Biochimica et Biophysica Acta, 1176 (1993) 37-42 37 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13336

Activation of K+/C1 - cotransport in human erythrocytes exposed to oxidative agents

Oliviero Olivieri a, Manuela Bonollo b, Simonetta Friso a, Domenico Girelli a, Rober to Corrocher a and Luciano Vet tore b

" Institute of Medical Pathology, Chair oflnternal Medicine, University of Verona, Verona (Italy) and b Chair of Clinical Methodology, University of Verona, Verona (Italy)

(Received 8 May 1992)

Key words: Oxidant; Potassium/chloride cotransport; Potassium ion channel, calcium-activated; SH group; Sickle-cell anemia; (Human erythrocyte)

Activation of K+/C1 cotransport was studied after exposure of normal human erythrocytes to the oxidative action of acetylphenylhydrazine (APH), menadione sodium bisulfite (MSB), hydrogen peroxide ( H 2 0 2) or phenazine metasulfate (PMS). In order to better define the relative contributions of K+/CI - cotransport on ouabain and bumetanide-resistant (OBR) K + effiux induced by oxidation, we used (dihydroindenyl)oxyalkanoic acid (DIOA) and carbocyanine as specific inhibitors, respectively, of cotransport system and Ca2+-activated K + channel. APH, MSB and - to much less extent - H 2 0 2 promoted a K + effiux pathway with features corresponding to those of K+/CI - cotransport. This pathway showed: (i) kinetics of effiux compatible with a specific cation transport system; (ii) requirement for chloride anion; (iii) resistance to ouabain, bumetanide and carbocyanine inhibition; (iv) stimulation by hypotonic challenge; (v) susceptibility to inhibition by DIOA. Dithiothreitol (Dq'T) or 2-mercaptoethanol (2-ME) decreased K+/C1 - eotransport activation, suggesting that oxidative mechanisms affected crucial SH groups of the transporter. These data suggest that oxidation represents a factor capable of modulating activation of K+/C1 - cotransport. Its possible contribution in situations with high oxidative risk, such as sickle-cell anaemia or/3 thalassemia, is discussed.

Introduction

Recently, membrane physiologists and hematologists have devoted increasing attention to a powerful ouabain- and bumetanide-resistant (OBR) K + trans- port pathway, the K + / C I - cotransport, first described by Kregenow in duck red cells (1971) [1] and exten- sively studied in several other species of erythrocyte including humans and sheep [2-4]. This cation trans- port system, by catalysing outward cotransport move- ments of K + and C1- ions with consequent water loss and cell shrinkage, is thought to be involved in some pathophysiological conditions characterized by abnor- mal cell ion and water content, namely Hb C [5,6] and Hb S [6,7].

This pathway is rather quiescent under physiological conditions, but is stimulated by cell swelling, induced by hypotonic and acid pH media containing CI - (but not NO~), and by exposure to the thioalkylating agent

Correspondence to: O. Olivieri, Istituto di Patologia Medica, Poli- clinico Borgo Roma, 37134 Verona, Italy.

N-ethylmaleimide (NEM) [2,8]. Recently, a specific in- hibitor, the (dihydroindenyl)oxyalkanoic acid (DIOA), has been described [9,10].

Two factors are known to modulate the magnitude of K+/C1TM cotransport activation: cell age and hemoglobin structure. It is well known that the activity of cotransport is higher in reticulocytes than in mature erythrocytes [11,12], but the reasons for this difference are not yet clear.

The role of the hemoglobin structure in modulating cotransport activity has been demonstrated in a recent paper: positive charge modifications of /3 globin (like Hb S or C) induce activation of K+/C1 - cotransport only if the mutations occur a t /36 or/37 residues of the globin chain [13]. In the same report, erythrocytes from /3 thalassemia intermedia also showed an increased K + / C I - cotransport activity independently from reticulocyte count. As oxidative phenomena are rele- vant to the pathophysiology of both sickle-cell anaemia and /3 thalassemia [14,15], we hypothesize that oxida- tive mechanisms modulate K+/C1 - cotransport activa- tion. Several published reports give evidence that ox- idative damages (induced through different methods)

38

are associated with an increased membrane OBR K ÷ leak [16-22]; however, no implications as to the in- volvement of K+/CI cotransport have been sug- gested.

The aim of our study was to investigate whether a mild oxidative stress, induced in vitro by oxidative agents such as acetylphenylhydrazine (APH), mena- dione sodium bisulfite (MSB) and H 2 0 2, increases the K+/C1- cotransport activity of normal human erythro- cytes.

The results demonstrate that, in addition to cell age and Hb mutations, cell oxidation is another major factor which can modulate activation of the cotransport system.

Materials and Methods

Experiments were performed on erythrocytes from adult, healthy, volunteer workers from our laboratory. Blood was collected in heparinized tubes and pro- cessed immediately.

Plasma and buffy coat were removed after centrif- ugation at 1200 x g for 10 min and the cells washed four times with a choline washing solution (CWS) containing 150 mM choline chloride, 1 mM MgC12,10 mM tris(hydroxymethyl)aminomethane-3-(N-morphol- ino)propanesulfonic acid (Tris-Mops) (pH 7.40) at 4°C.

Two ml of packed red cells were resuspended in 30 ml of a similar isotonic medium (final Ht 6%) where choline was substituted for sodium or potassium as cation and oxidants were added in adequate concentra- tions (oxidative solution).

In preliminary dose-effect experiments, we deter- mined for each oxidant the concentrations inducing the larger OBR K + effluxes without significant hemolysis (less than 2-3%). These concentrations were: APH 20 mM, MSB 1.5 mM, phenazine methosulphate (PMS) 1 mM and H202 1 mM. In experiments with H202, cells were also pretreated with sodium azide (1 mM) in order to inhibit catalase activity.

Control and oxidants-treated erythrocytes were in- cubated in a shaking water bath at 37°C for 40 min. During the first 10 min, tubes were flushed with hu- midified 0 2 to allow complete oxygenation of Hb.

After incubation, red cells were washed thrice with cold CWS and a 40% suspension was obtained. An aliquot of this suspension was used to measure Ht and Hb. Another aliquot (0.5 ml) was added to 10 ml of a solution (efflux medium) containing 140 mM NaC1, 10 mM glucose, 1 mM MgC12, 0.1 mM ouabain, 10 mM bumetanide and 10 mM Tris-Mops at 37°C and at variable pH (see Results). After incubation at 37°C, tubes were transferred at established times in ice-water bath for 5 min and spun at 3000 X g in a refrigerated centrifuge. Supernatant was removed and used for K ÷ measurements. All the measurements were performed

20 OBR K+ Efflux (mEq/L cells/h)

10

I -a- APH CI I -~ APH N

D

,~0 2~0 Min

Fig. 1. Time-course over 180 rain of OBR K* effiuxes (mEquiv . / l cells per h) in APH-treated erythrocytes in isotonic conditions (pH 7.40), in presence of CI (C1) or NO~ (N) anions. Flux measure- ments were calculated by normalizing the overall K ÷ ions lost at any time per min and per volume of packed cells and then multiplying for 60 min. The figure is representative of one of three similar

experiments.

on triplicate samples with an Eppendorf flame pho- tometer, using adequate standards according to pub- lished methods [13].

Since intracellular chloride is needed for the activity of K+/CI - cotransport [23], the K + effiux was meas- ured in the presence of either sodium chloride or sodium nitrate (NaNO 3) 140 mM in the efflux medium. The substitution of NO 3 for Cl - in effiux media does not allow the K + efflux mediated by K+/C1 - cotrans- port because NO~ quickly replaces (during the first minute of the flux measurement) intraceIlular C1 via the anion exchange. Therefore chloride-dependent K + effiux (as measure of the cotransport activity) was calculated by the difference of the values obtained in the both media (C1- effiux minus NO~- effiux).

When the effect of hyposmolarity was investigated, the effiux media were modified by reducing NaC1 (or NaNO 3) to 100 mM (220 mosM).

All experiments were performed by evaluating K + efflux at 20 min because the OBR oxidant-mediated K + efflux, previously evaluated over 180 min, had shown a linear increase within the first 20-25 min (Fig. 1), according to the reported kinetics of the K+/C1 - cotransport system [5].

In order to better define the relative contribution of K+/C1 - cotransport on OBR K ÷ efflux induced by oxidation, the specific inhibitor DIOA was added at concentration of 100 mM in effiux media. Similarly, in experiments designed to evaluate the relative contribu- tion of Ca2+-activated K + channel, the specific in- hibitor carbocyanine [24] was used at a concentration of 10 mM.

In order to investigate a possible involvement of thiol groups in modulating OBR K ÷ effiux, control and oxidants-treated cells were washed with CWS at 4°C

39

and reincubated for an additional 15 min at 37°C in isotonic choline chloride solution containing either dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), both at concentrations of 1 mM. After further washes with CWS at 4°C, cells were finally used for K ÷ effiux measurements , as described above.

Density of the red cell population was measured according to the method described by Danon and Marikovsky [25].

Glutathione (GSH) cell content was measured ac- cording to the published method [26].

R e s u l t s

The O B R K ÷ effiux induced by oxidants was largely dependent on the presence of C1- in the efflux media (Fig. 1). The kinetic curve of this OBR K ÷ effiux showed a fast linear increase and a subsequent plateau, suggesting the presence of a saturable specific carrier (Fig. 1). Oxidative t reatment induced a several-fold larger K ÷ effiux on OBR C1--dependent fraction in comparison to control erythrocytes in both isotonic and hypotonic conditions. This effect was different in rela- tion to the oxidant agents employed: A P H and MSB were more effective than PMS and H 2 0 2 (Fig. 2). Hyposmolari ty significantly stimulated the OBR C1-- dependent K ÷ efflux (Fig. 2), showing an increase of K ÷ effiux by at least 60%, as compared to isotonic conditions.

These results suggested an involvement of the K + / C I - cotransport; however, the mechanism trig- gered by oxidants could be mediated by cell swelling via a non-selective sodium and water passive leak and not by the direct interaction with the cotransporter. In order to exclude the former possibility, the density of

CI-dependent K+efflux (mEq/L cells/h)

14'

ai 1 APH 12'

m MSB

10' n PMS

8" a H20 2

6' t CONTROL

4"

2'

0" I 6,7 7 7,25 7,4 7,7 8

Fig. 3. pH dependency of oxidant-mediated Cl--dependent K ÷ ef- fluxes (mEquiv./I cells per h). The figure is representative of one of

five similar experiments.

cyte density were observed (data not shown), a role of cell swelling in increasing activity of the K+/C1 - co- transport was ruled out.

APH, MSB and H 2 0 2 slightly stimulated OBR Cl- -dependent K ÷ effiuxes at pH 7.00, as described for the K+/C1 - cotransport system [5-7]; however, the largest K ÷ effiux was observed at pH 7.70-8.00 (Fig. 3). For PMS-treated erythrocytes, no remarkable dif- ferences of K ÷ efflux were observed at different pH (Fig. 3).

Both in isotonic and hypotonic conditions D I O A strongly inhibited OBR a - - d e p e n d e n t K ÷ effiux in- duced by APH, MSB or H202 , but not by PMS (Fig. 4). Carbocyanine partially inhibited PMS-mediated K +

CI- dependent K+ E, fflux (mEq/I cell/h)

.i oi '°t I11 II ~1 Isotonic+ DIOA

1 II II D. o,onic 81 II I I • Hypotonic+DIOA

12

10 [ ] 6 Isotonic

[ ] Hypotonic 8 4

6

4

2" j 0-

i ! I

erythrocytes as an index of cell swelling was measured according to Danon and Marikovsky [25] in the pres- ence of either sodium or potassium chloride in oxida- tive solutions. As no significant variations in erythro-

El - dependent K+ Efflux (mEq/L cells/h)

i i

Control A P H M S B PMS H 2 0 2

Fig. 2. Cl--dependent K + effiuxes (mEquiv./l cells per h) induced by different oxidants in isotonic and hypotonic conditions (pH 7.40) (means :1: S.D., n = 5). * Significant difference (P < 0.05) between

isotonic and hypotonic values (paired t-test).

< ,-r

Fig. 4. Cl--dependent K + effiuxes (mEquiv./l cells per h) induced by different oxidants in isotonic (iso) and hypotonic (hypo) conditions

(pH 7.40), with or without 100 mM DIOA (means _+ S.D., n = 5).

40

15 O-dependent

K+ efflux [ ] without Cy (mEq/L cells/h)

I0 • with Cy

5

0

E E E E

u ~- m ~ o

< ~

Fig. 5. CI -dependent K + effiuxes (mEquiv./l cells per h) induced by different oxidants in hypotonic conditions (pH 7.40), with or

without 10 mM carbocyanine (Cy) (means +_ S.D., n = 5).

efflux in hyposmolarity, whereas it had no effect on the K + effluxes induced by the other oxidants (Fig. 5).

After the oxidative step, addition of SH donors (DTT and 2-ME) partially inhibited the OBR C1--de- pendent K + effluxes produced by APH and MSB, either in isotonic or hypotonic conditions. DTT, the dimeric form of 2-ME, showed an inhibition about 2-fold more powerful as compared to 2-ME. K + efflux produced by HeO2was inhibited by DTT but not by 2-ME. Neither DTY nor 2-ME remarkably affected the K + effiux in PMS-treated cells (Table I).

The reduction of cell GSH content was around 90-92% after APH, 80-84% after MSB and 20% after H202, in comparison with control erythrocytes (2.84 + 0.3 mmol /ml cells); cell GSH content was negligible (2-3% of control) after exposure to PMS.

Discussion

We investigated the possibility that exposure of nor- mal human erythrocytes to a mild oxidative stress, produced by different agents, enhanced the activation of the K+/C1 - cotransport system. Although all the

TABLE I

Effects of SH donors

Oxidant-mediated Cl--dependent OBR K + effiuxes (mEquiv./1 cells per h, means~g.D., n = 4) with or without addition of 1 mM DTT or 2-ME. Results .are reported for hypotonie conditions (220 mOsM) to shoW the effect on maximal stimulation of efflux.

Oxidant +DTT % inhibition +2-ME % inhibition

APH 10.6±2.9 3.7±0.7 65 7.6±0.8 28 MSB l l .7±2.6 3.2±0.3 72 7.9±0.6 32 PMS 4.4±0.9 3.8±0.8 15 4.4±0.4 0 H20 e 3.2±0.7 1.6±0.5 48 3.2±0.3 0

oxidants used induced a OBR K ~ loss, only APH. MSB and -to much less e x t e n t - H 2 0 2 promoted a K" pathway with the features of K+/C1 cotransport, which were: (i) a kinetics of effiux compatible with a specific cation transport system; (ii) requirement for chloride anion; (iii) resistance to ouabain, bumetanide and carbo- cyanine inhibition; (iv) stimulation by hypotonic challenge; (v) susceptibility to inhibition by D1OA.

The OBR K + effiux induced by PMS was very slightly or not at all affected by DIOA, in isotonic and hypotonic conditions, respectively; conversely, carbo- cyanine partially inhibited PMS-induced K + effiux. These results suggested that the mechanism involved in this case might be different from K+/C1 - cotransport.

As SH donors, DTT and 2-ME, decreased oxidant- mediated K+/CI - cotransport, oxidation probably af- fects SH groups crucial for the efficiency of the co- transporter. Interestingly, stoichiometric relationships with SH groups of the cotransporter (or with function- ally correlated structures) were suggested by an in- hibitory capacity ratio 1 : 2 for 2-ME to DTI'.

Several suggestions have been made in the literature on the role of thiols in the control of K+/CI - cotrans- port both in human and sheep red cells [2,8,27-31] and a theoretical model describing the interactions between divalent cations, protons and regulatory SH sites of transporter has been advanced by Lauf [29]. Lauf first indicated that biochemical perturbations of SH groups induced activation of the cotransport. The demonstra- tion of the role of sulfhydryl groups mainly lies in the stimulation of K+/C1 - cotransport following exposure to the thio-alkylating agent N-ethylmaleimide (NEM) [2,4,8]. Until now, oxidative mechanisms affecting thi- ols and K+/C1 - cotransport have been suggested for APH in human [16,19] and for diamide in sheep ery- throcytes [32].

Pretreatment with low concentrations of H 2 0 2 (315 IxM) has been also reported to increase NEM-activated K + effiux in human erythrocytes [33]. This would imply an independent activating effect of H 202 on the trans- porter. Our results confirm and extend all these find- ings.

An increase of K + effiux proportional to the de- crease of GSH cell content has been described after diamide [32], suggesting a cause-effect relationship be- tween the two phenomena. However, in our results, the near complete disappearance of cell GSH following PMS exposure was not associated with activation of K+/CI - cotransport. On the other hand, a rough rela- tionship was observed between GSH decrease and stimulation of cotransport activity by the other oxi- dants, as GSH content was slightly affected by H 2 0 : and strongly reduced by APH and MSB. Therefore,

oxidative agents seem to affect the K ÷ membrane permeability and specifically the K+/C1 - cotransport in different ways, which are not merely related to the specific redox capacity of the single compound.

It is well known that cytoplasmic acidification in- duces cell swelling as a consequence of ions and water entry due to Donnan effect; on the contrary, alkaline pH was described as inhibiting cotransport in normal, not oxidized, cells [3]. However, a loss of inhibition (or a true stimulation) of cotransport induced by alkalin- ization was observed in SS, CC and AA least dense cells after NEM treatment or removal of internal Mg 2÷ [34], or also after isoosmotic cell swelling by the nys- tatin procedure [3]. In our experiments two different peaks of OBR K ÷ loss were observed at acidic and alkaline pH, but exposure to pH 7.70-8.00 resulted in a stronger activation of K÷/C1 - cotransport than at acidic pH. As Mg 2÷ was added in our incubation media, lack of intracellular Mg 2+ had to be ruled out and a thiol-dependent mechanism similar to that in- duced by NEM appeared more plausible.

Two factors have been demonstrated to play an important role in determining the magnitude of the K+/C1 - cotransport activation: cell age [3,11,12] and presence of /36 or /37 positively charged mutations on haemoglobin molecule [13].

In a recent work by Fabry et al. [35], expression of cotransport activity was demonstrated to be heteroge- neously distributed in SS cell population and the ma- jority of cells (80%) exhibiting cotransport, although young, were not reticulocytes. The Authors established that at least 21% of all reticulocytes did not show cotransport activity, while other less young ceils did. Therefore, in spite of the presence of the same Hb mutation, differences in cotransport expression cannot be explained only by cell age; additional factor(s) has (have) to play some further role in producing heteroge- neous expression of the transport system.

In a recent paper, erythrocytes from /3 thalassemia intermedia have been shown to possess an increased K÷/C1 - cotransport activity (independently of reticu- locyte count) in comparison with normal AA cells [13]. In this situation, a role of oxidative events might be likely, because neither cell age nor the presence of /36 or /37 haemoglobin mutations were involved. Although further works are needed to better define the role of K÷/C1 - cotransport in the pathophysiology of tha- lassemia, the present data may offer an interpretative key to that result.

Oxidation may represent a third factor capable of modulating activation of K+/CI - cotransport and con- tributing to its heterogeneous expression in situations such as sickle cell anaemia or /3 thalassemia.

Finally, the present results suggest the application of APH or MSB as a new and simple model to study K+/C1 - cotransport activation in human erythrocytes;

41

in comparison with NEM treatment, it is less drastic, probably closer to the erythrocyte pathophysiology and gives the advantage of the reversibility of effect.

Acknowledgements

This work was supported by grants from National Research Council No. 89.02512.04, Ministry of Univer- sity and of Scientific and Technologic Research 60%, and from the Health Assessorship of the Venetian Region.

References

1 Kregenow, F.M. (1971) J. Gen. Physiol. 58, 372-395. 2 Lauf, P.K., Adragna, N.C. and Garay, R.P. (1984) Am. J. Physiol.

246, C385-C390. 3 Brugnara, C. and Tosteson, D.C. (1987) Am. J. Physiol. 252,

C269-C276. 4 Dunham, P.B. and Ellory, J.C.(1981) J. Physiol. 318, 511-530. 5 Brugnara, C., Kopin, A.S., Bunn, H.F. and Tosteson, D.C. (1985)

J. Clin. Invest. 75, 1608-1617. 6 Canessa, M., Spalvins, A. and Nagel, R.L. (1986) FEBS Lett. 200,

197-202. 7 Brugnara, C., Bunn, H.F. and Tosteson, D.C. (1986) Science 232,

388-390. 8 Lauf, P.K. and Theg, B.E. (1980) Biochem. Biophys. Res. Com-

mun. 92, 1422-1428. 9 Garay, R.P., Nazaret, C., Hannaert, P.A. and Cragoe, Jr., E.J.

(1988) Mol. Pharmacol. 33, 696-701. 10 Vitoux, D., Olivieri, O., Garay, R.P., Cragoe, Jr., E.J., Galac-

teros, F. and Beuzard, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 4273-4276.

11 Hall, A.C. and Ellory, J.C. (1986) Biochim. Biophys. Acta 858, 317-320.

12 Canessa, M., Fabry, M.E., Blumenfeld, N. and Nagel, R.L. (1987) J. Membr. Biol. 97, 97-105.

13 Olivieri, O., Vitoux, D., Galacteros, F., Bachir, D., Blouquit, Y., Beuzard, Y. and Brugnara, C. (1992) Blood 79, 1-5.

14 Hebbel, R.P., Eaton, J.W., Balasingam, M. and Steinberg, M.H. (1982) J. Clin. Invest. 70, 1253-1259.

15 Rachmilewitz, E.A., Lubin, B.H. and Shohet, S.B. (1976) Blood 47, 495-505.

16 Orringer, E.P. and Parker, J.C. (1977) Blood 50, 1013-1021. 17 Snyder, L.M., Sauberman, N., Condara, H., Dolan, J., Jacobs, J.,

Szymanski, I. and Fortier, N.L. (1981) Br. J. Haematol. 48, 435-444.

18 Maridonneau, I., Braquet, P. and Garay, P. (1983) J. Biol. Chem. 3107-3113.

19 Orringer, E.P. (1984) Am. J. Hematol. 16, 355-366. 20 Van der Zee, J., Dubbelman, T.M.A.R. and Van Steveninck, J.

(1985) Biochim. Biophys. Acta 818, 38-44. 21 Deuticke, B., Heller, K.B. and Haest, C.W.M. (1986) Biochim.

Biophys Acta 854, 169-183. 22 Claster, S., Quintanilha, A., Schott, M.A., Chiu, D. and Lubin, B.

(1987) J. Lab. Clin. Med. 109, 201-210. 23 Brugnara, C., Van Ha, T. and Tosteson, D.C. (1989) Am. J.

Physiol. 256, C994-C1003. 24 Simons, T.J.B. (1976) Nature (London) 264, 467-469. 25 Danon, D. and Marikovsky, Y. (1964) J. Lab. Clin. Med. 64,

668-674. 26 Beutler, E. (1984) in Red Cell Metabolism: A Manual of Bio-

chemical Methods (Beutler, E., ed.), Grune and Stratton, New York.

42

27 Lauf, P.K. (1983) J. Membr. Biol. 73, 237-246. 28 Lauf, P.K. (1984) J. Membr. Biol. 82, 167-178. 29 Lauf, P.K. (1985) J. Membr. Biol. 88, 1-13. 30 Lauf, P.K. (1985) Mol. Physiol. 8, 215-234. 31 Ryu, K.H. and Lauf, P.K. (1990) Mol. Cell. Biochem. 99, 135-140. 32 Lauf, P.K. (1988) J. Membr. Biol. 101, 179-188.

33 Sheerin, H.E., Snyder, L.M. and Fairbanks, G. (1989) Biochim Biophys. Acta 983, 65-76.

34 Brugnara, C. and Tosteson, D.C. (1987) Blood 70, 1810-1815. 35 Fabry, M.E., Romero, J.R., Buchanan, I.D., Suzuka, S.M., Stam-

atoyannopoulos, G., Nagel, R.L. and Canessa, M. (1091) Blood 78, 217-225.