456
Btochlrrlca ~r lTiophysica Acre. 1073 (I ~lg"t) 456-462 1991 Elsevier Science Publishers B.V. 0304-4165/91/$03.513 A D O N I S 0304416591C0"1184
BilAGEN 23469
Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes Zhong Ming Qian and Evan H. Morgan Department of Physiology, The Universityo/Western A~trali~ Nedtands, WesternAustralia (dustraha)
(Received 8 August 1990) Key words: Membrane transport; Iron transport; Active transport; Non-transferdn-bound iron; Transfcrrin; Rcticuloeytc;(Rabbit) The relationship between transrerrin-free iron uptake and cdlular metabolism was investigated using rabbit retieulucytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimyein A, 2,4-dinitropbenal, NaCN, NaN~ sad rotenone) or substrates. Measurments were made o | cellular A T P concentration and the rate o | uptake of Fe(ll) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into eyfosolie and stromal fractions of the cells and A T P levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vms but had no effect on the K m values for this process, it is concluded that the uptake of traasferrin-free iron by reticalneyles is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated Umlsport process. Additional studies were performed using translerrin-beund iron. The metabolic inhibitors also reduced the uptake oi this form of iron hut the inhibition could he accounted for entirely by reduction in the rate of ~ronsferriu e n d a ~ t o s i s . Introduction
Mo~t cells of vertebrate animals obtain iron from the plasma iron-transport protein, transferdn, b y receptormediated endocytosis [1,2]. During this process the iron is released from transferrin within the endocytotic vesicles,but how it then passes across the vesicular membrane into the eytosol is an unresolved problem. Previous experiments from this laboratory have shown that rabbit reticulocytes can take up transferrin-frce iron and incorporate it into haemoglobin [3]. The process is rapid, saturable and competitively inhibited b y certain other divalent metal ions saeh as Z n 2÷, Co 2+, Mn 2÷ and Ni 2+, On the basis of this and other evidence it was considered to be dependent on carrier-mediated transport and to occur independently o[ any involvement of tran~ferrin [3]. The experiments described in this paper were designed to investigate the relationship between the uptake of transferrin-free ferrous iron and cellular metabolism in rabbit reticulocytes and, hence, to provide evidence A'0bre~iatio~s: Fe(ll). transferrin-freeiron; TI'-Fu, transferrin-bound iron. Correspondence: E.H. Morgan, Department oI Physiology,The University of Western Australia, Nedlands, Western Australia+ 6009 Australia.
as to whether the transport process is an active or a passive one. Cellular metabolism was altered by the use of metabolic iahibitors and substrates+ a n d A T P concentrations were measured so that any correlation between this index of cellular metabolism and the rate of iron uptake could be determined. The effects of the inhibitors on the rates of transferrin endocytosis and the uptake of transferrin-bound iron were also measured for comparison with those obl~dned with transfertin-free iron. The transferrin-free and transferrin-boond forms of irgn used in these experiments will be referred to as Fe(II) and Tf~Fe, respecliw'ly. Materials and Methods Materials S~Fe (FeC13) a n d 12sI (Nal) were purchased from Amersham International. Amersham, U.K. The metabolic inhibitors, metabolic substrates a n d reagents to assay A T P were from Sigma Chemicals, St. Louis, M e , U.S.A. Pronase was obtained from Boehringer Mannheim, Manaheim, Y.R.G. Rabbit transferrin was isolated from plasma and labelled with t2Sl and 5~Fe as previously described [4]. It was used in the diferric form. The solutions of N a C N and N a N 3 were prepared by dissolving the compounds in 0.15 M NaCI, while the other in.hibitors (2,4-dinitrophenol, roteaone and antimyeln A) were dissolved in dimethylsulphoxide at 100-
457 times the final concentration. When these inhibitors were used dimethylsulphoxide was added to the cnmrot incubations in the same concentration as that present in the incubation media containing the inhibitors (1%, v/v).
Ceils Reticuloeyte-rieh blood was obtained from rabbits with phenylhydrazine-~nduced baemolytic anaemia, 3-5 days after the last dose of phenylhydrazine [4]. The cells were washed four times with 0.15 M NaCI, then centrifuged at 2000 × g for 30 rain at 4°C, the huffy coat removed and the top one-quarter of the red cell layer which was enriched in re:~eulocytes was collected and suspended at an haematocrit of 10-15% in 0.27 M sucrose. The reticuloeyte count varied from 48 to 91,%. but, for brevity, the cells will be referred to as reticulocytes since the mature ceils present in the suspension take up very little transferrin-free [3] or lransferrinbound iron [1], although they could eoraribute towards the cellular ATP levels and explain why they were well above zero even in the presence o[ high concentrations of inhibitors (see Results).
Measurement of iron uptake The methods for preparation of the radiolabelled Fe(II) solution and measurement of Fe(lI) uptake were as previously described [3]. Briefly, the F¢(II) solution was prepared by adding ~FeCI3, ~OFeSO,, and 2-mereaptoethanol (50-fold molar excess relative to Fe) to 0.27 M sucrose to give a final concentration of 62.5 #M. The incubations were performed at 370C in a shaking water bath using 0.1 ml aliquots of retieulocytes and 4.9 m | of incubation medium which consisted of 0.27 M sucrose buffered to p H 6.4-6.~ with 4 mM Pipes (1,4piperazinediethanesulphonic acid). Metabolic inhibitors or substrates were added to this medium as required. The cells were incubated in the presence or absence (conifers) of the inhibitors a n d snbstrates for 30 rain, the F¢([I) solution added in amounts required to give the desired final iron concentration a n d the incubations continued for varying periods of time. The cell suspension was then centrifuged at 1000 × g for 10 rain at 4°C and the cells washed three times with ice-cold 0.15 M NaCI, transferred to new test tubes, haemolyzed and separated into haem, cytosulie a n d stromal fractions as in earlier work [3]. Each fraction was counted for radioactivity. For the measurement of Tf-Fe uptake the same general incubation procedure was used except that diferric ~ZSl-~gFe-labelled tranferrin replaced the Fe(II) and the incubation solution was 0.15 M NaCI buffered to p H 7.4 with 4 mM Hepes (4-(2-hydroxyethyl)-l-piperazineethane sulphonic acid) or Hanks and Wallace balanced salt solution 15] as described below. In addition, a different procedure was employed to fraetionate the
washed radiolabelled cells so that the rate of trausferrin endocytosis as well as iron uptake could he measured [6]. The cells were incubated with pronase (1 m g / m l ) for 30 min at 4°C. This leads to the release of receptorbound transferrin on the outer cell membrane and allows separation of membrane-bound and intracenular radioactive transferrln and iron. In this p~',~eedure the intracellular fraction contains the cytosol as well as intracellular nrganeltes. In contrast, the fraetionation procedure used after the incubations with F e t i d yields cytosol which contained the haem (as haemoglobin) bat was devoid of cell membranes and intracellular organelles, and a stromal fraction which eonslm~ of plasma membrane, intracelhilar organelles and any ~gFe bound to these cellular components.
Analytical methods The mticul0cyte count was deterrmned by staining with new Methylene blue and the packed cell volume by the microhaematoerit method. Haem was extracted by the method of Thnnell [7~. Radioactivity was measured in a three-channel "t-scintillation counter (LKB-Wallac 1282 Compa-garnma). The method described by Jaworek and Welsch [8] was used for ATP assay. For these assays samptes of reticulocytes were incubated with metabolic inhibitors and substrates at 37°C for 30 rmn under identical conditions to those used for preincubafinn before iron uptake determinations were performed. The cells were then sedimented by centrifugation at 1000 × g for 10 rain at 4°C, suspended in 0_7 ml 0.15 M NaCI and e~tracted with 1.0 mi 12% ( w / v ) triehloroacetic acid, fo~owed by eentrifugation at 1000 × g for 10 min. The supernatant was then neutralized with 2 M K O H a n d used for the A T P assays. Results
Effects of metabolic inhibRors on Fe(ll) uptake The inhi.bitors used were antimycin A, 2,~.dinitrophenol, NaCN, N a N z and rotenone. Each produced a concentration-dependem inhibition of Fe(ll) uptake, as illustrated for N a N 3 in Fig. 1. It can be seen that the rate of F e t i d uptake into the cymsoi was linear for at least 20 min in the absence or the presence of NAN,. Similar results were obtained with the other inhibitors, and with incorporation into the haem and stromal fractions of the ceils. Therefore, the rate of iron uptake can be determined by using a single incubation time of 20 rain a n d calculating the rate of uptake in terms of rime! iron per ram. This was used in subsequent experiments. The results for F¢(1I) uptake rate into the three eell fractions in the absence and presence of varying concentrations of the inhibitors are summarized in Table I. With all of the inhihitors uptake into the cytosolic, haem and stmmal fractions of the cells was inhibited in a concentration-dependent manner, incorporation into
458 haem being most sensitive and that into the stromal fraction least sensitive to the inhibitors.
8O E o
60
LII
~ 40 I--
~ 20' n',
1 '0 1 '~. 2 '0 INCUBATION TIME (min) Fig. 1, Effects of NaN3 on uptake of F¢01) into the cyloso| of rabbit retiealocytes. The cells were preir~eubated at 37 ° C "with0 (e~, liJO(a) or 10130p.M (£3)Nalq3 for 30 mln before the addition of :iSF~ll) and measurement of iron uptake to the eytosol as dcscribe,,d in the text.
TABLE I
E//eel of metabolic inhibitor~- on ATP concentration and F~(II) uptake by reticuloLytes The results show the cellular ATP concentrat/ons and rates of Fe([]) uptake into the ¢ytosolie, hacm and stromal f,,:ctions of reticufoeytes which had been pnnlneubated for 30 mm in the absence or presence at" the inhibitors before measurement of ATP or incubation with 1 ~M Fe([1). The results are expressed per ml ceils or retieulocyles, those for Fe(tI) representing the uptake during a 20 mix incubntio~a period. Each value is the mean {+S.E.) of six mCasuremenu, each on a separate celt preparation. Inhibitor
(~M)
ATp (/*real/ nil cells)
F¢(III uptake (nmole/nd retiealocytcs) ~ytosol h a c m stroma
Rotertone
0 1.35=[;0.10 34.7-1-4.3 12.3:1-2,2 35.9:1_5.1 0.0l 1.12.[=0.04 22.54-1.1 8.3±0.7 32.6:t-2.4 03 0.85±0.03 16.7+1.3 2.0+0.3 29.44.1.7
AntimyeinA
0 1.504-0.10 39.7±3.5 11.34.3.5 38.7±5.1 0.05 1.004-0.05 19.l:t0.5 3.8+1.1 26.0+1.1 O.5 O.654-0.02 14,9±1,2 2,9±0,2 19.7+1.2
2,4dinit~ophenol
NaCN
0 It) loa 1000
1.35:1:0.16 35,8:k2,8 O,Sl:k13.0619,9±1.7 0.69:1:0.03 16.9:t_0.7 0,42-t:0.06 3,3:t-0,5
9.4-1-2.3 34.7+3.3 %6+0,6 22.1+2,5 5.34-0.3 16.74-0.8 0.54-0.1 7.9+1.1
200 500 1O0O
1,314-0.07 ~.9±4.:1 1.08-t-0.03 30.9+2.1 1.004.0.04 25.7+1.4 0.545:0.03 13.74.L2 0.46~:(].0] 12.5:50.8
9.9:1:0.8 2.7t-0.2 1.64-0.] l.l:l:0.] 0,9±0.2
43,9±3.7 30.2+1.8 31.5:1:2.5 29.2~:1.5 20.0+0.9
0 10O 5~o 1000
1.31±1L13 33.94-:~.7 1.23±0.07 25.3+1,9 0.82±0.0~ 14,7±1,5 0.73~0.05 12.1--0.6
12.2.-1.1 11.2±0.8 5.6±0,6 4.54-0,3
39.1+5.1 33.1±4.0 27.3+1,4 2d..14-l,6
0 1O0
NaN
Relationship between celhdar A T P concentration and rate of Fe(ll) uptake The reductions in rate o f F e ( l l ) uptake which resulted from pre-incubation with metabolic inhibitors were accompanied by decreases in the cellular concentrations o f A T P {Table I). As with Fe(lI) uptake these reductions varied with the irdaibitor concentrations. I n other experiments attempts were made to raise '.he cellular A T P concentrations by incubation of retieuIocytes for 30 mix at 3 7 ° C in Hanks a n d Wallace solution c o n t a i n i n g a mixture of metabolic substrates {sodium pyruvate, 4 m M ; inosinc, 4 raM; adenosine, 4 m M ; sodium phosphate, 20 raM). Control cells were preineubated with H a n k s and Wallace solution only or were given n o preinenbation. T h e cells were then centrifuged and resuspended in the sucrose incubation medium prior to measurements o f rate of F e ( l l ) ttptake o r cellular A T P level. The results ~tre summarized in Fig. 2. Preincubation with the substr,ttes led to a significant increase in the mean cellular A T P level ( P < 0.05) and the rate of F e ( l l ) uptake into the cytosolic fraction of the cells ( P < 0.05). T h e relationship between the rate of Fe(II) uptake into the cytosolic a n d stromal fractions of the reticuIocytes a n d their A T P concentrations was determined
150 ta~" ~tU
'~ Z.J OO
100
0
ATP
CYTO
HEME STROMA IRON UPTAKE
Fig, 2. Effect of addition of malabolic substratcs to the incubation medium on reticulocyte ATP concentrations and rate of F¢(ll) apta.ke ;nto th,~ cytosolic, hacm and slromal fractions of tba cells. The cells were suspended in Hanks and Wallace solulion and received no w©incubation (open bars) or were plcineubaled for 30 mln at 37°C in Ha~ks and Wallace solution alone (closed bars) or Hanks and Wallace solution containing sodium pyruvate (4 raM), inn;the {4 raM), adenosine (4 raM) and sodium phosphate (20 raM) (striped bars). prior to measurement of cellular ATP concentrations or F¢(ll) uptake. The results arc the meaas~S.E. (wrtical bars) of ilxree expcrimertts and are e×pte*sod as percent ol th~ values obtained with i.:1oe.onaol cells which rec¢ivedno preiacabatinn.
459
,00]A
150- B
N 100~
/
m m *00
ICJ._I
i...> o..,j Zre o z~
..l
o~
. ~ I L~ *
_m o
o
50
0 0 ATP (% CONTROL VALUES)
5'0
1 (~0
ATP (% CONTROL VALUES)
Fig. 3. Relationship between rate of Fell[) uptake into the cyto*oli¢ (A) and slromat LB} fractions and c¢llufar ATP concentration of rabbit reticuloeytes. The cells ~verepreineubated for 311rain at 37°C before measurement of FellI) uptake, with metabolic sabstrates as in Fig. 2 (ell or with the metabolic iahibitors rotcnone (0), anlim3~cinA (G), X4-dimtrophenol (a,), NaCN (~.) and NaN~ (m).The results are expressed as pe~ent of control values obtained with cells preincubated in the absence of zubstra~es or inhJbitors_ "t~-e equations for the regression lines are A: v-l.08x-15.2(r=0.845) andB: y 0.985.~+6.24(r=O,772L
by plotting the values for these two measurements expressed as percent control values against one another (Fig. 3). These were highly significant correlations for both the cytosolie ( r = 0.845; P < 0.0005) and stromal ( r = 0.772: P < 0.0005) fractions.
Effecr of metabolic h~hibitors an V,,~ and K~ values ]or Fe(ll) uptake As was found previously [3,91, when rctieulocytes were incubated with increasing concentrations of Fe(ll)
80-
i
6o-
vm
40-
z 0
20 ¸
Effeczs of metabolic inhibitom on rates of Tf-Fe uptake and lransferrin endocytosis
=_ -,0
0.0
the rate of uptake of Fe(II) into the cell cytosol showed evidence of saturation at a n iron concentration of 1 - 2 txM (Fig. 4). At higher Fe(ll} concentrations the uptake continued to increase in a linear manner. Hence, there is evidence for a sa*.urable, probably carrier-mediated, uptake process plus a non-saturable process. In the presence of the metabolic inhibitors the saturable process was inhibited b u t there was little change in the rate of uptake by the non-saturable process (Fig. 4). T h e results for several experiments of the type illustrated in Fig. 4 were analysed using a Newton-Raphson non-linear curve fitting p r o g r a m m e to determine the m a x i m u m rates of Fe(II) uptake, V~,~, and the Michaelis constants, K=, of the saturable components of the uptake process. All the inhibitors reduced the Vm~ values for Fe(II) incorporation into eytosol, h a e m lind stroma, but h a d no significant effects on the KEn values (Table ll). As before (Table I) the effects were greatest for incorporation into h a e m and Mast for incorporation into the stromal fractions.
.
.
.
w
.
i
".
1.0 2.0 3.0 4.0 IRON CONCENTRATION {pmoletl)
Fig. 4. Effect of Fe concentration on rate of F¢(ll) uptake by rabbit reticuloc~cs in the gbs~nce at inhihltors .(e,)ox th© presence of 100 t~M NaN~ (o), 10 pM 2,4 dimtrophcnol (41, 100 /AM 2,4-dinitrophenol (A)mad t mM NaCN(ra).
It has been shown previously that preincubation of rabbit reficulocytes with metabolic inhibitors leads to a reduction in the rate of uptake of Tf-Fe [10-13] which is proportional to the reduction in cellular A T P concentration [13]. However, the effects of the inhibitors on the rate of transferrin endocytosis was not investigated. Therefore, in the present work the effects of 2,4-dinitro-
460 TABLE n Effect of metabolic intubiwrs on Vm~~ und K m eatues fer specific Fe~lO uptake The re.suits show the maximal rates ( Vma~ ) and Miehaclis constants { Kin) for the zpceifie (saturable) component of Fe(II) uplake ~nto the eytosolie, haem and stromal fractions of reticuloeytes preincubated [or 30 rain in the absence or premnee of the inhibitors prior to incubation ¢vlth
concenlratinnsof t:e(lll varying frora 0.1 to 4.0 gM. Each value is the mean (4- S.E.} of four mea.smemcnts. Conditions
Vm~, (nmol/ml reticvloeytes per 20 rain)
K~ OtM)
cytoso]
hacm
slroma
cytosol
haem
slroma
Contm]
37.32.7.0
19,8 + 4 A
40,0 i 12. ]
0,24 .L 0.04
0.21 + 0.1)7
0.31 _+OI07
Antimy¢in A 0i~ g M
]6,~ ± 2,5
'9,5 i 0 , 8
2 t . 0 ± 2.5
0.24+0.04
0.20 =[:0.02
D.32.+ 0.04
Rotenone 0.01 #M
]2.44-4.1
4.3 i 018
39.94- 9,0
0.254-0.02
0.23±0.04
0.32 ± 0.01
2,4-dinitrupheaol 100.aM 1 ~ 0 VM
13.54-4,4 2.0 + l , l
7,6 £3.1 0.63 ~ 0.4
~ . 0 ~ 6.0 4.02- 1.2
O,2a ~ 0.05 0.24+0.06
0.23±0,09 0.22+0.07
0.30::1:0.03. 0.31+0.07
NaN~ 100/~M 1000 ~M
24.04-6,1 9.8±2,5
16,9 4-4.6 7,6 4-37
32.8± 8.3 24,1+ 7.0
0.25 + 0A)3 0,244- 0.02
0.22±0,07 0.23+0.07
0,304-0.04 f130 ± 0.05
NaCN 1000 # M
22,•±5.2
t.~ ±0.2
38.0+ 6.5
0.23+0.03
0,19 4- 0,04
0.304-0.01
phenol, NaCN and rotenone on ~he rates of both Tf-Fe uptake and trartsferrin endocyto ;is were determined as previously described [61. The resahs are summarized in Fig. 5 which shows that the inhibitors reduced the rates of iron uptake and transferrin endocytosis to a similar degree. Overall, a very close and highly significant correlation (r= 0.982; P < 0.0005) between these values was observed. The slope of the regression line indicates that approximately two atoms of icon were acquired by the cells for each molecule of transferrin which was endocytosed in both the absence or the presence of the metabolic inhibitors. In other words, the degree of inhibition of the rate of iron uptake which occurred after preincubation with the inhibitors could be
o'1
.o/
0.6 tltl~
.//.
0' I
0.00"1 ~ 0.0
.
.
.
.
.
.
.
I
0,1 0.2 0.3 0.4 TRANSFERRIN ENDOCYTOSIS {nmole/ml/minl
Fig, 51 Relationship between the rate of iron uptake ~lld rl~t¢ of transferrin endoeytosis by rabbit reticulocytes incubated with SgFe12~l-lahelled fiiferfie transferrin in the absence of inhibitors (Q) or in th= presence of OiOI (o)1 Oil (1) or LO (.~1 /JM rotenono; t mM NaCN ([2); and 0.5 (l} and 110 ( * ] mM 2,4-dinitrophenol. The equation for the regression line is ? = 1.64x 4 0.052 (r = 0.952).
aocounted for by a comparable reduction in the rate of transferrin endocytosis. Discussion These experiments confirm earlier observations [3,9] that Fe(ll) dissolved in 0.27 M sucrose at p H 6.5 is readily taken up b y rabbit reticulocytes. The uptake displayed saturation kinetics and resulted in iron incorporation into haem, supporting the conclusion [3] that reticulocytes can transport non-transferrin bound iron across the plasma membrane into the eytosol by a carrier-mediated process. In the present work the uptake of Fe.(II) was showlt to be inhibited by five inhibitots which affect oxidative phosphorylation in the mitochondria in different ways, while incubation of the cells with metabolic substrates enhanced the uptake. Moreover, a close correlation was observed between the rate of Fe(II) uptake and the cellular concentration of ATP in the presence or absence of the inhibitors and the ~ubstrates. Hence, the results strongly suggest that the transport of Fe(I1) into reticulocytes under the conditions used in these experiments is an active process in which ATP is involved. All of the inhibitors produced reduction of the V.,~ values for Fe(II) uptake but did not affect the K m values significantly. These results provide evidence that the inhibition is of non-competitive type. Since inhibition of cellular energy production is one of the more common causes of non-competitive inhibition of active membrane transport processes [14] the results support the conclusion that the Fe(ll) is transported into reticuIocytes by an energy-dependent, active transport mechanism. Uptake of Fe(II) into the cytosolic, haem and stromal fractions of the cell were all reduced by the action of
461 the metabolic inhibitors. As discussed above, the results with respect to the cytosolic fraction are probably the result of inhibition of an energy-dependent transmembrahe carrier mechanism. In experiments of the type described in this paper ~gFe incorporation into haem must depend on iron being supplied by transport across the cell membrane. Hence, it would be expected 1o decline if the ecU entry process was inhibited. However, iron uptake into haem was even more sensitive to the effects of the inhibitors than was that into the eytosoL This may be because the various steps in the synthesis of haem require metabolic energy as well as a supply of iron. Iron uptake by the stroma would be expected to consist of two main components, one representing iron bound by the plasma membrane during incubation in the Fe(lI)-eontaining medium and the other iron which had passed through the plasma membrane and cytosol to intraecliular organelles such as mitochondria which are included in the stromal fraction of the ceils. The first component may consist of specific (e,g,, binding to a membrane carrier) and non-specific processes, and may not he dependent on cell metabolism. The second component, however, would be affected by the same factors which influence iron transport into the cytosol and would therefore be reduced by metabolic inhibitors. Overall, the effect of these inhibitors on iron uptake by the stroma would be expected to be less than their effects on iron uptake to the cytosol or haem, as was observed. Several investigators have reported that teticulocytes [15-201, liver cells [21-23] and various types of cultured cell lines [24] can utilize non-transferrin-bound iron. However, in these investigations the iron was bound by various iron complexing agents which may have acted as vehicles for iron entry into the cells, In contrast, the iron stablizing agent used in the present work. sucrose, is unable to enter reticulocytes [3] and probably acts only as a weak iron chelator which maintains the iron in solution in a form which can be assimilated by the cells. Since the form of iron taken up by retieuloeytes is not sucrose-iron and no other chelators were used it is likely that the iron is transported into the cells in the ionic state, probably tn the form of Fe(tI), although the possibility Jf participation of iron ehelators derived from the cells, such as ATP. cannot be entirely excluded. An important aim of research on iron entry into reticuloeytes is 1o determine the mechanism by which Tf-Fe, the normal source of iron for these cells, passes into the cytosol. The experiments on the effects of the metabolic inhibitors on Tf-Fe uptake were undertaken with this aim in view, to see whether the uptake was inhibited in a similar manner to that of Fe(lI) and hence whether the same carder mechanism could be involved in both processes. Many pre~ous investigators
have shown that TGFe uptake is reduced when reticulocytes are incubated by metabolic inhibitors [10-14], but the mechanism of this effect has not been studied in any detail_ The results confirmed the reduction in the rate of iron uptake but showed that this was accompanied by equivalent reduction in the rate of transferrin endocytosis. Hence, all of the reduction in iron uptake could be accounted for by the change in the rate of endocytosis which was therefore the rat~-limiting step for Tf-Fe uptake in the presence of the inhibitors as it is in norma~ redculocytes [1t. As a consequence, these results do not provide any evidence as to whether the iron carrier responsible for Fe(II) uptake is also involved in the transport of Tf-Fe ink,, ,he cytosol after the iron is released from transferrin in the endosomes. The incubation conditions and the metabolic inhibitots used in these experiments were chosen for specific reasons. The uptake of Fe(ll) by reticulocyles is maximal at pH 6.5 and in solutions of low ionic strength [3] while that of Tf-Fe is maximal at about p H 7.4 and physiological ionic strength [25]. Hence, the studies with Fe(ID were performed in sucrose solution and pH 6.5 and those with Tf-Fe in salt solutions buffered at pH -/.4, Only respiratory chain inkibitors were used because ATP production in reticulocytes is very largely dependent on respiration [26]. In addition, the commonly used inhibitors of anaerobic glycolysis, sodium fluoride and sodium fluoroaeetate are ionized molecules which must be used at concentrations [12,13] which would inhibit Fe(lI) uptake simply by their effect on the ionic strength of the incubation solution, Aeknuwledgenmnt This research was supported by a grant from the Australian Research Council. References
1 2 3 6
Morgan,E.H. (1981) Mol. AspectsMad, 4, 1-123. Huebers, H,A. and Finch, C.A. (1987~ Physiol. Rev.67, 520-SS2. Morgan, E.H.(1988) Binehim. Biophys. Aeta 943, 428-439, Hemmaplardh.D, and Morgan, E.H. {1974) Bie-!_irn. Bioph3/s. Acts 373, 84--99. 5 Hanks.J.H, and Wallace, R,E. (19g9) Proe. So=, Exp. BioL Mad, 71, 196-2IKI, 6 [acopetta, BJ. and Morgan, E.H. (19831J.Biol, Chem. 258,91089115. 7 Thun¢]|,S. (196~)Clin, Chem. Acta It. 321-333, 8 3aworek, D. and WeLsclx J. (1983) in Methods of Enzymatic Analysis, Third Edn.. Vol. 7 (Bergmeyer,J~ and Grasl, M., eds.L pp. 3~3-346, VerlagChemle,Weinl~elm. 9 Qian, Z.M. and Morgan. F_,H. (1990) Biochem. Pharma¢oL,in press. 10 Jandl. J.N. lnman, &K.,Simmons,R.L. and Allen,D.W. (1959)J. Clin. Invest.3S, 161-185. 11 Morgan, EH, (19~4)Br. J. l-]aernatol, t0, aa2-452. 12 Morgan, E.H. and Baker, E. 0969) Biochim. Biophys. Acts 184. 4,12-454.
462 13 Kailis, S.G. and Morgan. E.H. (]977) Biochim. Biophys, Acla 464. 389-398. 14 N¢ame, K.D. and Pdchards. T.G. (1972) Elementary Kinetic~ of Membrane Carrier Transport, pp. 69 70. Blackwell. Oxford. 15 Young, $.P. and Gomperts. B.D. (1977] Biochim. Biophy:~. Acta 469, 281 291. t6 Ponka, P., Schulman, H.M. mad Wilc~nska. A. (1982) Bio~htim. Biophy~. Acta 718, 151-156. 17 Ponka, P. and Sehulman, IteM, 0985) L Bioh Chem. 260, 1471714721. ]8 Ponka, P. and Sehutman, H.M. (1985) Blood 65, 850-857. 19 Egycd, A. (1988) Br. J. HaematoL 68, 483-486, 20 Fuehs, O.. Boro¢a, J., ~radil~L A. and Neawirt, J, (1988) B i ~ china. Eqophys. Aeta 969, 158-165.
21 Brissot, P.. Wright, T.L., Ma, W.-L and Weisiger. R.A. (19E~51J. Clin. Invest. 76, 1463-1470. 22 Wright, T.L., Bfis,,iot, P, Ma~ W.-L. and Weisigar. R.A. (1986) L Biol, Chem. 261. 10909-10914. 23 Wright. T.L.. Fits. LG. and Weisiger, R.A. (1988) J. Biol. Chem. 263, 1842-1847. 24 Sturroek, A., Alexander, J., Lamb. J., Craven. C.M. and Kaplan, J. (19~0) J. BioL Chem. 265, 3139-3145. 25 Bowen, B.J, and Morgan. EM. (1988) J', Cell. Physiol. 134. 1-12. 26 Rapoport. S.M. (1986) The Reticulocyte, pp. 105-117, CRC Press, Boca Ralon.
Btochlrrlca ~r lTiophysica Acre. 1073 (I ~lg"t) 456-462 1991 Elsevier Science Publishers B.V. 0304-4165/91/$03.513 A D O N I S 0304416591C0"1184
BilAGEN 23469
Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes Zhong Ming Qian and Evan H. Morgan Department of Physiology, The Universityo/Western A~trali~ Nedtands, WesternAustralia (dustraha)
(Received 8 August 1990) Key words: Membrane transport; Iron transport; Active transport; Non-transferdn-bound iron; Transfcrrin; Rcticuloeytc;(Rabbit) The relationship between transrerrin-free iron uptake and cdlular metabolism was investigated using rabbit retieulucytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimyein A, 2,4-dinitropbenal, NaCN, NaN~ sad rotenone) or substrates. Measurments were made o | cellular A T P concentration and the rate o | uptake of Fe(ll) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into eyfosolie and stromal fractions of the cells and A T P levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vms but had no effect on the K m values for this process, it is concluded that the uptake of traasferrin-free iron by reticalneyles is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated Umlsport process. Additional studies were performed using translerrin-beund iron. The metabolic inhibitors also reduced the uptake oi this form of iron hut the inhibition could he accounted for entirely by reduction in the rate of ~ronsferriu e n d a ~ t o s i s . Introduction
Mo~t cells of vertebrate animals obtain iron from the plasma iron-transport protein, transferdn, b y receptormediated endocytosis [1,2]. During this process the iron is released from transferrin within the endocytotic vesicles,but how it then passes across the vesicular membrane into the eytosol is an unresolved problem. Previous experiments from this laboratory have shown that rabbit reticulocytes can take up transferrin-frce iron and incorporate it into haemoglobin [3]. The process is rapid, saturable and competitively inhibited b y certain other divalent metal ions saeh as Z n 2÷, Co 2+, Mn 2÷ and Ni 2+, On the basis of this and other evidence it was considered to be dependent on carrier-mediated transport and to occur independently o[ any involvement of tran~ferrin [3]. The experiments described in this paper were designed to investigate the relationship between the uptake of transferrin-free ferrous iron and cellular metabolism in rabbit reticulocytes and, hence, to provide evidence A'0bre~iatio~s: Fe(ll). transferrin-freeiron; TI'-Fu, transferrin-bound iron. Correspondence: E.H. Morgan, Department oI Physiology,The University of Western Australia, Nedlands, Western Australia+ 6009 Australia.
as to whether the transport process is an active or a passive one. Cellular metabolism was altered by the use of metabolic iahibitors and substrates+ a n d A T P concentrations were measured so that any correlation between this index of cellular metabolism and the rate of iron uptake could be determined. The effects of the inhibitors on the rates of transferrin endocytosis and the uptake of transferrin-bound iron were also measured for comparison with those obl~dned with transfertin-free iron. The transferrin-free and transferrin-boond forms of irgn used in these experiments will be referred to as Fe(II) and Tf~Fe, respecliw'ly. Materials and Methods Materials S~Fe (FeC13) a n d 12sI (Nal) were purchased from Amersham International. Amersham, U.K. The metabolic inhibitors, metabolic substrates a n d reagents to assay A T P were from Sigma Chemicals, St. Louis, M e , U.S.A. Pronase was obtained from Boehringer Mannheim, Manaheim, Y.R.G. Rabbit transferrin was isolated from plasma and labelled with t2Sl and 5~Fe as previously described [4]. It was used in the diferric form. The solutions of N a C N and N a N 3 were prepared by dissolving the compounds in 0.15 M NaCI, while the other in.hibitors (2,4-dinitrophenol, roteaone and antimyeln A) were dissolved in dimethylsulphoxide at 100-
457 times the final concentration. When these inhibitors were used dimethylsulphoxide was added to the cnmrot incubations in the same concentration as that present in the incubation media containing the inhibitors (1%, v/v).
Ceils Reticuloeyte-rieh blood was obtained from rabbits with phenylhydrazine-~nduced baemolytic anaemia, 3-5 days after the last dose of phenylhydrazine [4]. The cells were washed four times with 0.15 M NaCI, then centrifuged at 2000 × g for 30 rain at 4°C, the huffy coat removed and the top one-quarter of the red cell layer which was enriched in re:~eulocytes was collected and suspended at an haematocrit of 10-15% in 0.27 M sucrose. The reticuloeyte count varied from 48 to 91,%. but, for brevity, the cells will be referred to as reticulocytes since the mature ceils present in the suspension take up very little transferrin-free [3] or lransferrinbound iron [1], although they could eoraribute towards the cellular ATP levels and explain why they were well above zero even in the presence o[ high concentrations of inhibitors (see Results).
Measurement of iron uptake The methods for preparation of the radiolabelled Fe(II) solution and measurement of Fe(lI) uptake were as previously described [3]. Briefly, the F¢(II) solution was prepared by adding ~FeCI3, ~OFeSO,, and 2-mereaptoethanol (50-fold molar excess relative to Fe) to 0.27 M sucrose to give a final concentration of 62.5 #M. The incubations were performed at 370C in a shaking water bath using 0.1 ml aliquots of retieulocytes and 4.9 m | of incubation medium which consisted of 0.27 M sucrose buffered to p H 6.4-6.~ with 4 mM Pipes (1,4piperazinediethanesulphonic acid). Metabolic inhibitors or substrates were added to this medium as required. The cells were incubated in the presence or absence (conifers) of the inhibitors a n d snbstrates for 30 rain, the F¢([I) solution added in amounts required to give the desired final iron concentration a n d the incubations continued for varying periods of time. The cell suspension was then centrifuged at 1000 × g for 10 rain at 4°C and the cells washed three times with ice-cold 0.15 M NaCI, transferred to new test tubes, haemolyzed and separated into haem, cytosulie a n d stromal fractions as in earlier work [3]. Each fraction was counted for radioactivity. For the measurement of Tf-Fe uptake the same general incubation procedure was used except that diferric ~ZSl-~gFe-labelled tranferrin replaced the Fe(II) and the incubation solution was 0.15 M NaCI buffered to p H 7.4 with 4 mM Hepes (4-(2-hydroxyethyl)-l-piperazineethane sulphonic acid) or Hanks and Wallace balanced salt solution 15] as described below. In addition, a different procedure was employed to fraetionate the
washed radiolabelled cells so that the rate of trausferrin endocytosis as well as iron uptake could he measured [6]. The cells were incubated with pronase (1 m g / m l ) for 30 min at 4°C. This leads to the release of receptorbound transferrin on the outer cell membrane and allows separation of membrane-bound and intracenular radioactive transferrln and iron. In this p~',~eedure the intracellular fraction contains the cytosol as well as intracellular nrganeltes. In contrast, the fraetionation procedure used after the incubations with F e t i d yields cytosol which contained the haem (as haemoglobin) bat was devoid of cell membranes and intracellular organelles, and a stromal fraction which eonslm~ of plasma membrane, intracelhilar organelles and any ~gFe bound to these cellular components.
Analytical methods The mticul0cyte count was deterrmned by staining with new Methylene blue and the packed cell volume by the microhaematoerit method. Haem was extracted by the method of Thnnell [7~. Radioactivity was measured in a three-channel "t-scintillation counter (LKB-Wallac 1282 Compa-garnma). The method described by Jaworek and Welsch [8] was used for ATP assay. For these assays samptes of reticulocytes were incubated with metabolic inhibitors and substrates at 37°C for 30 rmn under identical conditions to those used for preincubafinn before iron uptake determinations were performed. The cells were then sedimented by centrifugation at 1000 × g for 10 rain at 4°C, suspended in 0_7 ml 0.15 M NaCI and e~tracted with 1.0 mi 12% ( w / v ) triehloroacetic acid, fo~owed by eentrifugation at 1000 × g for 10 min. The supernatant was then neutralized with 2 M K O H a n d used for the A T P assays. Results
Effects of metabolic inhibRors on Fe(ll) uptake The inhi.bitors used were antimycin A, 2,~.dinitrophenol, NaCN, N a N z and rotenone. Each produced a concentration-dependem inhibition of Fe(ll) uptake, as illustrated for N a N 3 in Fig. 1. It can be seen that the rate of F e t i d uptake into the cymsoi was linear for at least 20 min in the absence or the presence of NAN,. Similar results were obtained with the other inhibitors, and with incorporation into the haem and stromal fractions of the ceils. Therefore, the rate of iron uptake can be determined by using a single incubation time of 20 rain a n d calculating the rate of uptake in terms of rime! iron per ram. This was used in subsequent experiments. The results for F¢(1I) uptake rate into the three eell fractions in the absence and presence of varying concentrations of the inhibitors are summarized in Table I. With all of the inhihitors uptake into the cytosolic, haem and stmmal fractions of the cells was inhibited in a concentration-dependent manner, incorporation into
458 haem being most sensitive and that into the stromal fraction least sensitive to the inhibitors.
8O E o
60
LII
~ 40 I--
~ 20' n',
1 '0 1 '~. 2 '0 INCUBATION TIME (min) Fig. 1, Effects of NaN3 on uptake of F¢01) into the cyloso| of rabbit retiealocytes. The cells were preir~eubated at 37 ° C "with0 (e~, liJO(a) or 10130p.M (£3)Nalq3 for 30 mln before the addition of :iSF~ll) and measurement of iron uptake to the eytosol as dcscribe,,d in the text.
TABLE I
E//eel of metabolic inhibitor~- on ATP concentration and F~(II) uptake by reticuloLytes The results show the cellular ATP concentrat/ons and rates of Fe([]) uptake into the ¢ytosolie, hacm and stromal f,,:ctions of reticufoeytes which had been pnnlneubated for 30 mm in the absence or presence at" the inhibitors before measurement of ATP or incubation with 1 ~M Fe([1). The results are expressed per ml ceils or retieulocyles, those for Fe(tI) representing the uptake during a 20 mix incubntio~a period. Each value is the mean {+S.E.) of six mCasuremenu, each on a separate celt preparation. Inhibitor
(~M)
ATp (/*real/ nil cells)
F¢(III uptake (nmole/nd retiealocytcs) ~ytosol h a c m stroma
Rotertone
0 1.35=[;0.10 34.7-1-4.3 12.3:1-2,2 35.9:1_5.1 0.0l 1.12.[=0.04 22.54-1.1 8.3±0.7 32.6:t-2.4 03 0.85±0.03 16.7+1.3 2.0+0.3 29.44.1.7
AntimyeinA
0 1.504-0.10 39.7±3.5 11.34.3.5 38.7±5.1 0.05 1.004-0.05 19.l:t0.5 3.8+1.1 26.0+1.1 O.5 O.654-0.02 14,9±1,2 2,9±0,2 19.7+1.2
2,4dinit~ophenol
NaCN
0 It) loa 1000
1.35:1:0.16 35,8:k2,8 O,Sl:k13.0619,9±1.7 0.69:1:0.03 16.9:t_0.7 0,42-t:0.06 3,3:t-0,5
9.4-1-2.3 34.7+3.3 %6+0,6 22.1+2,5 5.34-0.3 16.74-0.8 0.54-0.1 7.9+1.1
200 500 1O0O
1,314-0.07 ~.9±4.:1 1.08-t-0.03 30.9+2.1 1.004.0.04 25.7+1.4 0.545:0.03 13.74.L2 0.46~:(].0] 12.5:50.8
9.9:1:0.8 2.7t-0.2 1.64-0.] l.l:l:0.] 0,9±0.2
43,9±3.7 30.2+1.8 31.5:1:2.5 29.2~:1.5 20.0+0.9
0 10O 5~o 1000
1.31±1L13 33.94-:~.7 1.23±0.07 25.3+1,9 0.82±0.0~ 14,7±1,5 0.73~0.05 12.1--0.6
12.2.-1.1 11.2±0.8 5.6±0,6 4.54-0,3
39.1+5.1 33.1±4.0 27.3+1,4 2d..14-l,6
0 1O0
NaN
Relationship between celhdar A T P concentration and rate of Fe(ll) uptake The reductions in rate o f F e ( l l ) uptake which resulted from pre-incubation with metabolic inhibitors were accompanied by decreases in the cellular concentrations o f A T P {Table I). As with Fe(lI) uptake these reductions varied with the irdaibitor concentrations. I n other experiments attempts were made to raise '.he cellular A T P concentrations by incubation of retieuIocytes for 30 mix at 3 7 ° C in Hanks a n d Wallace solution c o n t a i n i n g a mixture of metabolic substrates {sodium pyruvate, 4 m M ; inosinc, 4 raM; adenosine, 4 m M ; sodium phosphate, 20 raM). Control cells were preineubated with H a n k s and Wallace solution only or were given n o preinenbation. T h e cells were then centrifuged and resuspended in the sucrose incubation medium prior to measurements o f rate of F e ( l l ) ttptake o r cellular A T P level. The results ~tre summarized in Fig. 2. Preincubation with the substr,ttes led to a significant increase in the mean cellular A T P level ( P < 0.05) and the rate of F e ( l l ) uptake into the cytosolic fraction of the cells ( P < 0.05). T h e relationship between the rate of Fe(II) uptake into the cytosolic a n d stromal fractions of the reticuIocytes a n d their A T P concentrations was determined
150 ta~" ~tU
'~ Z.J OO
100
0
ATP
CYTO
HEME STROMA IRON UPTAKE
Fig, 2. Effect of addition of malabolic substratcs to the incubation medium on reticulocyte ATP concentrations and rate of F¢(ll) apta.ke ;nto th,~ cytosolic, hacm and slromal fractions of tba cells. The cells were suspended in Hanks and Wallace solulion and received no w©incubation (open bars) or were plcineubaled for 30 mln at 37°C in Ha~ks and Wallace solution alone (closed bars) or Hanks and Wallace solution containing sodium pyruvate (4 raM), inn;the {4 raM), adenosine (4 raM) and sodium phosphate (20 raM) (striped bars). prior to measurement of cellular ATP concentrations or F¢(ll) uptake. The results arc the meaas~S.E. (wrtical bars) of ilxree expcrimertts and are e×pte*sod as percent ol th~ values obtained with i.:1oe.onaol cells which rec¢ivedno preiacabatinn.
459
,00]A
150- B
N 100~
/
m m *00
ICJ._I
i...> o..,j Zre o z~
..l
o~
. ~ I L~ *
_m o
o
50
0 0 ATP (% CONTROL VALUES)
5'0
1 (~0
ATP (% CONTROL VALUES)
Fig. 3. Relationship between rate of Fell[) uptake into the cyto*oli¢ (A) and slromat LB} fractions and c¢llufar ATP concentration of rabbit reticuloeytes. The cells ~verepreineubated for 311rain at 37°C before measurement of FellI) uptake, with metabolic sabstrates as in Fig. 2 (ell or with the metabolic iahibitors rotcnone (0), anlim3~cinA (G), X4-dimtrophenol (a,), NaCN (~.) and NaN~ (m).The results are expressed as pe~ent of control values obtained with cells preincubated in the absence of zubstra~es or inhJbitors_ "t~-e equations for the regression lines are A: v-l.08x-15.2(r=0.845) andB: y 0.985.~+6.24(r=O,772L
by plotting the values for these two measurements expressed as percent control values against one another (Fig. 3). These were highly significant correlations for both the cytosolie ( r = 0.845; P < 0.0005) and stromal ( r = 0.772: P < 0.0005) fractions.
Effecr of metabolic h~hibitors an V,,~ and K~ values ]or Fe(ll) uptake As was found previously [3,91, when rctieulocytes were incubated with increasing concentrations of Fe(ll)
80-
i
6o-
vm
40-
z 0
20 ¸
Effeczs of metabolic inhibitom on rates of Tf-Fe uptake and lransferrin endocytosis
=_ -,0
0.0
the rate of uptake of Fe(II) into the cell cytosol showed evidence of saturation at a n iron concentration of 1 - 2 txM (Fig. 4). At higher Fe(ll} concentrations the uptake continued to increase in a linear manner. Hence, there is evidence for a sa*.urable, probably carrier-mediated, uptake process plus a non-saturable process. In the presence of the metabolic inhibitors the saturable process was inhibited b u t there was little change in the rate of uptake by the non-saturable process (Fig. 4). T h e results for several experiments of the type illustrated in Fig. 4 were analysed using a Newton-Raphson non-linear curve fitting p r o g r a m m e to determine the m a x i m u m rates of Fe(II) uptake, V~,~, and the Michaelis constants, K=, of the saturable components of the uptake process. All the inhibitors reduced the Vm~ values for Fe(II) incorporation into eytosol, h a e m lind stroma, but h a d no significant effects on the KEn values (Table ll). As before (Table I) the effects were greatest for incorporation into h a e m and Mast for incorporation into the stromal fractions.
.
.
.
w
.
i
".
1.0 2.0 3.0 4.0 IRON CONCENTRATION {pmoletl)
Fig. 4. Effect of Fe concentration on rate of F¢(ll) uptake by rabbit reticuloc~cs in the gbs~nce at inhihltors .(e,)ox th© presence of 100 t~M NaN~ (o), 10 pM 2,4 dimtrophcnol (41, 100 /AM 2,4-dinitrophenol (A)mad t mM NaCN(ra).
It has been shown previously that preincubation of rabbit reficulocytes with metabolic inhibitors leads to a reduction in the rate of uptake of Tf-Fe [10-13] which is proportional to the reduction in cellular A T P concentration [13]. However, the effects of the inhibitors on the rate of transferrin endocytosis was not investigated. Therefore, in the present work the effects of 2,4-dinitro-
460 TABLE n Effect of metabolic intubiwrs on Vm~~ und K m eatues fer specific Fe~lO uptake The re.suits show the maximal rates ( Vma~ ) and Miehaclis constants { Kin) for the zpceifie (saturable) component of Fe(II) uplake ~nto the eytosolie, haem and stromal fractions of reticuloeytes preincubated [or 30 rain in the absence or premnee of the inhibitors prior to incubation ¢vlth
concenlratinnsof t:e(lll varying frora 0.1 to 4.0 gM. Each value is the mean (4- S.E.} of four mea.smemcnts. Conditions
Vm~, (nmol/ml reticvloeytes per 20 rain)
K~ OtM)
cytoso]
hacm
slroma
cytosol
haem
slroma
Contm]
37.32.7.0
19,8 + 4 A
40,0 i 12. ]
0,24 .L 0.04
0.21 + 0.1)7
0.31 _+OI07
Antimy¢in A 0i~ g M
]6,~ ± 2,5
'9,5 i 0 , 8
2 t . 0 ± 2.5
0.24+0.04
0.20 =[:0.02
D.32.+ 0.04
Rotenone 0.01 #M
]2.44-4.1
4.3 i 018
39.94- 9,0
0.254-0.02
0.23±0.04
0.32 ± 0.01
2,4-dinitrupheaol 100.aM 1 ~ 0 VM
13.54-4,4 2.0 + l , l
7,6 £3.1 0.63 ~ 0.4
~ . 0 ~ 6.0 4.02- 1.2
O,2a ~ 0.05 0.24+0.06
0.23±0,09 0.22+0.07
0.30::1:0.03. 0.31+0.07
NaN~ 100/~M 1000 ~M
24.04-6,1 9.8±2,5
16,9 4-4.6 7,6 4-37
32.8± 8.3 24,1+ 7.0
0.25 + 0A)3 0,244- 0.02
0.22±0,07 0.23+0.07
0,304-0.04 f130 ± 0.05
NaCN 1000 # M
22,•±5.2
t.~ ±0.2
38.0+ 6.5
0.23+0.03
0,19 4- 0,04
0.304-0.01
phenol, NaCN and rotenone on ~he rates of both Tf-Fe uptake and trartsferrin endocyto ;is were determined as previously described [61. The resahs are summarized in Fig. 5 which shows that the inhibitors reduced the rates of iron uptake and transferrin endocytosis to a similar degree. Overall, a very close and highly significant correlation (r= 0.982; P < 0.0005) between these values was observed. The slope of the regression line indicates that approximately two atoms of icon were acquired by the cells for each molecule of transferrin which was endocytosed in both the absence or the presence of the metabolic inhibitors. In other words, the degree of inhibition of the rate of iron uptake which occurred after preincubation with the inhibitors could be
o'1
.o/
0.6 tltl~
.//.
0' I
0.00"1 ~ 0.0
.
.
.
.
.
.
.
I
0,1 0.2 0.3 0.4 TRANSFERRIN ENDOCYTOSIS {nmole/ml/minl
Fig, 51 Relationship between the rate of iron uptake ~lld rl~t¢ of transferrin endoeytosis by rabbit reticulocytes incubated with SgFe12~l-lahelled fiiferfie transferrin in the absence of inhibitors (Q) or in th= presence of OiOI (o)1 Oil (1) or LO (.~1 /JM rotenono; t mM NaCN ([2); and 0.5 (l} and 110 ( * ] mM 2,4-dinitrophenol. The equation for the regression line is ? = 1.64x 4 0.052 (r = 0.952).
aocounted for by a comparable reduction in the rate of transferrin endocytosis. Discussion These experiments confirm earlier observations [3,9] that Fe(ll) dissolved in 0.27 M sucrose at p H 6.5 is readily taken up b y rabbit reticulocytes. The uptake displayed saturation kinetics and resulted in iron incorporation into haem, supporting the conclusion [3] that reticulocytes can transport non-transferrin bound iron across the plasma membrane into the eytosol by a carrier-mediated process. In the present work the uptake of Fe.(II) was showlt to be inhibited by five inhibitots which affect oxidative phosphorylation in the mitochondria in different ways, while incubation of the cells with metabolic substrates enhanced the uptake. Moreover, a close correlation was observed between the rate of Fe(II) uptake and the cellular concentration of ATP in the presence or absence of the inhibitors and the ~ubstrates. Hence, the results strongly suggest that the transport of Fe(I1) into reticulocytes under the conditions used in these experiments is an active process in which ATP is involved. All of the inhibitors produced reduction of the V.,~ values for Fe(II) uptake but did not affect the K m values significantly. These results provide evidence that the inhibition is of non-competitive type. Since inhibition of cellular energy production is one of the more common causes of non-competitive inhibition of active membrane transport processes [14] the results support the conclusion that the Fe(ll) is transported into reticuIocytes by an energy-dependent, active transport mechanism. Uptake of Fe(II) into the cytosolic, haem and stromal fractions of the cell were all reduced by the action of
461 the metabolic inhibitors. As discussed above, the results with respect to the cytosolic fraction are probably the result of inhibition of an energy-dependent transmembrahe carrier mechanism. In experiments of the type described in this paper ~gFe incorporation into haem must depend on iron being supplied by transport across the cell membrane. Hence, it would be expected 1o decline if the ecU entry process was inhibited. However, iron uptake into haem was even more sensitive to the effects of the inhibitors than was that into the eytosoL This may be because the various steps in the synthesis of haem require metabolic energy as well as a supply of iron. Iron uptake by the stroma would be expected to consist of two main components, one representing iron bound by the plasma membrane during incubation in the Fe(lI)-eontaining medium and the other iron which had passed through the plasma membrane and cytosol to intraecliular organelles such as mitochondria which are included in the stromal fraction of the ceils. The first component may consist of specific (e,g,, binding to a membrane carrier) and non-specific processes, and may not he dependent on cell metabolism. The second component, however, would be affected by the same factors which influence iron transport into the cytosol and would therefore be reduced by metabolic inhibitors. Overall, the effect of these inhibitors on iron uptake by the stroma would be expected to be less than their effects on iron uptake to the cytosol or haem, as was observed. Several investigators have reported that teticulocytes [15-201, liver cells [21-23] and various types of cultured cell lines [24] can utilize non-transferrin-bound iron. However, in these investigations the iron was bound by various iron complexing agents which may have acted as vehicles for iron entry into the cells, In contrast, the iron stablizing agent used in the present work. sucrose, is unable to enter reticulocytes [3] and probably acts only as a weak iron chelator which maintains the iron in solution in a form which can be assimilated by the cells. Since the form of iron taken up by retieuloeytes is not sucrose-iron and no other chelators were used it is likely that the iron is transported into the cells in the ionic state, probably tn the form of Fe(tI), although the possibility Jf participation of iron ehelators derived from the cells, such as ATP. cannot be entirely excluded. An important aim of research on iron entry into reticuloeytes is 1o determine the mechanism by which Tf-Fe, the normal source of iron for these cells, passes into the cytosol. The experiments on the effects of the metabolic inhibitors on Tf-Fe uptake were undertaken with this aim in view, to see whether the uptake was inhibited in a similar manner to that of Fe(lI) and hence whether the same carder mechanism could be involved in both processes. Many pre~ous investigators
have shown that TGFe uptake is reduced when reticulocytes are incubated by metabolic inhibitors [10-14], but the mechanism of this effect has not been studied in any detail_ The results confirmed the reduction in the rate of iron uptake but showed that this was accompanied by equivalent reduction in the rate of transferrin endocytosis. Hence, all of the reduction in iron uptake could be accounted for by the change in the rate of endocytosis which was therefore the rat~-limiting step for Tf-Fe uptake in the presence of the inhibitors as it is in norma~ redculocytes [1t. As a consequence, these results do not provide any evidence as to whether the iron carrier responsible for Fe(II) uptake is also involved in the transport of Tf-Fe ink,, ,he cytosol after the iron is released from transferrin in the endosomes. The incubation conditions and the metabolic inhibitots used in these experiments were chosen for specific reasons. The uptake of Fe(ll) by reticulocyles is maximal at pH 6.5 and in solutions of low ionic strength [3] while that of Tf-Fe is maximal at about p H 7.4 and physiological ionic strength [25]. Hence, the studies with Fe(ID were performed in sucrose solution and pH 6.5 and those with Tf-Fe in salt solutions buffered at pH -/.4, Only respiratory chain inkibitors were used because ATP production in reticulocytes is very largely dependent on respiration [26]. In addition, the commonly used inhibitors of anaerobic glycolysis, sodium fluoride and sodium fluoroaeetate are ionized molecules which must be used at concentrations [12,13] which would inhibit Fe(lI) uptake simply by their effect on the ionic strength of the incubation solution, Aeknuwledgenmnt This research was supported by a grant from the Australian Research Council. References
1 2 3 6
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