The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner Ralph N. Watts and Des R. Richardson The Iron Metabolism and Chelation Group, The Heart Research Institute, Camperdown, Sydney, New South Wales, Australia Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724–4732]. We hypothesized that GSH completes the coordination shell of an NO–Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59 Fe-autoradiography shows that NO decreased ferritin- 59 Fe levels in cells prelabelled with [ 59 Fe]transferrin. In prelabelled cells, ferritin- 59 Fe levels increased 3.5)fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin- 59 Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59 Fe on route to ferritin, and indirectly facilitates removal of 59 Fe from the protein. Studies using the GSH-depleting agent, L -buthionine-(S,R)- sulphoximine, indicated that the reduction in ferritin- 59 Fe levels via NO was GSH-dependent. Competition experi- ments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentra- tion gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization. Keywords: chelators; ferritin; glutathione; iron; nitrogen monoxide. Many of the diverse biological effects of nitrogen monoxide (NO) are mediated through its binding to iron (Fe) in the haem prosthetic group of soluble guanylate cyclase [1–3]. Indeed, the high affinity of NO for Fe is a well-characterized branch of coordination chemistry [2]. Apart from the regulatory role of NO, its cytotoxic actions are found when it is produced in large quantities by cells such as activated macrophages [3]. Interestingly, NO produced by such systems inhibits the proliferation of intracellular pathogens and tumour cells [3–5]. These effects can be explained by the reactivity of NO with Fe in the [Fe–S] centres of critical proteins, including aconitase and complex I and II of the electron transport chain [4–6]. The high affinity of NO for Fe probably results in both the removal of Fe from [Fe–S] centres and the formation of dinitrosyl Fe species within [Fe–S] proteins (reviewed in [7]). It has already been shown that NO forms complexes with a range of Fe-containing proteins including ferritin [8], ribonucleotide reductase [9], haem-containing proteins [10–12], and ferrochelatase [13]. Further, it has been suggested that ferritin can act as a store of NO [8], and NO-mediated Fe release from isolated and purified ferritin has been demonstrated [14]. When activated macrophages are cocultured with tumour cells, this inhibits target cell DNA synthesis and results in the release of 64% of cellular 59 Fe within 24 h [15]. This loss of Fe may be due to the NO-mediated release of Fe from enzymes such as mito- chondrial aconitase [4,16–18]. Others have suggested that NO can also target loosely bound pools of nonhaem Fe [19]. Nonetheless, the identification of Fe–nitrosyl complexes (Fe–dithiol dinitrosyl complexes and haem–nitrosyl com- plexes) by EPR spectroscopy in activated macrophages and their tumour cell targets show the importance of the Fe–NO interaction [17–23]. Apart from the above effects, NO can also increase the RNA-binding of iron-regulatory protein 1 (IRP1), that plays an important role in regulating intracellular Fe homeo- stasis (reviewed in [3,24]). The effect of NO on IRP1-RNA binding activity occurs via two main mechanisms, a direct effect on the [4Fe)4S] cluster and Fe mobilization from Correspondence to D. R. Richardson, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, New South Wales, 2050 Australia. Fax: + 61 2 9550 3302, Tel.: + 61 2 9550 3560, E-mail: d.richardson@hri.org.au Abbreviations:BSO, L -buthionine-[S,R]-sulphoximine; BSS, balanced salt solution; DFO, desferrioxamine; GSH, reduced glutathione; GSNO, S-nitrosoglutathione; HMPS, hexose monophosphate shunt; IRP1, iron-regulatory protein 1; MEM, minimum essential medium; NAP, N-acetylpenicillamine; PIH, pyridoxal isonicotinoyl hydrazone; SNAP, S-nitroso-N-acetylpenic- illamine; Sper, spermine; SperNO, Spermine-NONOate; Tf, transferrin; 311, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; DTPA, diethylenetriaminepentaacetic acid. (Received 14 February 2002, revised 29 April 2002, accepted 6 May 2002) Eur. J. Biochem. 269, 3383–3392 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02987.x cells [25–29]. Our previous studies have shown that a range of NO-generators [e.g. S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and spermine NONOate (SperNO)], could mobilize 59 Fe from prelabelled cells as, or more, effectively than the clinically used Fe chelator desferrioxamine (DFO) [29]. In contrast, the precursor compounds of these latter NO-generators, namely N-acetylpenicillamine (NAP), glutathione (GSH), and spermine (Sper), respectively, had no effect [29]. Previous studies have suggested that NO may be released from cells as a complex composed of NO, Fe, and thiol- containing ligands such as cysteine or GSH [23,30,31]. Considering this and the other data described above, we recently examined the energy-dependency of NO-mediated Fe release from cells [32]. Our investigation showed that metabolism of D -glucose potentiates NO-mediated Fe efflux from a variety of cell types. Further, we demonstrated that the metabolism of D -glucose by the hexose monophosphate shunt (HMPS) and the maintenance of GSH levels was essential for NO-mediated Fe mobilization [32]. However, we are not proposing a direct coupling between glucose import/metabolism and NO metabolism. Rather, our ex- periments suggested that the generation of GSH after incubation with D -glucose could result in GSH acting as a ligand which together with NO would complete the coordi- nation shell of Fe [32]. Such a Ômixed Fe complexÕ with both NO and GSH ligands bound to Fe may provide an appro- priate lipophilic balance to allow diffusion through the membrane and/or transport by a carrier. In fact, we showed that NO-mediated 59 Fe release was both temperature- and energy-dependent, suggesting a membrane transport mech- anism could be involved [32]. However, the intracellular site of NO-mediated Fe release was not established. In this investigation we have extended our knowledge of NO-mediated Fe mobilization. For the first time, we demonstrate using a cellular system that NO intercepts Fe before being incorporated into ferritin in a similar manner to Fe chelators. Further, NO facilitates removal of 59 Fe from ferritin probably by an indirect mechanism. This process of depleting ferritin-bound 59 Fe was dependent on cellular GSH. Our studies also indicate that cellular metabolism was required for NO-mediated Fe mobilization which appears to be an active rather than a passive process. These results may be important in understanding the cytotoxic actions of NO produced by activated macrophages. EXPERIMENTAL PROCEDURES Cell treatments and reagents The NO-generator SNAP was synthesized by established techniques [33] from the precursor compound NAP (Sigma Chemical Co.). Apotransferrin (apoTf), L -buthionine-(S,R)- sulphoximine (BSO), diethylenetriaminepentaacetic acid (DTPA), E ` DTA, GSH, GSNO, horse spleen ferritin and Sper were obtained from Sigma. SperNO was obtained from Cayman Chemicals. Eagle’s minimum essential medium (MEM) was obtained from Gibco BRL. DFO was obtained from Novartis Pharmaceutical Co. Pyridoxal isonicotinoyl hydrazone (PIH) and its analogue, 2-hydroxy-1-naphthylal- dehyde isonicotinoyl hydrazone (311), were synthesized by standard techniques [34]. Both PIH and 311 are strong Fe- binding ligands [34] and were used as positive Fe chelation controls. Apolactoferrin was from Calbiochem. A polyclonal rabbit anti-(human ferritin) Ig was from Roche Diagnostics. All other chemicals were of analytical reagent quality. The NO-generators and other compounds were dissolved in media immediately prior to an experiment [29,35]. Cell culture Human SK-N-MC neuroepithelioma cells, SK-Mel-28 melanoma cells, and MCF-7 breast cancer cells were from the American Type Culture Collection. The mouse LMTK – fibroblast cell line was from the European Collection of Cell Cultures. The BE-2 neuroblastoma cell line was a gift from G. Anderson, Queensland Institute of Medical Research (Brisbane, Australia). All cell lines were grown in MEM containing 10% foetal calf serum (Gibco), 1% (v/v) non- essential amino acids (Gibco), 100 lgÆmL )1 streptomycin (Gibco), 100 UÆmL )1 penicillin (Gibco), and 0.28 lgÆmL )1 fungizone (Squibb Pharmaceuticals). Cells were grown in an incubator (Forma Scientific) at 37 °C in a humidified atmosphere of 5% CO 2 /95% air and subcultured as described previously [36]. Cellular growth and viability were assessed by phase contrast microscopy, cell adherence to the culture substratum, and Trypan blue staining. Nitrite determination The accumulation of nitrite in cell culture supernatants is commonly used as a relative measure of NO production [25,29,35]. Nitrite was assayed using the Griess reagent that gives a characteristic spectral peak at 550 nm [37]. Protein preparation and labelling Apotransferrin was labelled with 59 Fe (Dupont NEN) or 56 Fe to produce Fe 2 -Tf using established procedures [36]. Efflux assay of 59 Fe from prelabelled cells Standard techniques were used to examine the effect of NO and other agents on the efflux of 59 Fe from prelabelled cells [29,32,34]. Briefly, cells were labelled with 59 Fe-Tf (0.75 l M ) for 3 h at 37 °C in MEM. After this incubation, the cell culture dishes were placed on a tray of ice, the medium aspirated, and the cell monolayer washed four times with ice-cold balanced salt solution (BSS). The cells were then reincubated for various incubation times up to 240 min at 37 °C. After this incubation, the overlying supernatant (efflux medium) was transferred to c-counting tubes. The cells were removed from the petri dishes after adding 1 mL BSS and by using a plastic spatula to detach them. Radioactivity was measured in both the cell pellet and supernatant using a c-scintillation counter (LKB Wallace 1282 Compugamma, Finland). Determination of intracellular iron distribution using native-PAGE- 59 Fe-autoradiography Native-PAGE- 59 Fe-autoradiography was performed using standard techniques in our laboratory [38]. Bands on X-ray film were quantified by scanning densitometry using a Laser Densitometer and analysed by BIOMAX I software (Kodak Ltd). 3384 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002 Glutathione assay GSH was measured as described previously [39]. Cellular GSH levels were reduced using the GSH synthesis inhibitor, BSO (0.1 m M ). This latter agent is a potent and selective inhibitor of the enzyme c-glutamylcysteine synthetase that is involved in GSH synthesis [40]. A 20-h incubation with BSO at a concentration of 0.1 m M was used, as these conditions were shown in our previous studies to markedly deplete GSH levels without affecting cellular viability [32]. Assay for examining the ability of NO or iron chelators to bind 59 Fe from cell lysates Cells grown to near confluence in T75 culture flasks were labelled with 59 Fe-Tf (0.75 l M ) for 3 h at 37 °C, placed on a tray of ice, the medium decanted and the cell monolayer washed six times with ice-cold BSS. The cells were lysed by one freeze-thaw cycle and then detached from the flask using a Teflon spatula in the presence of the nonionic detergent Triton X-100 (1.5%) at 4 °C. These samples were then centrifuged at 21 300 g for 30 min at 4 °Candthe cytosol removed and assessed for radioactivity using the c-counter described above. The cytosolic samples were then incubated for 3 h at 37 °C with DFO (0.5 m M )orGSNO (0.5 m M ). The generation of nitrite by GSNO was used as a control to ensure that the NO-donor was producing NO in the lysate. After this incubation, the samples were then subjected to centrifugation at 4 °C through a 5-kDa M r exclusion filter (Vivaspin 500, Sartorius AG). After centri- fugation, the eluent, eluate, and membrane were taken to estimate 59 Fe levels. Examination of 59 Fe levels on the membrane were considered important to assess the possi- bility of adsorption of the 59 Fe-complex. Statistics Experimental data were compared using Student’s t-test. Results were considered statistically significant when P <0.05. RESULTS The effects of NO on intracellular iron distribution: NO decreases ferritin- 59 Fe levels Considering our previous studies demonstrating that incu- bation with NO results in intracellular Fe mobilization [29,32], it was important to determine the source of the Fe mobilized. For these experiments we used native PAGE- 59 Fe-autoradiography that has proved useful in examining the intracellular distribution of 59 Fe in our previous studies [38,41]. Cells were prelabelled with 59 Fe-Tf for 3 h at 37 °C, washed on ice, and then reincubated for up AB Cellular Iron Released (% Total ) 0 10 20 30 40 50 60 Control GSNO GSH SNAP NAP SperNO Sper DFO 311 Control GSNO GSH SNAP NAP SperNO Sper DFO 311 59 Fe-Ferritin Levels Relative Density (% Control) 0 25 50 75 100 125 150 Anti-Ferritin Antibody Control GSNO GSH SNAP NAP SperNO Sper DFO 311 Ferritin- 59 Fe Low M r 59 Fe Anti-Ferritin Antibody Supershifted Band Fig. 1. A variety of NO-generating agents (GSNO, SNAP, SperNO) and Fe chelators (DFO, 311) increase 59 Fe release from prelabelled cells (A), and decrease intracellular 59 Fe–ferritin levels (B). (A) SK-N-MC neuroepithelioma cells were labelled for 3 h at 37 °Cwith 59 Fe-transferrin (0.75 l M )and washed four times on ice. The cells were then reincubated for 3 h at 37 °C with control media, GSNO (0.5 m M ), GSH (0.5 m M ), SNAP (0.5 m M ), NAP (0.5 m M ), SperNO (0.5 m M ), Sper (0.5 m M ), DFO (100 l M ), 311 (25 l M ) or polyclonal anti-human ferritin antibody (1 : 10 dilution). The overlying media and cells were then separated and the 59 Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE- 59 Fe- autoradiography (see Materials and methods). (B) Native PAGE- 59 Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of three performed. The data shown in (B) are a representative experiment of three performed. Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3385 to 240 min at 37 °C in the presence or absence of the agents to be tested. The cells were then lysed and subjected to native PAGE- 59 Fe autoradiography. Cells prelabelled with 59 Fe were incubated with a variety of NO-generating agents, including GSNO, SNAP and SperNO at a concentration of 0.5 m M (Fig. 1A). The effects of these NO donors were compared to their respective control compounds without the NO group, namely GSH, NAP, and Sper. We also compared NO-mediated 59 Fe mobilization to the efficacy of the well-characterized Fe chelators, DFO and 311 [34,41]. Each of the NO-generators resulted in the release of 18–24% of total cellular 59 Fe, whereas the relevant control compounds were no more effective than media alone which released 3 ± 1% of 59 Fe (Fig. 1A). The NO-generators were more effective at mobilizing cellular 59 Fe than DFO, but less active than the potent Fe chelator 311 [34,41] (Fig. 1A). Examining intracellular 59 Fe distribution in SK-N-MC cells (Fig. 1B), the most pronounced band identified comi- grated with purified horse spleen ferritin (data not shown). Experiments incubating the lysate with an anti-ferritin antibody demonstrated that only this band could be super- shifted (Fig. 1B), again indicating that it was ferritin. As described previously in neoplastic cells [41], a faint and very diffuse band below ferritin was present which comigrated with low M r Fe complexes ( 59 Fe-citrate) (Fig. 1B). We previously showed that this low M r Fe appeared to be 59 Fe bound from the lysate by the low M r chelators in the gel running buffer e.g. Tris [41]. In the present study, the low M r band will not be considered in detail as this component remains undefined and its relevance uncertain. In each case, GSNO, SNAP and SperNO, decreased ferritin- 59 Fe levels to 55–63% of the control while their respective control compounds (GSH, NAP, and Sper) had either little effect or increased ferritin- 59 Fe levels (Fig. 1B). The effect of GSH or NAP at increasing ferritin- 59 Fe was not a consistent finding in repeat experiments. In addition, the chelators DFO and 311 decreased ferritin- 59 Fe levels (Fig. 1B) to 69% and 51% of the control, respectively (Fig. 1B). As all NO generators had a similar effect, subsequent studies examining the effect of NO on cellular Fe metabolism were performed using GSNO because of its potential physiological importance. The effect of GSNO concentration and reincubation time on ferritin- 59 Fe levels: NO intercepts 59 Fe before it reaches ferritin To determine the efficacy of NO on 59 Fe mobilization, the effect of GSNO concentration (0.01–1 m M )on 59 Fe release from prelabelled cells (Fig. 2A) and ferritin- 59 Fe levels (Fig. 2B) was examined. These experiments showed that GSNO appreciably increased 59 Fe mobilization from pre- labelled cells at a GSNO concentration of 0.05 m M ,and then plateaued at 0.5 m M (Fig. 2A). When assessing the effect of NO on intracellular 59 Fe distribution, a GSNO concentration of 0.05 m M decreased ferritin- 59 Fe levels to 26% of the control value (Fig. 2B). Higher concentrations of the NO-donor were no more effective at reducing ferritin- 59 Fe levels (Fig. 2B). Studies were performed to determine the effect of reincubation time in the presence and absence of GSNO on 59 Fe mobilization (Fig. 3A) and ferritin- 59 Fe levels (Fig. 3B). As in the studies above, cells were prelabelled with 59 Fe-Tf for 3 h at 37 °C, washed on ice, and then reincubated in the presence of control media or GSNO (0.5 m M ) for 30–240 min at 37 °C. In the control samples < 3% of total cellular 59 Fe was released, while in GSNO- treated cells 59 Fe mobilization increased linearly from 30 to 180 min and then plateaued at 240 min (Fig. 3A). AB GSNO Concentration (mM) 0.0 0.2 0.4 0.6 0.8 1.0 Cellular Iron Released (% Total) 0 3 6 9 12 15 18 GSNO Concentration (mM) 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 120 59 Fe-Ferritin Levels Relative Density (% Control) Control 0.01 mM 0.05 mM 0.1 mM 0.5 mM 1 mM GSNO Concentration Fig. 2. The effect of GSNO concentration on (A) the mobilization of 59 Fe from prelabelled cells and on (B) intracellular ferritin- 59 Fe levels. (A) SK-N- MC neuroepithelioma cells were labelled for 3 h at 37 °Cwith 59 Fe-transferrin (0.75 l M ), washed four times on ice and then reincubated for 3 h at 37 °C with increasing concentrations of GSNO (0.01–1 m M ). The media and cells were separated and the 59 Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE- 59 Fe-autoradiography (see Materials and methods). (B) Native PAGE- 59 Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of four performed. The data shown in (B) are from a representative experiment of three performed. 3386 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002 Examining the intracellular distribution of 59 Fe in control cells, ferritin- 59 Fe increased as a function of reincubation time (Fig. 3B). In fact, after a reincubation time of 240 min in control media, ferritin- 59 Fe levels were 3.5-fold that found after 30 min (Fig. 3B). These results in control cells demonstrated that there was some redistribution of 59 Fe between compartments, with 59 Fe incorporation into ferritin gradually increasing as a function of reincubation time (Fig. 3B). In contrast, in cells treated with GSNO, ferritin- 59 Fe levels decreased after 90 min of reincubation. In fact, after 240 min, ferritin- 59 Fe levels were 10-fold less than those of control cells at the same reincubation time (Fig. 3B). These data suggest that NO directly or indirectly results in 59 Fe mobilization from ferritin and also intercepts 59 Fe before it is deposited within this protein. NO reduces ferritin- 59 Fe levels in a variety of cell types TheeffectofNOatreducingferritin- 59 Fe levels was found in a number of cell types including SK-N-MC neuroepi- thelioma cells, MCF-7 breast cancer cells, LMTK – fibro- blasts, BE-2 neuroblastoma cells and SK-Mel-28 melanoma cells. However, there was marked variation in the effect of NO between cell types, with a 15–74% decrease in ferritin- 59 Fe being observed (data not shown). Depletion of intracellular GSH prevents the NO-mediated decrease in ferritin- 59 Fe levels Our previous studies showed that NO-mediated 59 Fe efflux was GSH-dependent [32]. Considering this, we examined the effect of a 20-h incubation of both LMTK – and SK-N- MC cells with the specific GSH synthesis inhibitor, BSO (0.1 m M ) [40], on the ability of GSNO to increase 59 Fe mobilization (Fig. 4A) and reduce ferritin- 59 Fe levels (Fig. 4B). As shown previously, preincubation with BSO markedly decreased NO-mediated 59 Fe mobilization from both cell types (Fig. 4A). Examining intracellular 59 Fe distribution, NO decreased ferritin- 59 Fe levels, while BSO treatment totally prevented this decrease (Fig. 4B). These results indicate that GSH is required for the effect of NO at decreasing ferritin- 59 Fe levels. It is of interest that incubation of BSO-treated LMTK – and SK-N-MC cells with GSNO resulted in an increase in the amount of ferritin- 59 Fe (Fig. 4B). These data were in contrast to the decrease observed after treatment of control cells with GSNO (Fig. 4B). Cell membrane-impermeable or -permeable iron chelators do not increase NO-mediated 59 Fe efflux It was possible that passive diffusion may be involved in NO-mediated 59 Fe release from cells. Previous studies have shown that Fe mobilization in the absence of NO is increased by incubation with apoTf and extracellular chelators, presumably due to the ability of these agents to act as an extracellular Fe ÔsinkÕ to increase the concentration gradient across the cell membrane [42–44]. Considering this, and the fact that a NO–Fe–GSH complex may be released from cells [32], experiments were designed to investigate the effects of 0.1 mgÆmL )1 of apoTf, apolactoferrin, or BSA (as a protein control) on 59 Fe mobilization during incubation with increasing concentrations of GSNO (0.025–0.5 m M ). However, apoTf, apolactoferrin and BSA only very slightly increased 59 Fe release from prelabelled cells at a GSNO concentration of 0.5 m M (Fig. 5A). However, compared with the BSA protein control, there was no significant effect of apoTf or apolactoferrin on 59 Fe mobilization from Control GSNO Control GSNO Control GSNO Control GSNO 30 90 180 240 Time (min): AB Time (min) 0 60 120 180 240 Cellular Iron Released (% Total) 0 2 4 6 8 10 12 14 Control GSNO Time (min) 0 60 120 180 240 0 2 4 6 8 10 Control GSNO 59 Fe-Ferritin Levels Relative Density (Arbitrary Units) Fig. 3. The effect of GSNO on (A) the mobilization of 59 Fe from cells, and (B) ferritin- 59 Fe levels in prelabelled cells as a function of reincubation time. (A) SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith 59 Fe-transferrin (0.75 l M ), washed four times on ice, and then reincubated with control media or media containing GSNO (0.5 m M ) for 30–240 min at 37 °C. The media and cells were separated and the 59 Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE- 59 Fe-autoradiography (see Materials and methods). (B) Native PAGE- 59 Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of two performed. The data shown in (B) are a representative experiment of three performed. Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3387 SK-N-MC cells (Fig. 5A). Studies combining GSNO (0.5 m M ) with increasing concentrations (0.03–1 m M )of the extracellular chelators, EDTA or DTPA, also demon- strated no potentiation of 59 Fe mobilization from prela- belled cells (Fig. 5B). As NO acted like an Fe chelator to mobilize 59 Fe from prelabelled cells [29,32], studies were performed to deter- mine if the same Fe pool bound by the permeable chelators, DFO (Fig. 6A) or PIH (Fig. 6B), was bound by NO. In these studies, cells were prelabelled with 59 Fe-Tf for 3 h at 37 °C, washed, and then reincubated for 3 h at 37 °Cwith either increasing concentrations of DFO (0.05–1 m M )or PIH (1–50 l M ) or these chelators combined with GSNO (0.5 m M ). The addition of increasing concentrations of PIH or DFO to GSNO had no significant effect on cellular 59 Fe mobilization (Fig. 6A and B). These results suggested that (A) (B) Cellular Iron Released (% Total) 0 5 10 15 20 25 Control Control GSNO BSO Control GSNO LMTK – Control Control GSNO BSO Control GSNO SK-N-MC Control GSNO Control GSNO Control BSO LMTK – Control GSNO Control GSNO Control BSO SK-N-MC 59 Fe-Ferritin Levels Relative Density (% Control) 0 40 80 120 160 Fig. 4. The depletion of GSH prevents (A) NO-mediated 59 Fe mobilization from prelabelled cells, and (B) the decrease in intracellular ferritin- 59 Fe levels seen in the presence of GSNO. (A) SK-N-MC neuroepithelioma cells and LMTK – fibroblasts were pretreated for 20 h at 37 °C in the presence or absence of the specific GSH inhibitor BSO (0.1 m M ). The cells were then prelabelled for 3 h at 37 °Cwith 59 Fe-transferrin (0.75 l M ), washed four times on ice, and then reincubated with control media or media containing GSNO (0.5 m M )for3hat37°C. The media and cells were separated and the 59 Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE- 59 Fe-autoradiography (see Materials and methods). (B) Native PAGE- 59 Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of seven performed. The data shown in (B) are a representative experiment of five performed. Fig. 5. The extracellular high affinity Fe-binding proteins, apoTf and apolactoferrin, and the extracellular Fe chelators, DTPA and EDTA, do not promote NO-mediated 59 Fe mobilization from prelabelled cells. (A) SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith 59 Fe- transferrin (0.75 l M ), and washed four times on ice. The cells were then reincubated with: control media or media containing GSNO (0.025– 0.5 m M ) and either apoTf (0.1 mgÆmL )1 ), apolactoferrin (0.1 mgÆmL )1 )orBSA(0.1mgÆmL )1 )for3hat37°C. (B) SK-N-MC cells labelled and washed as in (A) were then incubated for 3 h at 37 °C with control media or media containing EDTA or DTPA (0.03–1 m M ). The overlying media and cells were then separated and the 59 Fe levels in each assessed (see Materials and methods). These results are mean ± SD (three replicates) in a representative experiment of three performed. 3388 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002 GSNO and the chelators were acting on the same intracel- lular compartment of 59 Fe. In contrast, when added without GSNO, increasing concentrations of DFO or particularly PIH, resulted in enhanced 59 Fe mobilization from cells (Fig. 6A,B). Examination of the direct effect of NO and iron chelators on iron pools in cytosolic lysates: comparison with intact cells As NO could form an intracellular low M r Fe–dithiol dinitrosyl complex [23], experiments were performed to determine if NO or DFO could mobilize 59 Fe from lysates prepared from cells labelled with 59 Fe-Tf for 3 h at 37 °C (Fig. 7A). The lysates were centrifuged to obtain cytosols and then incubated for 3 h at 37 °C with DFO (0.5 m M )or GSNO (0.5 m M ). The cytosol was then subjected to centrifugation at 4 °C through a 5-kDa M r exclusion filter. In five experiments, only DFO significantly (P <0.009) increased the amount of 59 Fe that was passing through the membrane, while GSNO had no significant effect (Fig. 7A). In contrast with the lysates, when cells were prelabelled with 59 Fe-Tf and reincubated with DFO or GSNO under the same conditions, GSNO was significantly (P < 0.00001) more effective than DFO or media at mobilizing cellular 59 Fe (Fig. 7B). These results suggest that intact cellular metabolism was required for NO-mediated 59 Fe mobiliza- tion. Fig. 6. There is no potentiation on NO-mediated 59 Fe mobilization from prelabelled cells upon combination of GSNO with permeable Fe chelators. SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith 59 Fe-transferrin (0.75 l M ), washed four times on ice, and then reincubated with increasing concentrations of desferrioxamine (DFO; 0.05–1 m M ) or pyridoxal isonicotinoyl hydrazone (PIH; 1–50 l M ) either alone or in the presence of GSNO (0.5 m M ) for 3 h at 37 °C. The overlying media and cells were then separated and the 59 Fe levels in each assessed (see Materials and methods). These results are mean ± SD (three replicates) in a representative experiment of three performed. Fig. 7. GSNO, in contrast to DFO, does not mobilize 59 Fe from (A) cytosolic lysates, derived from prelabelled cells, while (B) GSNO is more effective than DFO at mobilizing 59 Fe from prelabelled intact cells. (A) Cells were labelled with 59 Fe-Tf for 3 h at 37 °C and washed four times on ice and lysates prepared. The lysates were centrifuged to obtain cytosols and then incubated for 3 h at 37 °C with DFO (0.5 m M )orGSNO(0.5m M ). The cytosols were then subjected to ultracentrifugation through a 5-kDa cut-off filter. (B) Cells were prelabelled with 59 Fe-Tf (0.75 l M )for3hat37 °C, washed four times on ice, and then reincubated for 3 h at 37 °C with DFO (0.5 m M )orGSNO(0.5m M ). The overlying media and cells were then separated and the 59 Fe levels in each assessed (see Materials and methods). The results in (A) are mean ± SD of five experiments. The results in (B) are mean ± SD (three replicates) in a representative experiment of three performed. Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3389 Further experiments examined the effect of incubation of lysates derived from 59 Fe-labelled cells with either DFO, 311, SNAP, NAP, GSNO, GSH, or GSH in the presence of GSNO. The lysates were then subjected to native PAGE- 59 Fe-autoradiography (Fig. 8). In these studies examining the direct effect of the chelators and NO on the lysate, no significant effect was observed on ferritin- 59 Fe levels. In addition, in the presence of both GSH and GSNO no effect was apparent (Fig. 8). This indicated that intact cellular metabolism was required for the NO-mediated effect on this molecule, and that NO did not directly remove substantial 59 Fe from the protein (Fig. 8). DISCUSSION Previous studies have clearly demonstrated that NO has a marked effect on cellular Fe metabolism [25–27,32]. Indeed, NO-mediated Fe depletion of tumour target cells by activated macrophages could play an important role in immune surveillance [3–5,15,16,45]. Our previous studies have shown that NO-mediated Fe mobilization is potenti- ated by incubating cells with D -glucose due to the subsequent generation of GSH [32]. In the present study we have significantly extended our knowledge of this process. For the first time, we demonstrate in a cellular system that NO intercepts Fe before it is incorporated into ferritin and appeared to indirectly mobilize Fe from this protein. NO could remove Fe from ferritin by two possible mechanisms: (a) by directly chelating ferritin-bound Fe, or (b) by chelating a cellular Fe pool which leads to ferritin releasing its Fe. Of these two possibilities our evidence favours the second mechanism, as NO could not remove 59 Fe from ferritin in cellular lysates (Fig. 8). Furthermore, the processes resulting in cellular Fe mobilization and Fe release from ferritin were dependent on cellular metabolism (Fig. 7) and the generation of GSH (Fig. 4 and [32]). These latter observations indicate that active cellular metabolism was required for Fe mobilization rather than direct chela- tion of ferritin-Fe by NO. A previous in vitro study by Reif & Simmons [14] using isolated horse spleen ferritin showed that NO generated by sodium nitroprusside could remove some Fe from this protein. Our current data are obviously different, and these inconsistent results may relate to the very different experi- mental systems being used. Lee and colleagues [8] have reported, using isolated ferritin, that NO forms a complex with Fe in its core, and have suggested that ferritin could act as a store of NO. Again, it is difficult to compare this latter study to our present experiments, as we have examined the effect of NO using intact cells or cellular lysates. It is significant that we have shown that NO not only releases Fe from ferritin indirectly (Figs 1B, 2B, 4), but can also intercept Fe on route to this molecule (Fig. 3B). At present the precise molecular mechanism(s) involved in the intra- cellular trafficking and delivery of Fe to ferritin remain unclear, although intermediates of low M r [46] or high M r (e.g. metal-binding chaperones) [47] could be involved. Nevertheless, our results demonstrate that both permeable Fe chelators (e.g. PIH and DFO) and NO can intercept the same intermediary pool of Fe (Fig. 6). It is of interest that the ability of NO to induce Fe mobilization is dependent on GSH while that for chelators is independent of GSH [32]. This suggests that NO by itself does not have the capacity to remove Fe from intermediates and could require the reducing capacity of GSH. Alternat- ively, or in combination with this latter mechanism, GSH may form a mixed Fe complex with NO that has the appropriate lipophilicity and charge to diffuse or be transported from the cell. Previous studies using EPR spectroscopy have demonstrated the presence of dithiol dinitrosyl–Fe complexes within cells [23,30]. Further, Rogers and Ding [48] have shown that L -cysteine is necessary for the removal of dinitrosyl–Fe complexes from [Fe–S]-containing proteins in Escherichia coli. Interestingly, these authors showed that GSH was able to perform the same function but Con DFO 311 SNAP NAP GSNO GSH GSH + GSNO Fig. 8. The direct effect of incubating GSNO and Fe chelators on 59 Fe-containing molecules in cytosolic lysates derived from prelabelled cells. SK-N-MC neuroepithelioma cells were labelled for 3 h at 37 °C with 59 Fe-transferrin (0.75 l M ) and washed four times on ice. Cells were then lysed and the cytosols incubated with DFO (0.5 m M ), 311 (50 l M ), SNAP (0.5 m M ), NAP (0.5 m M ), GSNO (0.5 m M ), GSH (0.5 m M ), or GSNO (0.5 m M )andGSH(0.5m M ) for 3 h at 37 °C. These samples were then subjected to native PAGE- 59 Fe-autoradio- graphy (see Materials and methods). The results are a representative experiment of three performed. Cell Membrane ADP GS-Fe-NO Transporter ? ? GS-Fe-NO Protein ? ATP TCA HMPS Glucose G-6-P GSH Glucose Transporter Fe-Protein NO NO NO-Fe-Protein Transporter Diffusion Fig. 9. 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 for the production of ATP and by the HMPS for the generation of reduced GSH. Nitrogen monoxide (NO) either diffuses or is transported into cells where it intercepts and binds Fe bound to proteins or Fe on 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 lig- ands or may complete the Fe coordination shell along with the NO ligand(s). This complex may then be released from the cell by an active process requiring a transporter (see text for details). 3390 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002 not as efficiently as L -cysteine [48]. Hence, these results provide support for the possible mechanism of action of GSH in our experimental system. However, at present, we cannot exclude that GSH has other roles. Indeed, recently the S-nitrosylated form of GSH has been suggested to act as a transport molecule for NO which increases its half-life and allows effective biological activity [49,50]. Considering that a low M r Fe–NO–GSH complex may be released from cells and that a concentration gradient across a membrane can facilitate diffusion [44], we examined whether NO-mediated Fe release could be potentiated by strong extracellular Fe chelators (Fig. 5). In these studies, high concentrations of both physiological chelators (apolactoferrin and apoTf) and synthetic chelators (EDTA and DTPA) were used, and for all ligands no significant potentiation of 59 Fe mobilization was observed upon combination with NO. These studies suggest that enhance- ment of the concentration gradient across the cell membrane did not alter NO-mediated Fe release. Considering this, it is of note that intact cellular metabolism was required for Fe mobilization by NO (Fig. 7), and NO-mediated Fe release was prevented by metabolic inhibitors and at 4 °C [32]. Collectively, these data suggest that an energy-dependent mechanism was required to enable efflux of the NO–Fe complex. Based upon the results presented in this and our previous study [32], we suggest in Fig. 9 a 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 HMPS for the generation of GSH. NO diffuses or is transported [51] into cells where it intercepts Fe on route to ferritin and binds Fe bound to proteins (Fig. 9). The high affinity of NO for Fe [2] results in the formation of an NO-Fe complex and GSH may either be involved as a reductant to remove Fe from endogenous ligands [48] or may complete the Fe coordination shell along with NO [17,20,23,30]. This complex may then be transported out of the cell by an energy- dependent transporter such as ferroportin 1 [52], or alternatively, the ATP-binding cassette (ABC) transporter family (e.g. glutathione-S-conjugate export pump), which are known to mediate the efflux of glutathione-conjugates [53,54] (Fig. 9). Further studies aimed at identifying the exact molecular nature of the Fe released by NO and the transporter involved are underway. 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