Uptake of Non-Transferrin-bound Iron by Both Reductive and ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269,No. 23,Issue of June 10,pp. 16046-16053,

1994 Printed in U.S.A.

Uptake of Non-Transferrin-boundIron by Both Reductive and Nonreductive ProcessesIs Modulated by Intracellular Iron* (Received for publication, February 15, 1994)

Edward

W.Randell$, Joel G. ParkesS, Nancy F. Olivieri0, and Douglas M.TempletonSnll

From the $Department of Clinical Biochemistry, University of Ibronto, Toronto, Ontario, Canada M5G 1L5 and the and Wepartment of Biochemistry, Hospital for Sick Children, Toronto, Ontario $Division of HematologylOncology -~ M5G 1x8,Canada ”

Non-transferrin-boundiron (NTBI)uptake occurs in a This is achieved by two processes: transport that controls iron variety of cells bya saturable, specific and temperature- uptake and storage that sequesters iron in Fenton-inactive sensitive process. Our previous studies indicated that forms such as ferritin and hemosiderin. Translationally coNTBI uptake by cardiac myocytes and Hep 6 2 cells was regulated processes utilizingan iron-responsive element couple reversibly up-regulated by iron deposition. In the pre- receptor-mediated uptake of plasmatransferrin-boundiron sent work we have characterized this up-regulation and and intracellular iron storage in ferritin (1).However, an alterexamined its mechanism by comparing the uptake of native low molecular mass species of plasma iron, non-transoxidized (FeS+)and ascorbate-reduced (Fez+)forms of ferrin-bound iron (NTBI),’ has been described (2, 3) that may iron. Iron loading markedly enhanced the uptakeof iron attain significant concentrationsunder conditions of transboth in the presence and absence of ascorbate, but the ferrin saturation, such as occurs in iron overload (4, 5). In increment was greater when ascorbate was absent. This recent years, several laboratories (6-16) have demonstrated up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein uptake of NTBI by a variety of mammalian cell types to be a synthesis. Uptake by the iron-loaded cells wasless sen- temperature-sensitive saturableprocess distinct from those resitive to thiol-alkylating agents and competing metal sponsible fortransport of transferrin-bound iron and consistent ions, but was moresensitive to proteolysis. Iron loading with a specific NTBI carrier. A candidate iron transport protein and V , for uptake of both has been identifiedas a 160-kDa trimer in brush border plasma causes an increase in both K,,, Fez+and Fes+,although the values differ, suggesting dis- membranes of the upper small intestine(171, although such a tinct rate-limiting steps for uptake of Fez+and Fe3+. Con- carrier remains tobe identified in other tissues. The mechanism of NTBI transport and the means by which sistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing it is modulated in mammalian cells are poorly understood. metal ions and monensin. The Fez+-specific chelators Kaplan and co-workers (9, 12) have demonstrated that NTBI bathophenanthroline disulfonate and ferrozine mark- uptake can be increased within minutes by extracellular iron, edly inhibited iron uptake whether ascorbate was pre- and this increase is sustained in the presence of high concensent or not, indicating that Fes+uptake isdependent on trations of extracellular iron. We have observed up-regulation reduction to the ferrousstate. This requirement for re- of NTBI uptake in proportion to increasesin intracellular iron duction was independent of the iron statusof the cells, achieved by several days of iron loading in Hep G2 cells’ and demonstrating that the process of up-regulation is not rat cardiac myocytes (161, in contrast todecreased transport of due to theappearance of a new mechanismfor translo- diferric transferrin under similar circumstances. Thus, there cation of Fe3+without reduction. Taken together, the evi- appear to be two phenomena regulating NTBI transport, one dence favors a model of NTBI transport where an obli- associated with increased intracellular iron developing over gate and rate-determining reduction of FeS+occurs prior several days and the other responding rapidly to extracellular to or during uptake, followed by translocation through iron. Up-regulated NTBI transport afterlong-term iron loading an Fez+carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate sug- is reversible by chelation (6,16); further supportinga regulatory role of intracellular iron and establishinga link between gest that exogenous Fez+does not access the carrier available to the nascent ferrous ion derived from the the chelatable iron pool and the process of NTBI uptake. Alreductase and isconsistent with close coupling between tered numbers or affinities of putative NTBI transporters, or a transporter activity, all remain candidate the reduction and the translocation processes. In iron- events unrelated to loaded cells with increased rates of NTBI transport, a regulatory mechanisms. Another feature of the NTBI uptake process is its possible similar mechanism prevails. relationship to accumulation of other metalions. Previous studies haveshown that a number of divalent metal cations inhibit The requirement of mammalian cells for iron necessitates competitively the uptakeof iron by cells. Inman andWesslinghighly regulated processes to avoid iron-catalyzed generation of Resnick (14)showed that uptake of the Fe3+-nitrilotriacetate DNA. free radicalsthat modify proteins, membrane lipids, and Theabbreviations used are: NTBI, non-transferrin-boundiron; 1,2-dimethyl-3-hy* This work was supported by an operating grant from the Medical BPS, bathophenanthrolinedisulfonate;CP020, DFO, deResearch Councilof Canada. The costs of publication of this article were droxypyrid-Cone;CP094 1,2-diethyl-3-hydroxypyrid-4-one; defrayed in part by the payment of page charges. This article must feroxamine; 2,4-DNP, 2,4-dinitrophenol; FAC,ferric ammonium citrate; “advertisement” in accordance with 18 MEM, minimal essential medium;NTA, nitrilotriacetate; PBS, phosthereforebeherebymarked phate-buffered saline;NEM, N-ethylmaleimide; PCMBS, p-chloromerU.S.C. Section 1734 solely to indicate this fact. 11 To whomcorrespondenceshouldbe addressed: Dept. of Clinical curibenzenesulfonic acid; DTNB,5,5’-dithiobis(2-nitrobenzoicacid). J. G. Parkes, E. W. Randell, N. F. Olivieri, and D. M. Templeton, Biochemistry, University of Toronto, 100 College St., Toronto, Ontario submitted for publication. M5G 1L5, Canada. Tel.: 416-978-3972; Fax: 416-978-5650.

16046

Iron in Dansport (Fe-NTA) complex by K562 cells was specifically inhibited by Cd2+and Cu2+,whereas Mn2+,Co2+,and Ni2+were ineffective. Sturrock et al. (9) found that Mn2+, Cu2+, Zn2+,and Cd2+,inhibited Fe3+uptake by HeLa cells to varyingdegrees. In fibroblasts, Cuz+and Zn2+could inhibit Fe3+uptake, whereasCd2+and Mn2+ were ineffective. In media containing ascorbate or amino acids, Cu2+and Zn2+were also ineffective. In contrast, the uptakeof Fez+by liver was inhibitedby MnZ+,Co2+,Cu2+,and Zn2+(191, and its uptake by rabbit reticulocytes was inhibited by Mn2+,Co2+, Ni2+,and Zn2+(20).Previous studies havealso shown that NTBI uptake depends on the presence of Ca2+(9, 12). In neutral aqueous solutions Fe3+forms insoluble ferric hydroxides which hamper free movement of iron into cells. In contrast, Fez+is freely soluble in these environments(21) but is oxidized to Fe3+in thepresence of oxygen (22). Several studies (23-26) have shown that inSaccharomyces cerevisiae Fe3+uptake is preceded by reduction. Reduction may aslo be required for uptake by other micro-organisms (27) and higher plants (28, 29) and for transport in the intestine (30). NTBI uptake has been less widely studied in mammalian systems, but recently it has been suggested that a reduction step may be required for the uptake of Fe3+by K562 cells (14).A reductive mechanism has also been suggested for transferrin-bound iron uptake by hepatocytes (31), possibly mediated by a plasma membrane bound NADH:diferric transferrin reductase(321, and a similar activity for release of iron from internalized transferrin has been proposed in endosomes (33). These studies with transferrin have been criticized on methodological grounds; intervention with Fez+-specificchelators may favor reductive dissociation from transferrin by shifting theequilibrium t o unmask an otherwise insignificant process (34). Nevertheless, recent work suggests two mechanisms of transfenin-bound iron uptake by melanoma cells, one of which may require an oxidoreductase (10, 35). The present investigation was undertaken to gain further insight into themechanism of up-regulated NTBI transport in iron-loaded mammalian cells and to characterize theprocesses that modulate Fez+ and Fe3+uptake. The results are consistent with the involvement of a rate-limiting reductive step prior to or during Fe3+ entry into human hepatocarcinoma (Hep G2) cells, regardless of iron loading. They also suggest that Fez+ generated by extracellular ascorbate andFez+produced by the endogenous reductase have distinct routes of entry into the cell. EXPERIMENTAL PROCEDURES

Hep G2 Cells

16047

Uptake of NTBI NTBI transport was measured as described previously.2 Briefly, total 1 or 2.5 PFeIFe-NTA complex NTBI uptakewasmeasuredusing (1:2.5 molar ratio) prepared by mixing appropriate volumes of 1.0 mM [59Fe]FeC13(20 mCi/mg) and a-MEM containing 2.5 m NTA and 20mM HEPES (a-MEM-HEPES), pH7.4. For studiesof Fez+ uptake,CY-MEMHEPES containing 1 mM ascorbate was used. In preliminary experiments we measured the absorbance of the Fe2+-ferrozinecomplex at 562 nm (36) and found that under these conditions more than 90% of the after 15 min, the end iron was reduced and remained in the Fez+ state point of the uptake experiments. Therefore, in the presence of 1 mM ascorbate theobserved NTBI uptake was due to ferrous ion. Nonspecific NTBI uptake was measured in the presence of a 1000-fold excess of unlabeled Fe-NTA prepared by diluting appropriatevolumes of 1M NTA and 1 M FeCI, (in 0.1M HCl) in a-MEM-HEPES 1 mM ascorbate. The pH of this solution was adjusted to 7.4 with 1 M NaOH prior to the addition of r5'Fe]Fe-NTA. The specific radioactivity of each solution was adjusted to give 2-4 x lo5 cpdml(80-160 cpdpmol iron). To ensure that transferrin, ferritin, and NTBI were a t a minimum concentration in the culture medium, Hep G2 cultures were washed twice with 2 ml of prewarmed phosphate-buffered saline (PBS) and incubated for 60 min in serum-free a-MEM-HEPESa t 37 "C. This medium was thenremoved, and thecells were washed a third time with 2 ml of a-MEM-HEPES prior to the addition of L5'Fe1Fe-NTA. Triplicate wells each received 0.5 ml of [59Fe]Fe-NTAand were incubated for 15 min at 37 "C. The serum-free incubation is intended to allow time for depletion of cellular stores,e.g. through transferrin-transferrin receptor recycling, and the final wash removes any secreted products prior to the 15-min pulse labeling. Uptake was stopped by removing the labeling medium and washing thecells twice with 2 ml of ice-cold PBS. Monolayers were subsequentlydissolved in 1 ml of 1% (w/v) sodium dodecyl sulfate and the cell-associated 59Fe determined by counting gamma radiation. Specific uptake was calculated by subtracting the cell-associated radioactivity measured in the presence of a 1000-fold excess Fe-NTA (nonspecific) from the total uptake achieved with 1 or 2.5 p c5'FelFe-NTA. In addition, incorporation of radioactivity at zero time was measured and subtracted from each point. The protein content of each well was determined by the method of Lowry et al. (37) and the rate of NTBI uptake expressed as femtomoledpgof proteidmin. Kinetic parameters (K, and V,,) for specific Fez+ uptakeat 37 "C were determinedby varying the concentrationof i5'Fe1Fe-NTA in 1mM ascorbate. The data were expressed as femtomoledpg of proteidmin Michaelisand the parameters determined by iterative curve fitting ato Menten model ( u = V,;S/(K,,, + S ) )and by regression analysisof EadieHofstee plots.

*

Chelation Studies In some studies the uptake of NTBI was measured in the presence of the Fez+ chelators ferrozine or BPS, both at 1 m. To study chelatordriven efflux of iron, stock solutions of DFO, CP020, and CP094 were prepared in sterile PBSa t a concentration of 320 m, and appropriate dilutions were made in growth medium immediately before use. The amount of radioactivity released intothe medium or retainedby cells at each time interval was measured in triplicate wells.

Materials 1,2-Dimethyl-3-hydroxypyrid-4-one (CP020)and 1,2-diethyl-3-hydroxypyrid-4-one (CP094) were a gift of Prof. R. C. Hider, Department Modulators of NTBI Uptake of Pharmacy, King's College, London. Deferoxamine(DFO)was obAll NTBI uptake studies were performed as described above except tained from CibaGeigy, Canada. Fetalbovine serum, ferric ammonium that specific modulators were added as described below either during citrate (FAC: 13.75% ironby weight), chloroquine,colchicine, actinomy- preincubation with a-MEM-HEPES or coincident with the addition of cin D, cycloheximide, monensin, N-ethylmaleimide (NEM), iodoacetate, PFeIFe-NTA. Specific conditions used were as follows. p-chloromercuribenzenesulfonicacid (PCMBS),5,5'-dithiobis(2-nitroProteolysis-Trypsin was used under conditions mild enough toavoid benzoic acid) (DTNB), 2,4-dinitrophenol (2,4-DNP), soybean trypsin in- detachment of cells from the plate but sufficient to inhibit known Hep hibitor, verapamil, nifedipine, diltiazem, bathophenanthroline disulfonG2 cell surface functions such as low density lipoprotein receptor activity ate (BPS), 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-l,2,4-triazine (38). Thus, cells were incubated for 30 min at 0 "C in a-MEM-HEPES, (ferrozine), and the enzymes neuraminidase, chondroitinase ABC, hep- containing 100 pg trypsidml. The reaction was stopped by removing the arin-sulfate lyase I11 (EC 4.2.2.8), and trypsin were all from Sigma. trypsin solutionand washing the cells once with ice-cold a-MEM-HEPES ob- containing 1 mg/ml soybean trypsin inhibitor. Prior to the addition of [59FelFeC13was a product of DuPont NEN. ~-[4,5-~H]Leucine was tained from Amersham Corp. I5'Fe1Fe-NTA, cells were warmed by a 15-min incubation a t 37 "C in a-MEM-HEPES containing 1 mg/ml soybean trypsin inhibitor. Cell Culture a n d I r o n L o a d i n g Competing MetalZonsAompetition for NTBI uptake by metal ions Hep G2 cells, obtained from the American Type Culture Collection was measured by the addition of 20 p Ni", Co2+,Cu2+, Cd2+, and Zn2+ (Rockville, MD; ATCCHB 8065),were grown toconfluence in a humidi- to 1 PM PFeIFe-NTA. All metals were used as their chloride salts and fied atmosphere of 95% air, 5% CO, at 37 "C in a-minimum essential were of analytical reagent grade or equivalent. medium (a-MEM) supplemented with 10% (v/v) fetal bovine serum, Sulfhydryl-alkylatingAgents-Cells were treatedfor 15 min at 37 "C penicillin G (100 unitdml), and streptomycin (100 pg/ml) (growth me- with a-MEM-HEPES containing 5 mM DTNB, NEM, iodoacetate, or dium). Iron loadingof confluent Hep G2 cells was achieved by culture PCMBS. The thiol-active reagents wereremoved and the cells washed for 1-7 days in growth medium containing 20 pg/ml Fe3+as FAC, as once with a-MEM-HEPES before measuring NTBI uptake. described previously.2 FAC was omitted from control cultures. Calcium Channml Blockers-Cells were treated for 60 min at 37 "C

Iron Dansport in Hep G2 Cells

"1

0

Iron-loaded Cells

"

TABLEI Effectsof iron loading on response to acute additionof ascorbate and FAC Cells were incubated for2 h at 37 "C in a-MEM-HEPESin the presence or absence of 1m~ ascorbate or 40 pg irodml (FAC). Therate of Fe3+ uptake was measured by incubating cells with 1p~ 59Fe-NTA for 0 and 15 minat 37 "C in a-MEM-HEPES. Valuesare the mean * S.D. of triplicate determinations and are expressed as percentage of Fe3+ uptake in control cells withouttreatment (1.55 2 0.08 fmol/pg of proteid rnin). Treatment

None Ascorbate FAC Ascorbate + FAC FIG.1. Concentration dependence of rate of Fez+uptake by control and iron-loaded Hep 6 2 cells. Control (0) or iron-loaded cells ( 0 )were incubated with various concentrationsof 69Fe-NTAin the at 37 "C in a-MEM-HEPES.Data presence of 1m~ ascorbate for 15 min points are the mean* S.D. of triplicate determinations.

Fe3+uptake

Iron-loaded

Control

100 * 5 108 t 6 105 -c 1 9"153

% control

261 2 19" 319 * 14",* 203 f 21"* 245

* 16"

Significantly different fromuntreated control cells (p < 0.001). * Significantly different from untreated iron-loaded cells ( p < 0.05).

studied the uptakeprocess in the presence of the Fez+-specific chelators BPS and ferrozine. In the presence of ascorbate, these agents decreased iron uptake in control cells by 95 2 1% and with a-MEM-HEPES containing20 p~ diltiazem, verapamil,or nifedi- 100 2 0.01% (mean 2 S.D., n = 3), respectively, demonstrating pine added from 10mM stock solutions prepared in DMSO; control cells the involvement of Fez+under these conditions. Although the received the same concentration of DMSO (0.2%, vh). incremental stimulation of Fez+transport by iron loading (Fig. Metabolic and EndocyticInhibitors-For these studies cellswere treated for 60 minat 37 "C with the indicated concentrationof inhibitor, 1)was much less than the severalfold increase we observed and priorto determiningNTBI uptake, the agentwas removed andthe previously with Fe3+,2the uptake rates of both ions weresimilar cell monolayer washed once with a-MEM-HEPES. The potential role of in the loaded cells. The greater increase in Fe3+uptake with subcellular processing on NTBI uptake was determined using chloro- iron loading seemed to be due to its muchslower transport in quine (200 p ~ ) a, drug that disrupts lysosome function,or colchicine (20 control cells (compare Fig. 1 and Ref. 161, and this was conp), a microtubule disrupting agent. ATP production wasinhibited by 5 firmed in a single experiment performed for internal consism~ 2,4-DNP or KCN. Inhibitors ofprotein Synthesis and Secretion-% measure the effects tency: uptake rates of Fez+ andFe3+in control cells were 2.01 2 of de now protein synthesison NTBI uptake, Hep G2 cells were incu- 0.06 and 0.39 2 0.03 fmol/pg proteidmin, respectively. Therebated for 20 h with growth medium containing actinomycinD (3.7 p ~ ) , fore, we wondered if Fe3+ uptake by the control cells might cycloheximide(350 p ~ ) or , monensin (5 p ~ ) Protein . synthesis was involve reduction. Both Fez+ chelators decreased uptake of Fe3+ measured by the incorporation of [3Hlleucine into trichloroacetic acid- nearly as much as they did for Fez+(BPS, 83 2%; ferrozine, 94 precipitable material as described previously2Briefly, cells were incuinvolvement of reduction in ferric ion (1 2 l%), demonstrating the bated at 37"Cfor 1 h ingrowthmediumcontainingt3HIleucine pCi/ml). Incorporation was stopped by removing the labeling medium transport and suggesting that extracellular reduction of Fe3+ 1ml of ice-cold PBS followed may be rate-limiting for its uptake by Hep G2 cells. and washingthe cell monolayer twice with m e r incubation on icefor 15 min, the We next asked if these samedependencies applied t o uptake by 1ml of 5% trichloroacetic acid. trichloroacetic acid-insoluble material was treated twice more with1ml in iron-loaded cells. Ferrozine and BPSdecreased Fe3+uptake of trichloroacetic acid on ice for 5 min each. The final trichloroacetic to 2.4 2 0.4% and 9 2 2%of control values, respectively, and acid-insoluble fraction was solubilized with 1 ml of 1 M NaOH. Cellassociated radioactivity was determinedby scintillation counting, and nearly abolished Fez+ transport. These results also indicate that the uncomplexed form of iron, rather than its chelate, is the protein concentration was measuredby the Lowry assay. Extracellular Polyanions-The ability of glycoconjugates containing transported in control and iron-loaded cells, because chelation polyelectrolytes (such as sialic acid, heparan sulfate, or chondroitin prevents uptake. Thisis in keeping with the impermeability of sulfate) to modulate iron uptake was examined by treating cells for 60 cells to the disulfonates. min at 37 "C with either neuraminidase (0.6 unitlml), heparin-sulfate The requirement for prolonged exposure to iron (>20 h) belyase 111 (0.15 unit/ml), or chondroitinase ABC (0.15 unitlml). fore an increased rate of Fe3+ uptake could be observed' is consistent witha time-dependent populatingof an intracellular Statistical Analysis cells is almost five Data were analyzed forstatistical significance using Student's t test regulatory ironpool. Since entry of Fez+ into times faster than entry of Fe3+, exposureof cells to increased for unpaired samples. concentrations of Fez+should result in more rapid up-regulaRESULTS tion of Fe3+ transport. In keeping with this suggestion, cells exposed to FAC (40 pg irodml) and 1 mM ascorbate for 2 h Effects of Iron Loading and Ascorbate on Iron UptakePrevious studies showed that Fe3+uptake by Hep G2 cells was exhibited a 53% greater rateof Fe3+uptake compared with that observed when either ascorbate or FAC were added alone to augmented by iron-loading in a dose-dependent manne? and that maximal stimulation was achieved after incubating with naive cells (Table I). In contrast, additionof ascorbate plusFAC FAC for several days.To determine if this transport process is to iron-loaded cells had no further effect on the already upregulated uptake of NTBI transport, indicating that additional dependent on the redox state of iron, ascorbate was used to effect reduction to Fez+.The uptakeof iron following the addi- loading was not significant. However, addition of ascorbate tion of 1 mM ascorbate to a solution of Fe-NTA is saturable in alone to the iron-loaded cells did increase the rateof uptake of both control and iron-loaded cells (Fig. 1).Uptake in thepres- NTBI by 22% (Table I; 261% untreated cells uersus 319% ascorence of a 1000-fold excess of unlabeled Fe-NTA was less than bate-treated), suggesting that, in the absence of extracellular 5%, demonstrating specificity (data not shown). In agreement FAC, ascorbate may reductively mobilize internal iron stores with ourprevious studies utilizing Fe3+,the uptakeof Fez+was into the regulatory pool. Effects of Fe3+ Chelators on Iron Bansport by Iron-loaded stimulated after 7 days of iron loading with FAC. To confirm that ascorbate wasachieving Fez+-dependentuptake of iron, we Cells-To gain insight into the mechanismby which intracel-

Iron Pansport in Hep G2 Cells

16049

higher concentrations and appears to saturate (Fig. 3). The apparent V, for Fez+ uptake increasedfrom 17.8 fmoVpg of proteidmin incontrol cells to 55 fmol/pg of proteidmin in those exposed to FAC for 7 days. The associated K,,, increased from 3.6 t o 8.3 p ~ These . parameters, derived from iterative least squares fits toa Michaelis-Menten model, are consistent with those calculated from Eadie-Hofstee plots of the data (control: K, = 4.1 p ~ V,, = 19 fmol/pg/min; iron-loaded: K, = 8.8 p ~ , V, = 59 fmol/pg/min), adding further support to themodel. htrd Effect of Proteolysis-The potential roles of reductase en+ CP020 zymes and protein carriers facilitating translocation prompted "c CP094 "c OF0 an investigation on the effect of proteolysis on NTBI uptake in control and iron-loaded cells. Mild trypsin treatmentdecreased uptakeunderall circumstances, with Fe3+ transport being somewhat more sensitive than Fez+, and iron-loaded cells showing larger absolute decreases than control cells (Table 11). This inhibition of iron transport by a n impermeable proteolytic enzyme suggests that iron uptake is dependent upon cell surface protein(s). Although a carrier would qualify as a proteolytic target, other extracellular rate-determiningenzyme activities such as ferric ion reduction cannot be ruled out. Indeed, the significantly greater decrease in Fe3+compared with Fez+ transport in control cells is consistent with a trypsin-sensitive reContrd ductase acting in concert with an Fez+ transporter protein. "e CPOZO -C CP094 Competitive Effects of Metal Ions-If a common transporter OF0 is used for Fe2+ andFe3+uptake, a similar pattern of competi0.0 4 tion by other metal ions would be expected. Therefore, we ex0 20 40 60 80 100 120 amined the ability of other divalent metals to inhibit the upTime (h) take by control and iron-loaded cells. Ni2+had no inhibitory FIG.2. Effect of chelators on the efflux of cellular iron(a)and effect upon either oxidation state in either cells (Table 111, the rate of FeS+ uptakeby iron-loaded Hep 6 2 cells ( b ) .a, cells conditions. Co2+was were loaded with 59Fe-FAC(20 pg/ml iron; 0.8 pCi/ml) for 7 days. Ex- whereas Co2+was inhibitory under all t o Fe3+uptake. Whereas tracellular iron was removed,and the release of 59Feradioactivity was somewhat more inhibitory to Fez+ than expressed as the percentage of cellular radioactivity remaining as a it attenuated the up-regulation of both Fe3+and Fez+transport, function of time after adding fresh medium in the absence (0) or pres- there was no significant effect of the other metal ions tested. ence of either CP020 ,).( CP094 (W), or DFO (A),all a t 100 p.b, the rate of 59Fe3+ uptake was determined after treating iron-loaded cells for Cd2+and Zn2+were especially effective in inhibiting Fez+ transvarious times with growth medium in the absence (0) or presence of port in control cells, decreasing uptake to 23 and 28% of coneither CP020 (01,CP094 (W), or DFO (A),again at 100 p ~ The . rate of trols exposed to no competing ion, respectively (Table 111, but uptake was determined by incubating cells with 1p 59Fe-NTA for15 they had no significant effect on Fe3+uptake. Cu2+competed to min at 37 "C in a-MEM-HEPES. Data points are the mean of triplicate the same degree for the transport of both iron species. Thus, determinations but the S.D. ( Zn2+)bears no obvious relation to have described a role for proteoglycans and mucins in the of iron (18,40,49-50). However, the inabilityof the observed pattern of inhibition since, for example, Ni2+and ing and uptake 111, or chondroitinase Cu2+ wereunable to compete with Fe2+,whereas Zn2+and Co2+ neuraminidase,heparin-sulfatelyase did. The loss of all antagonizing effects of Cd2+and Zn2+,but not ABC to significantly decrease the rate of Fe3+uptake is inconof Co2+,in iron-loaded cellswhere both Fe2+ Fe3+ and uptake are sistent with a polyelectrolyte-mediated uptake of NTBI in eiincreased, is striking and suggests a different rate-limiting ther control or iron-loaded cells, and our results, taken as a of Fe2+ transporters. process that is unaffected by these otherwise inhibitory ions. whole, are consistent with the operation In summary, the up-regulation of iron transport in relation to Since the up-regulation of Fe3+transport is determined by intracellular iron concentration (18), loss of Cd2+and Zn2+com- cellular iron loading occurs independently of the oxidation petition may be a function of conditions of the long-term iron state of iron, although Fe2+is transported more rapidly than loading imposed by the use of FAC. For instance, one might Fe3+in both loaded and unloaded cells. Reduction is a ratespeculate that in control cells Cd2+ and Zn2+gain access to limiting stepfor Fe3+transport, in bothcontrol and iron-loaded protein sites that are not accessible in iron-loaded cells due to cells. The nascent Fez+is derived from a reductase activity at chelators. a more stringent coupling between reductionand translocation. the cell surface andis accessible to extracellular Fe2+ The abilityof NEM to inhibit Fe2+and Fe3+uptake confirms It is nevertheless directedfor translocation by a pathway disthe findings of Inman and Wessling-Resnick (14) with K562 tinct from that availableto exogenous Fe2+derived by reduction

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