothelial cells may be taken up by smooth muscle cells through diffusion or active transport, e.g. via protein sulfide isomerase for instance [83]. (B) Where NO is used as a cytotoxic effector, the substantial quantities generated by inducible NOS (iNOS) of activated macrophages could lead to the efflux of a large proportion of Fe and GSH from tumor cell targets that would be cytotoxic. (Taken with permission from Ref. [89].)

Fig. 10. Schematic illustration of hypotheses proposing the consequences of MRPl-mediated DNIC efflux from cells. (A) The efficient efflux of DNICs by an active transport mechanism could be crucial where NO is produced in small quantities as a messenger molecule, e.g. in blood vessels where endothelial NOS (eNOS) generates NO. The ability of cells to actively transport and traffic NO overcomes the random process of diffusion that would be inefficient and non-targeted. The small quantities of GS-Fe—NO complexes released from endothelial cells may be taken up by smooth muscle cells through diffusion or active transport, e.g. via protein sulfide isomerase for instance [83]. (B) Where NO is used as a cytotoxic effector, the substantial quantities generated by inducible NOS (iNOS) of activated macrophages could lead to the efflux of a large proportion of Fe and GSH from tumor cell targets that would be cytotoxic. (Taken with permission from Ref. [89].)

Hence, at least in the presence of these inhibitors, GSH and 59Fe appear to be separate transportable entities [89].

Iron efflux from cells has been described for many years [11], and recently, ferroportin-1 has been shown to be a physiologically relevant Fe exporter [44—46]. It is probable that the role of MRP1 is quite different from that of ferroportin-1 and this is suggested by differences g 80

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