Co

v

CO + GSNO

Fig. 3. (A) The NO-donor GSNO but not CO gas results in 59Fe mobilization from prelabelled LMTK-fibroblasts. (B) Iron mobilization from LMTK- fibroblasts prelabelled for various times in the presence of GSNO, CO gas or a combination of both. (A) Cells were prelabelled with 59Fe-Tf (0.75 m-M) for 180 min at 37°C, washed, and then reincubated for up to 180 min at 37°C with GSNO (0.5 mM), GSH (0.5 mM) or 2% CO gas. Results are means of duplicate determinations in a typical experiment from 3 performed. (B) Cells were prelabelled with 59Fe—Tf (0.75 mM) for 15-180 min at 37°C, washed and then reincubated for 180 min with GSNO (0.5 mM), 2% CO gas or GSNO (0.5 mM) and 2% CO gas. Results are expressed as mean ± SD of triplicate determinations in a typical experiment of 3 experiments performed. (Taken with permission from Ref. [66].)

increased (Fig. 3B) [66]. This can be explained by the entry of Fe into less accessible cellular pools (e.g. ferritin) as the incubation time increased [80].

Recently, my laboratory has extensively examined the mechanism of NO-mediated Fe release from cells. These studies have demonstrated that it is GSH- and is energy-dependent and relies on the uptake and metabolism of D-glucose (d-G1c) [65,78]. In fact, only sugars that can be taken up and metabolized by cells were effective in increasing NO-mediated Fe release [65]. Fig. 4 is a schematic illustration summarizing a model of NO-mediated Fe release based upon these investigations [65,78]. Glucose enters the cell by the well-characterized family of glucose transporters [81] and is subsequently phosphorylated to glucose-6-phosphate (G-6-P; Fig. 4) [79]. Glucose-6-phosphate (G-6-P) is metabolized by two major pathways, either through glycolysis and/or the tricarboxylic acid cycle (TCA) to form ATP, or through the pentose phosphate pathway (PPP; that is also known as the hexose monophosphate shunt) to form reduced NADPH (e.g. for GSH synthesis) and pentose sugars (Fig. 4) [66].

Our studies demonstrated that D-Glc uptake and metabolism by the PPP was essential for NO-mediated Fe release [65]. Significantly, depletion of GSH using the specific

Fig. 4. Hypothetical model of d-Glucose-dependent NO-mediated Fe mobilization from cells. d-Glucose is transported into cells and is used by the tricarboxylic acid cycle (TCA) for the production of ATP and by the pentose phosphate pathway (PPP) for the generation of pentose sugars and NADPH. This reductant is involved in the production of reduced glutathione (GSH). Nitrogen monoxide (NO) either diffuses or is transported into cells where it intercepts and binds Fe bound to proteins or Fe in route to ferritin. The high affinity of NO for Fe results in the formation of an NO-Fe complex and GSH may either be involved as a reductant to remove Fe from endogenous ligands or may complete the Fe coordination shell along with NO. This complex may then be released from the cell by an active process requiring a transporter that has recently been identified as multi-drug resistance associated protein 1 (MRP1). (Modified from Ref. [78].)

Fig. 4. Hypothetical model of d-Glucose-dependent NO-mediated Fe mobilization from cells. d-Glucose is transported into cells and is used by the tricarboxylic acid cycle (TCA) for the production of ATP and by the pentose phosphate pathway (PPP) for the generation of pentose sugars and NADPH. This reductant is involved in the production of reduced glutathione (GSH). Nitrogen monoxide (NO) either diffuses or is transported into cells where it intercepts and binds Fe bound to proteins or Fe in route to ferritin. The high affinity of NO for Fe results in the formation of an NO-Fe complex and GSH may either be involved as a reductant to remove Fe from endogenous ligands or may complete the Fe coordination shell along with NO. This complex may then be released from the cell by an active process requiring a transporter that has recently been identified as multi-drug resistance associated protein 1 (MRP1). (Modified from Ref. [78].)

GSH synthesis inhibitor, buthionine sulfoximine (BSO) [82], prevented NO-mediated Fe release from cells [65,66,78]. In addition, Fe mobilization after GSH depletion could be reconstituted by incubation of cells with N-acetylcysteine that increased cellular GSH levels [65].

It is probable that the effect of D-Glc on stimulating NO-mediated Fe mobilization from cells was not just due to its effect on GSH metabolism. Indeed, our experiments showed that NO-mediated 59Fe release was temperature- and energy-dependent, suggesting a membrane transport mechanism could be involved [65]. As shown in Fig. 4, NO can enter cells through diffusion and there has been some evidence that this can occur by a transport molecule such as the protein disulfide isomerase that catalyses transnitrosylation [83]. A major intracellular target of NO appeared to be the Fe storage molecule ferritin [78]. NO prevented the uptake of Fe into ferritin and also appeared to indirectly mobilize Fe from this protein [78]. An indirect mechanism of Fe release from this protein was postulated, as NO-generating agents added to cellular lysates had no effect on ferritin Fe mobilization [78]. The efflux of Fe from cells and its removal from ferritin was GSH dependent, and could be inhibited using BSO [65,78]. It was speculated that GSH may assist in the removal of Fe from cells by either acting as a reducing agent or by filling the coordination shell of an Fe complex composed of NO and GSH ligands. This complex could be lipophilic enough to pass through the plasma membrane to exit the cell. However, our experiments using a variety of metabolic inhibitors showed that NO-mediated Fe removal from cells was an ATP-dependent event [65,78]. While NO appeared to act like a typical synthetic chelator (e.g. DFO or dipyridyl) in terms of its ability to mobilize cellular Fe, the mechanism was quite different, as chelator-mediated Fe release was not dependent on cellular GSH levels [65].

NITROGEN MONOXIDE MEDIATES IRON EXPORT FROM CELLS BY THE GSH TRANSPORTER, MRP1

As discussed above, NO-mediated Fe release from cells was a temperature- and ATP-dependent event suggesting the involvement of a transport system. Considering possible transport molecules responsible for NO-mediated Fe release, recently our attention became focused on the multi-drug resistance-associated protein 1 (MRP1 or ABCC1) [84]. MRP1 is an ABC transporter that is expressed ubiquitously in tissues [84]. Apart from the role of MRP1 as a detoxifying mechanism for the efflux of drugs from cells, this transporter also plays physiological roles where it is involved in the export of GSH and leukotriene C4 [84-88]. Earlier studies demonstrated that MRP1 can transport GSH coordinated to heavy metals such as As and Sb [85-88], but its role in Fe transport has not been previously proposed.

We examined NO-mediated 59Fe mobilization from several well-characterized cell models expressing high MRP1 levels, namely MCF7-VP cells [89]. These cells were compared to their wild-type parental counterparts (MCF7-WT) which do not express high MRP1 levels. To confirm functionality and expression of MRP1 in MCF7-VP cells we examined efflux of the classical substrate, tritiated-vincristine (3H-VCR) [90], and also MRP1 expression by RT-PCR and Western analysis. Fig. 5A shows that 3H-VCR efflux from MCF7-VP cells was significantly increased (p < 0.05-0.0001) compared to MCF7-WT parental cells. This suggested that MRP1 was expressed on the plasma membrane and was functional. These results were further confirmed in studies showing the higher levels of MRP1 mRNA and protein in MCF-VP cells compared to their WT counterparts (Fig. 5B). In addition, expression of several other potential GSH transporters, namely MRP2-4 or cystic fibrosis transmembrane conductance regulator (CFTR) [84] were not up-regulated in MCF7-VP cells compared to MCF7-WT. MCF7-VP cells are well-known to hyper-express MRP1 but not other drug transporters such as P-glycoprotein (multi-drug resistance protein 1; MDR1) [91,92]. Hence, these cells were implemented as an appropriate model to characterize 59Fe and GSH efflux via MRP1 after incubation with NO. They were also used in preference to several types of MRP1-transfected cells that do not express substantial functional MRP1 on the plasma membrane (data not shown).

Using the MCF7-VP and MCF7-WT cell lines, we showed [89] that MRP1 was involved in NO-mediated 59Fe and GSH efflux from our studies demonstrating that: (1) NO-mediated 59Fe release (Fig. 6A,B) and GSH efflux (Fig. 6D,E) were greater in MCF7-VP cells hyperexpressing MRP1 compared to MCF7-WT; (2) cellular 59Fe release (Fig. 7A,C) and GSH efflux (Fig. 7B) occurred by temperature- and metabolic energy-dependent mechanisms consistent with active transport; (3) the specific GSH inhibitor, BSO, that inhibits MRP1 transport activity [84] markedly prevented both NO-mediated 59Fe and GSH efflux from cells (Fig. 6A,D); (4) Well-characterized inhibitors of MRP1 such as MK571, difloxacin, verapamil and probenecid prevent NO-mediated 59Fe efflux (Fig. 8A,B); and (5) Potent inhibitors of MRP1 transport activity such as MK571 and probenecid result in an intracellular build-up of EPR-detectable DNICs (Fig. 9). Moreover, the extent of accumulation of

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