J. Phyeiol. (1978), 276, pp. 501-513 With 9 text-figure8 Printed in Great Britain
501
ANION TRANSPORT OF THE RED CELL UNDER NON-EQUILIBRIUM CONDITIONS
BY G. ORMOS AND S. MANYAI From the Department of Biochemistry, National Institute of Occupational Health, 1450 Budapest, Hungary
(Received 15 Mlarch 1977) SUMMARY
1. The exchange of sulphate for chloride across the human red cell membrane was measured in both directions, i.e. by sulphate influx and sulphate efflux. The influence of the concomitant transient pH changes was minimized by phosphate buffer and by choosing experimental conditions of moderate pH sensitivity (pH 6-4 and 7.8). Sulphate self exchange was determined in chloride-free erythrocyte suspensions. 2. The transport of external sulphate into red cells proceeded at a 15-fold greater rate if its initial concentration was raised from 5 to 95 mm. In contrast the velocity constant of sulphate efflux into sodium chloride medium increased only twofold when the intracellular sulphate concentration was increased. To explain this asymmetry it is proposed that external sulphate ions are more able to compete with chloride for the anion transport sites than those present in the cell interior. 3. The transient membrane potential due to the uneven distribution of sulphate and chloride was shown by the rapid introduction of chromate into the cells. When the erythrocytes contained chloride and the external anion was sulphate, the cells took up chromate 50 times (16.5 'C) or 100 times (3 TC) faster than with equilibrium chloride distribution. 4. Chloride efflux into sodium sulphate media was measured by a chloride-sensitive electrode. Under buffered conditions in neutral and alkaline media two kinetic components were observed as the result of chloride exchange against hydroxyl and sulphate ions. At pH 6-4, chloride efflux was characterized by a single velocity constant identical to that of sulphate movement in the opposite direction. The results show that under appropriate circumstances net chloride efflux measurements can provide comparative data on the anion permeability of the red cell membrane. INTRODUCTION
The exchange between chloride and bicarbonate ions through the red cell membrane is one of several successive processes involved in the removal of carbon dioxide. Its physiological significance prompted the study of the anion transport mechanism. Early techniques were based on the measurement of net concentration changes during exchange of different anion species (Dirken & Mook, 1931; Luckner, 1948; Schweitzer & Passow, 1953). The availability of radioisotopes was soon followed by diffusion rate measurements under conditions of equilibrium (Hahn & Hevesy,
502 0. ORMOS AND S. MANYAI 1942). Although the equilibrium exchange offers advantages by avoiding transient changes during the diffusion process (Passow, 1969; Gardos, Hoffman & Passow, 1969) it seems worthwhile to study the non-equilibrium exchange as well. It would be interesting to know whether the action of an anion on anion exchange requires interactions at both surfaces of the erythrocyte membrane, at either surface, or predominantly at one or other side of the membrane. Such investigations demand non-equilibrium distribution and the measurement of the net anion exchange. A further reason to study the non-equilibrium exchange is that it offers a simple way to observe the diffusion continuously with ion-sensitive electrodes. The purpose of our work was (1) to find reliable experimental conditions for net sulphate-chloride exchange measurement, (2) to study the anion permeability with non-equilibrium anion distribution and (3) to use the transient potential for rapid labelling of red cells with [51Cr]chromate. METHODS Blood stored in acid-citrate-dextrose solution for 1-7 days was washed 3 times and then incubated at 25 0C, 10 % haematocrit for 3 hr to establish equilibrium with the washing solution and obtain desired intracellular anion composition. The pH was set and controlled during this procedure by additions of 0-1 N hydrochloric acid and 0-1 N sodium hydroxide. These pH values are indicated on the Figures. The solutions for washing and pre-equilibrating were generally isotonic and contained sodium chloride, sodium sulphate and sodium phosphate buffer. In those cases when intracellular sulphate level was raised over 100 mm, cells were first suspended in a hypertonic solution containing 150 mm-sodium chloride and 150 mm-ammonium chloride. Ammonium ions pass the erythrocyte membrane by a special transport mechanism which is accompanied by charge-transferring chloride diffusion (Hunter, 1968). Next the intracellular chloride was replaced with sulphate by washing and equilibrating the cells with solutions containing sodium sulphate and ammonium sulphate. Care was taken that the ammonium concentration in the later steps was equal to that in the preceding chloride-loading solution. Changes in the intracellular chloride concentration were measured by analysis of haemolysates. Intracellular sulphate concentration was estimated by the distribution of 35SO4 between cells and medium. The cells with enhanced sulphate content were used for sulphate efflux measurements. In the pre-equilibrated cells sulphate transport was determined in inward and outward directions. When sulphate exchange for intracellular chloride was determined, pre-treated cells were again washed, this time with the incubating medium, to obtain the proper anion composition outside the cells. This step was done at 0 'C to prevent further exchange before the diffusion measurement. Determination of anion transport. Sulphate influx was started by resuspending cold cells in prewarmed medium containing 1-5 #sc 35SO4/ml. Samples of 1 ml. cell suspension were put on cooled (5 0C) Sephadex G-75 columns and separated as described by Till, Koehler & Loesche (1972). The effluent cells were collected for 35S activity determinations. Chromate influx was measured at an initial concentration of 0-05 mm. Samples of 0-5 ml. were added to 2 ml. ice-cold medium, centrifuged and aliquots of the supernatant were taken for 51Cr measurements. To determine sulphate efflux into sodium chloride media pre-equilibrated cells were first loaded with 35SO4. For this purpose erythrocytes were incubated at 50 % haematocrit in the original pre-equilibrating solution which contained 5S04 as well. The activity that remained outside at the end of the loading was removed by washing at 0 'C with the medium of the transport experiment. During the back exchange of ""SO, samples were processed as described for chromate uptake. If not stated otherwise, the indicated results are averages of triplicate measurements. Changes of water content and pH during sulphate-chloride exchange. The water content of the cells was measured before and after exchange of external sulphate for internal chloride. Slight shrinkage was observed but it never exceeded 5 %. These small differences were neglected and
ANION TRANSPORT OF THE RED CELL
503
the cell suspensions were characterized by the haematocrit values measured before the exchange. In unbuffered suspensions the medium became extensively acidified during exchange of external sulphate for internal chloride (Fig. 1). As sulphate transport has a strong pH-dependence (Passow, 1969; Schnell, 1972) addition of buffer was necessary. The incubating media contained small concentrations of buffer (generally 6-25 mM-NaPO4) to reduce the influence of a third anion. Mi8ceUaneou8. Chloride concentration of 20-fold diluted haemolysates and the efflux of chloride from cells was measured by chloride-sensitive electrode (Radelkis OP-Cl-711) connected to a voltmeter (Radelkis OP-203) and a recorder (Weinert eKN). pH was measured by combined glass electrode (Radelkis OP-8071-1/A). 85S activity was determined by an anticoincidence G.M. detector (Gamma NZ-305) and 51Cr was measured by means of an automatic gamma-counter (Gamma NZ-310).
I760 _
6-0
~~
~
5 0_
~
5*0
0
2
4
6
Time (min)
Fig. 1. Changes of the pH in the sodium sulphate medium after suspending red cells pre-equilibrated with sodium chloride. Temperature 25 'C; haematocrit 4-6 %; initial pH 7*4. Composition of medium: *, 100 mm sodium sulphate; 0, 95 mM sodium sulphate and 6-25 mm sodium phosphate buffer; A, 90 mmi sodium sulphate and 12 5 mm sodium phosphate buffer; A, 80 mm sodium sulphate and 25 mm sodium phosphate buffer.
[86S]sodium sulphate and [51Cr]sodium chromate were obtained from the Hungarian Isotope Institute (Budapest). SITS (4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid) was purchased from B.D.H. (Poole, England). Other chemicals were of reagent grade. Diffusion velocity constant, k, was derived from the initial rate of concentration change according to the equation AC/C = -k. At; and from the semilogarithmic plot of the fraction of the exchangeable amount of the anion, (a,0 - at)/aa, where a was determined by specific radioactivity or chloride concentration measurements. This representation gave straight lines when one type of anion exchange occurred whether it was a self exchange or a net one. The k values are considered suitable to compare the results obtained under the selected experimental conditions only, and not to calculate exact permeability constants.
0. ORMOS AND S. MANYAI
504
RESULTS
Inward and outward sulphate movement during sulphate-chloride exchange The efflux and influx of sulphate in exchange for chloride, at pH 6-4, are shown in Figs. 2 and 3 respectively. The sulphate diffusion rate was similar in the two directions at an initial concentration of 5 M (kout 0-0057 min-1, S.D. 000045; 1 0
08.
Time (min)
Fig. 2. Sulphate efflux from red cells into different media at pH 6-4. Temperature 20 0C; 6X25 mm phosphate buffer; haematocrit 82 %. Initial concentrations: *, inside: 4*8 mm sodium sulphate and 136 mm sodium chloride; outside: 143 mm sodium chloride; 0O inside: 95 mm sodium sulphate and 6 mm sodium chloride; outside: 143 mm sodium chloride; A, inside: 95 mm sodium sulphate and 6 mm sodium chloride; outside: 95 mM sodium sulphate. 1.0 8 -
8 1-
180 Time (min)
Fig. 3. Sulphate influx at pH 6-4 into red cells of different anion composition. Temperature 20 0C; 6-25 mm phosphate buffer; haematocrit 85 %. Internal anion composition was adjusted by pre-equilibrating the cells in solutions that beside phosphate contained: 0, 0, 143 mm sodium chloride; A, 95 mm sodium sulphate; A, 47.5 mM sodium sulphate and 71 mm sodium chloride. Composition of the extracellular medium: 0, 4-8 mmi sodium sulphate and 143 mm sodium chloride; 0, A, A, 95 mm sodium
sulphate.
ANION TRANSPORT OF THE RED CELL
F
002
I
0
a
I m.
a
N
I
100
50 Pre-equilibrating sulphate (mM)
Fig. 4. Efflux rate of sulphate from cells into sodium chloride medium at pH 7'8. Temperature 25 0C; 6-25 mM phosphate buffer; haematocrit 85%%. Velocity constants determined: *, from initial rate; 0, from semilogarithmic diagram.
0
006
I-
0*03
F
I
0
Fig.
50 Sulphate (mM)
I
iI 100
Dependence of the inward diffusion rate of sulphate on its initial concentration at pH 7-8. For experimental conditions and meaning of symbols see Fig. 4.
5.
505
G. ORMOS AND S. MANYAI kin = 0 0053 min-, S.D. 0.00030). Approximately a twofold increase of sulphate efflux rate was observed upon the elevation of the internal sulphate concentration to 95 mM (k0ut = 0-0096 min-, S.D. 0.00041). Sulphate self exchange proceeded at a similar rate (measured in both directions: kout= 00116 min-1, S.D. 0.00092; kin = 0-0102 min', S.D. 0.001 1). However, increasing the external sulphate concentration to 95 mm produced an asymmetrical, 15-fold increase of the velocity
506
0-40 r
x
v 0 20
0
0n
um
0
20
40
Time (min) Fig. 6. S04 efflux into sodium chloride medium at different intracellular sulphate level. Temperature 25 'C; 6-25 mm phosphate buffer pH 6-4; haematocrit, 8.9 %. Intracellular anion concentrations at the beginning of the diffusion measurement: *, 5-3 mm sulphate and 150 mm chloride; 0 91 mm sulphate and 11.8 mm chloride; A, 137 mm sulphate and 13-6 mm chloride. Ordinate: 35SO4 exit, fraction of the
equilibrium value.
constant of sulphate influx (kin = 00805 min-, S.D. 0.0065). These diffusion processes resulted in straight lines in the semilogarithmic representation. Only one experimental result deviated from linearity. In this case sulphate was the only external anion and the interior of the cells contained chloride and sulphate in comparable amounts. The exchange of chloride and sulphate was also measured at pH 7-8 at different initial sulphate concentrations (Figs. 4 and 5). In accordance with the findings at
ANION TRANSPORT OF THE RED CELL 507 pH 6-4 the elevation of intracellular sulphate produced a twofold increase of ko0t and the replacement of extracellular chloride by sulphate increased kin 15-fold. It should be noted that the concentrations of the pre-equilibrating solutions are indicated on Fig. 4. The actual intracellular sulphate concentration is smaller since at this pH the anion distribution ratio between the cells and suspending medium is less than unity (Funder & Wieth, 1966; Dalmark, 1975). Rate constants derived from both initial velocity and semilogarithmic representation were calculated to ascertain whether their values parallel the decrease of the initial sulphate concentration. Comparison of the data on Figs. 4 and 5 shows that 1.0
8
,
8
a
0.1 0
2
4
6
Time (min)
Fig. 7. Cl efflux into sodium sulphate media at pH 7-4. Temperature 30 'C; haematocrit 4*6 %. A, chloride efflux into a medium which contained 6-25 mm sodium phosphate buffer and 95 mm sodium sulphate; 0, chloride efflux, 0O sulphate influx, medium: 100 mm sodium sulphate. For chloride concentration calculation distinct points of the continuously monitored electrode-potential curves were taken.
it was the initial velocity that provided somewhat lower values. Similar findings were reported for anion transport under equilibrium conditions (Zaki, Fasold, Schumann & Passow, 1975), thus the deviations may rather reflect the time for perfect mixing and temperature equilibration than be a consequence of the non-
equilibrium exchange. The intracellular sulphate concentration was raised over 100 mm by means of ammonium chloride and ammonium sulphate (see Methods section). The results shown on Fig. 6 indicate that the elevation of sulphate concentration from 91 to 137 mm increased the velocity constant very little if at all. Efflux of chloride from red cells into sodium sulphate medium Fig. 7 shows the kinetics of the extracellular appearance of chloride at pH 7*4 determined by chloride-sensitive electrode. The diffusion of chloride depended
G. ORMOS AND S. MAN YAI 508 on the presence of buffer. A substantial part of chloride appeared at an enhanced rate when 6-25 mm phosphate buffer was added. In some experiments bicarbonate was excluded by using carbon-dioxide-free solutions and working under argon but the rapid efflux component was not diminished by this. When buffer was omitted chloride efflux followed first-order kinetics and its velocity constant agreed with that of sulphate transport in the opposite direction. It should be noted, however, that in the unbuffered medium the pH drops and therefore is quite different from that before the experiment. 10
8
T
Q
G
0.1 I
0
.
I
I
20
10
.
30
Time (min)
Fig. 8. Cl efflux into sodium sulphate medium at pH 6-4 and pH 7*0 and its inhibition by SITS. Temperature 26 0C; haematocrit 4.5 %; medium 95 mm sodium sulphate and 6-25 mm sodium phosphate. *, pH 6-4; 0, pH 7 0. Preincubation with SITS at 0 0C; pH 6-4 for 60 min: /, 0-25 ,umole/ml. cells; *, 2-5 /tmole/ml. cells.
The chloride efflux at pH 7-0 and at pH 6-4 in the presence of 6-25 mm phosphate buffer is shown on Fig. 8. The initial diffusion component was reduced at pH 7 0 and it disappeared at pH 6-4; thus chloride efflux could be characterized by a single velocity constant. At pH 6-4 0-25 molee SITS/ml. cells inhibited the chloride efflux by 92 %. Even a tenfold increase in the concentration of the reagent was unable to raise the level of inhibition. Effect on chromate influx of non-equilibrium distribution of anions The uptake of chromate was determined at chloride equilibrium and when sulphate was substituted for external chloride. Fig. 9 shows the results obtained at different temperatures. In parallel experiments sulphate influx was also determined. The velocity constant of sulphate transport together with the values obtained for chromate from the linear phases of the diagrams of Fig. 9 are listed in Table 1. The results show that at 30 00 sulphate influx was much faster than chromate diffusion whereas the situation was inverse at 16-5 and at 3 0C. Accordingly the original anion distribution changed significantly during the chromate uptake at high temperatures
ANION TRANSPORT OF THE RED CELL 100 80
0
Time (min) 60 0
30
30
60
60
120
509
60 40
20
.-o
0,0 0-0 0) .Y)
100 80 60
E
40
1655C
-
20
10 8 6 4
0
5
15 0 Time (min) Fig. 9. Comparison of the chromate uptake rate under 10
chloride/chloride and sulphate/ chloride distribution conditions at different temperatures. Haematocrit 20 %; pH 7-4; initial extracellular chromate concentration 0 050 mM. Cells were pre-equilibrated with 143 mm sodium chloride and 6-25 mm sodium phosphate. Media: 0O 143 mM sodium chloride and 6-25 mm sodium phosphate; *, 95 mm sodium sulphate and 6 25 mm sodium phosphate. TABLE 1. Comparison of chromate and sulphate transport into red cells at different temperatures Ion distribution between medium and cells n
eA
S04/Cl Temperature kso4*
(OC) 37 30 16-5 3
(min-') 0-257 0 014 0 00070
I
Cl/Cl kCrO*
kCrOn,j
(min-')
(min') 0-026 0-018 0 55 0-026
* Averages of duplicates. For details of the experimental conditions
0 025 0-017 0-0041 0 00049 see
Fig. 9.
510 5. ORMOS AND S. MA4NYAI and it was preserved at low temperatures. In line with this result at 30 and 37 TC only the initial section of chromate uptake was accelerated by the uneven anion distribution whereas at 16-5 and at 3 'C the uneven anion distribution greatly increased the entire process of chromate diffusion. It was attempted to remove the 51Cr-activity introduced into the cells at enhanced rate at 3 'C. 51Cr-tagged cells were suspended in 20 volumes of different washing solutions for 60 min. Washing at 0, 22, and 30 'C with isotonic saline resulted in 3-2, 3.9, and 4*1 % removal, whereas sodium sulphate medium produced 1 6, 2-4, and 4*1 % extracellular appearance of the incorporated 5lCr-activity. DISCUSSION
Characteristics of non-equilibrium sulphate transport The replacement of chloride by sulphate produced a large, asymmetric increase in the velocity constant of the transport of external sulphate into red cells compared to sulphate efflux. To interpret this finding we have to analyse first the possible experimental errors then to examine its relationship with the anion transport mechanism. Intracellular anion composition altered rapidly during anion translocations. This raised the possibility that an initial high velocity constant might have decreased during transport. However, the initial rate generally provided somewhat lower values for k than the long term exponential relationship. A rapid decrease of the initial sulphate level below a threshold concentration might also have produced asymmetric results. To examine this, sulphate efflux was measured also at 137 mm initial concentration. In this case more than 30 % of sulphate efflux occurred at sulphate concentrations above 95 mm without significant increase of Iout, so the differences between the two transport directions cannot be explained in this way. Transient pH changes occurred during sulphate movement due to the uneven anion distribution. The mobility of sulphate in the membrane is much less than that of chloride. Therefore, as an approximation, our selected experimental pH values were based on the pH sensitivity of sulphate self exchange. This is a bell-shaped curve with its maximum at pH 6-4 and flattening at more distant pH values (Schnell, 1972). Experiments were performed at pH 7-8 (limited pH sensitivity) and at pH 6-4. At the latter value any change could at most restrain the transport and so it could act against the increase of the velocity constant only. The asymmetry was observed at both of these pH values, suggesting its independence of the dissociation status of the intramembrane sites. Anion transport through the red cell membrane is strongly dependent on other anions present (Deuticke, 1967; Wieth, 1970). Schnell, Gerhardt & SchdppeFredenburg (1977) related the large increase of sulphate flux at increasing sulphate concentration to the release from a competitive inhibitory effect of chloride. The present findings suggest that external sulphate ions can more effectively compete for the anion transport sites than those present in the cell interior. There are also reports on the asymmetric inhibition of anion transport by some non-penetrating probe molecules from the external membrane side (Lepke & Passow, 1973; Schnell, Gerhardt, Lepke & Passow, 1973; Zaki et al. 1975). It is remarkable that an anion
ANION TRANSPORT OF THE RED CELL 511 for which the membrane is highly permeable also interacts with the transport sites asymmetrically. The unidirectional sulphate flux is the sum of different transport components. The effect of the membrane potential on them is not the same. The diffusion potential is determined by the permeability constants and the concentrations of ions on the two sides of the membrane (Goldman, 1943). In saline-washed red cells this potential is essentially equal to the chloride equilibrium potential. Sufficient changes in the concentrations and in the permeability of the individual ions, however, may alter the magnitude and the sign of the membrane potential. Changes due to increased cation permeability have been shown by micro-electrodes (Lassen, Pape & VestergaardBogind, 1976) and by fluorescent probes (Hoffman & Laris, 1974) and have been applied to load red cells with cations (Sarkadi, Szasz & Gardos, 1976). The potential is also influenced when the concentrations of permeating anions are altered on either side of the membrane. Since the exchange diffusion as defined by Ussing (1952) is electrically silent, the permeability constants of the chargetransferring diffusions are valid (Glynn & Warner, 1972). Accordingly, in a red cell suspension with unequally distributed chloride and sulphate the potential is maintained by that fraction of these anions that travel through ionic diffusion routes. Comparing the ionic movement and exchange rates of chloride (Hunter, 1971) and sulphate (Passow, 1974 a, b) one may assume that the ionic diffusions differ by a factor of 102-103 in favour of chloride. Therefore, the membrane potential is determined primarily by the uneven chloride distribution. The internal membrane side becomes positive when chloride, the anion of greater mobility, is in the cell interior. Chromate influx has been shown insensitive to the anion composition of the medium (Ormos & Manyai, 1974), hence changes in its uptake rate can be attributed to the alterations of the membrane potential. The enhanced diffusion rate of chromate when extracellular chloride is replaced by sulphate provides evidence that the membrane potential does change in the manner expected (inside more positive). In contrast to the electrically silent exchange the charge-transferring sulphate diffusion would be influenced by the membrane potential and it would contribute to the increase in the unidirectional flux. As the ionic sulphate diffusion is a small fraction of the total flux (Passow, 1974b) its contribution may be of minor importance. Although the main form of anion transport is generally attributed to potentialindependent exchange of anion-carrier complexes, it has frequently been put forward that even this main route may be under the influence of the membrane potential (Klocke, 1976; Passow, 1969, 1974b; Wieth, 1970). The results of our experiments do not exclude this possibility yet they do not offer a definitive answer to this question.
Chloride efflux into sodium sulphate medium Chloride efflux into media containing sodium sulphate and phosphate buffer consisted of two kinetic components at neutral and alkaline pH. The rapid component disappeared by lowering the pH or by omitting the buffer. Chloride self exchange is much faster than sulphate transport: therefore, at disequilibrium, chloride is likely to exchange with rapid penetrants if they are present. The rapid outflow of a portion of chloride may be the result of hydroxide influx/ chloride efflux. This supposition is consistent with the pH changes found as they also
512 5. ORMOS AND S. MANYAI involve the movement of hydroxyl ions as suggested by Scarpa, Cecchetto, & Azzone
(1970).
In the absence of chloride-hydroxyl exchange the velocity constants of chloride outflow and sulphate influx agreed. SITS, a specific inhibitor of anion transport (Knauf & Rothstein, 1971) inhibited this chloride movement. These observations support that the bulk of chloride efflux was a stoichiometric exchange against sulphate. There are reports on the determination of anion transport by the exchange of chloride against acetate, nitrate and halides (Gunn, Wieth & Tosteson, 1975), pyruvate (Motais, & Cousin, 1976) and bicarbonate (Klocke, 1976). The rate of exchange against monovalent inorganic anions is however too fast for convenient measurement at physiological temperatures whereas in the case of organic anions the contribution of non-ionic diffusion (Deuticke, 1973) may interfere with the exchange process. Measurements of sulphate-chloride exchange are not subject to these problems.
Rapid uptake of chromate at asymmetric anion distribution When sulphate was substituted for external chloride, chromate uptake proceeded 50 times (16.5 0C) or 100 times (3 0C) faster than in sulphate-free control. To obtain enhanced uptake rate it is important to keep the sulphate influx/ chloride efflux slower than chromate movement, otherwise the transient potential generated would act for only part of the period of chromate diffusion. Lowering the temperature has been shown to shift the relative transport velocities in favour of chromate diffusion as its activation energy is a third only of that of sulphate, 10 kcal/mole (Ormos & Manyai, 1974). The transient membrane potential could, accordingly, be applied to introduce chromate into the erythrocytes in a short time at low temperatures. 0 The skilful technical assistance of Miss Katalin Sarai is gratefully acknowledged. This work was supported in part by the Scientific Research Council, Ministry of Health, Hungary, no.
6-11-0401-03 1/MU. REFERENCES DAIMAEX, M. (1975). Chloride and water distribution in human red cells. J. Physiol. 250, 65-84. DEUTCKE, B. (1967). tVber die Kinetik der Phosphat-Permeation in den Menschen-Erythrocyten bei Variation von extracellularer Phosphat-Konzentration, Anionen-Milieu und Zell-Volumen. Pfliugers Arch. ges. Physiol. 2%, 21-38. DEuCKE, B. (1973). Transfer of monovalent organic anions across the red cell membrane: mechanism and experimental alterations. Erythrocytes, Thrombocytes, Leukocytes, ed. GERLACH, E., Mos8E, K., DEUTrscH, E. & WILMANNs, W., pp. 81-87. Stuttgart: Thieme. Dn&=q, M. N. J. & MooK, H. W. (1931). The rate of gas exchange between blood cells and serum. J. Physwol. 73, 349-360. FuNDER, J. & WIETH, J. 0. (1966). Chloride and hydrogen ion distribution between human red cells and plasma. Acta physiol. scand. 68, 234-245. GARDOS, G., HOFFMAN, J. F. & PAssow, H. (1969). Flux measurements in erythrocytes. Labora. story Techniques in Membrane Biophysics, ed. PASSOW, H. & STAMPFIJ, R., pp. 9-20. New York:
Springer. GLYNN, I. M. & WARNER, A. E. (1972). Nature of the calcium dependent potassium leak induced by (+ )-propranolol and its possible relevance to the drug's antiarrhythmic effect. Br. J. Pharmac. 44, 271-278.
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GoLDMAN, D. E. (1943). Potential, impedance and rectification in membranes. J. gen. Physiol. 27, 37-60. GuNN, R. B., WrETH, J. 0. & TosTEsoN, D. C. (1975). Some effects of low pH on chloride exchange in human red blood cells. J. gen. Physiol. 65, 731-749. HAE, L. & HEVESY, G. (1942). Rate of penetration of ions into erythrocytes. Acta phy8iol. Band. 3, 193-223. HOFFMAN, J. F. & LARIs, P. C. (1974). Determination of membrane potentials in human and Amphiuma red blood cells by means of a fluorescent probe. J. Physiol. 239, 519-552. HuINTEB, F. R. (1968). Kinetic analysis of the permeability of human erythrocytes to NH4CL. J. gen. Phy8iol. 51, 579-587. HUNTER, M. J. (1971). A quantitative estimate of the non-exchange restricted chloride permeability of the human red cell. J. Phyaiol. 218, 49-50P. KrLocER, R. A. (1976). Rate of bicarbonate-chloride exchange in human red cells at 37 'C. J. appt. Phy-&iol. 40, 707-714. KNAU-F, P. A. & ROTHsTsaN, A. (1971). Chemical modification of membranes. I. Effect of sulfhydril and amino-reactive reagents on anion and cation permeability of the human red blood cell. J. gen. Physiol. 58, 190-210. LAssEN, U. V., PAPE, L. & VESTERGAARD-BOGIND, B. (1976). Effect of calcium on the membrane potential of Amphiuma red cells. J. Membrane Biol. 26, 51-70. LEPKE, S. & PAssow, H. (1973). Asymmetric inhibition by phlorizin of sulphate movements across the red blood cell membrane. Biochim. biophy8. Acta 298, 529-533. LucKxNE, H. (1948). Die Temperaturabhangigkeit des Anionaustausches roter Blutkorperchen. Pfliuger8 Arch. gem. Physiol. 250, 303-311. MOTms, R. & CousIN, J. L. (1976). The inhibitoreffect ofprobenecid and structuralanalogues on organic anions and chloride permeabilities in ox erythrocytes. Biochim. biophy8. Acta 419,309-313. ORMos, G. & MAw&i, S. (1974). Chromate uptake by human red blood cells: comparison of permeability for different divalent anions. Acta biochim. biophy8. hung. 9, 197-207. PAssow, H. (1969). Passive ion permeability of erythrocyte membrane. Prog. biophy8. moles. Biol. 19, 423-467. PAssow, H. (1974a). Anion permeability of the red blood cell. Abdtr. Commun. 9th Meet. Fed Europ. Biochem. Soc., p. 205. Budapest: Hungarian Biochemical Society. PAssow, H. (1974b). Ionic diffusion versus exchange diffusion in the sulfate permeability of the human red cell. Proc. XXVI Int. Congr. Phyaiol. Sci., vol. xi, p. 35. New Delhi: Congress Organizations. SARKADI, B., SzAsz, I. & GARDos,SG. (1976). The use of ionophores for rapid loading of human red cells with radioactive cations for cation-pump studies. J. Membrane Biol. 26, 357-370. SCARPA, A., CEccHETmo, A. & AzzoxE, G. F. (1970). The mechanism of anion translocation and pH equilibration in erythrocytes. Biochim. biophy8. Acta 219, 179-188. ScHEELL, K. F. (1972). On the mechanism of inhibition of the sulfate transfer across the human erythrocyte membrane. Biochim. biophy8. Acta 282, 265-276. ScmHOLL, K. F., GERHARDT, S., LEPKE, S. & PAssow, H. (1973). Asymmetric inhibition by phlorizin of halide movements across the red blood cell membrane. Biochim. biophy8. Acta 318, 474-477. SCHNELL, K. F., GERHARDT, S. & SCHOPPE-FREDENBURG, A. (1977). Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts. J. Membrane Biol. 30, 319-350. SCiwIETZER, C. H. & PAssow, H. (1953). Kinetik und Gleichgewichte bei der langsamen Anionpermeabilitat roter Blutkorperchen. Pfluger8 Arch. gee. Phyeiol. 256, 419-445. TELL, U., KOERLER, W. & LoEsCHE, W. (1972). Rapid and complete separation of red cells from nP-orthophosphate containing medium. Acta biol. med. germ. 28, 51-62. USSING, H. H. (1952). Some aspects of the application of tracers in permeability studies. Advances in Enzymology, ed. NORD, F. F., vol. 13, pp. 21-65, New York: Interscience. Wrze-H, J. 0. (1970). Effect of some monovalent anions on chloride and sulphate permeability of human red cells. J. Phyeiol. 207, 581-609. ZAxI, L., FAsoiw, H., ScHuKAww, B. & PAssow, H. (1975). Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells. J. cell. Phyeiol. 86, 471-494. 17
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ANION TRANSPORT OF THE RED CELL UNDER NON-EQUILIBRIUM CONDITIONS
BY G. ORMOS AND S. MANYAI From the Department of Biochemistry, National Institute of Occupational Health, 1450 Budapest, Hungary
(Received 15 Mlarch 1977) SUMMARY
1. The exchange of sulphate for chloride across the human red cell membrane was measured in both directions, i.e. by sulphate influx and sulphate efflux. The influence of the concomitant transient pH changes was minimized by phosphate buffer and by choosing experimental conditions of moderate pH sensitivity (pH 6-4 and 7.8). Sulphate self exchange was determined in chloride-free erythrocyte suspensions. 2. The transport of external sulphate into red cells proceeded at a 15-fold greater rate if its initial concentration was raised from 5 to 95 mm. In contrast the velocity constant of sulphate efflux into sodium chloride medium increased only twofold when the intracellular sulphate concentration was increased. To explain this asymmetry it is proposed that external sulphate ions are more able to compete with chloride for the anion transport sites than those present in the cell interior. 3. The transient membrane potential due to the uneven distribution of sulphate and chloride was shown by the rapid introduction of chromate into the cells. When the erythrocytes contained chloride and the external anion was sulphate, the cells took up chromate 50 times (16.5 'C) or 100 times (3 TC) faster than with equilibrium chloride distribution. 4. Chloride efflux into sodium sulphate media was measured by a chloride-sensitive electrode. Under buffered conditions in neutral and alkaline media two kinetic components were observed as the result of chloride exchange against hydroxyl and sulphate ions. At pH 6-4, chloride efflux was characterized by a single velocity constant identical to that of sulphate movement in the opposite direction. The results show that under appropriate circumstances net chloride efflux measurements can provide comparative data on the anion permeability of the red cell membrane. INTRODUCTION
The exchange between chloride and bicarbonate ions through the red cell membrane is one of several successive processes involved in the removal of carbon dioxide. Its physiological significance prompted the study of the anion transport mechanism. Early techniques were based on the measurement of net concentration changes during exchange of different anion species (Dirken & Mook, 1931; Luckner, 1948; Schweitzer & Passow, 1953). The availability of radioisotopes was soon followed by diffusion rate measurements under conditions of equilibrium (Hahn & Hevesy,
502 0. ORMOS AND S. MANYAI 1942). Although the equilibrium exchange offers advantages by avoiding transient changes during the diffusion process (Passow, 1969; Gardos, Hoffman & Passow, 1969) it seems worthwhile to study the non-equilibrium exchange as well. It would be interesting to know whether the action of an anion on anion exchange requires interactions at both surfaces of the erythrocyte membrane, at either surface, or predominantly at one or other side of the membrane. Such investigations demand non-equilibrium distribution and the measurement of the net anion exchange. A further reason to study the non-equilibrium exchange is that it offers a simple way to observe the diffusion continuously with ion-sensitive electrodes. The purpose of our work was (1) to find reliable experimental conditions for net sulphate-chloride exchange measurement, (2) to study the anion permeability with non-equilibrium anion distribution and (3) to use the transient potential for rapid labelling of red cells with [51Cr]chromate. METHODS Blood stored in acid-citrate-dextrose solution for 1-7 days was washed 3 times and then incubated at 25 0C, 10 % haematocrit for 3 hr to establish equilibrium with the washing solution and obtain desired intracellular anion composition. The pH was set and controlled during this procedure by additions of 0-1 N hydrochloric acid and 0-1 N sodium hydroxide. These pH values are indicated on the Figures. The solutions for washing and pre-equilibrating were generally isotonic and contained sodium chloride, sodium sulphate and sodium phosphate buffer. In those cases when intracellular sulphate level was raised over 100 mm, cells were first suspended in a hypertonic solution containing 150 mm-sodium chloride and 150 mm-ammonium chloride. Ammonium ions pass the erythrocyte membrane by a special transport mechanism which is accompanied by charge-transferring chloride diffusion (Hunter, 1968). Next the intracellular chloride was replaced with sulphate by washing and equilibrating the cells with solutions containing sodium sulphate and ammonium sulphate. Care was taken that the ammonium concentration in the later steps was equal to that in the preceding chloride-loading solution. Changes in the intracellular chloride concentration were measured by analysis of haemolysates. Intracellular sulphate concentration was estimated by the distribution of 35SO4 between cells and medium. The cells with enhanced sulphate content were used for sulphate efflux measurements. In the pre-equilibrated cells sulphate transport was determined in inward and outward directions. When sulphate exchange for intracellular chloride was determined, pre-treated cells were again washed, this time with the incubating medium, to obtain the proper anion composition outside the cells. This step was done at 0 'C to prevent further exchange before the diffusion measurement. Determination of anion transport. Sulphate influx was started by resuspending cold cells in prewarmed medium containing 1-5 #sc 35SO4/ml. Samples of 1 ml. cell suspension were put on cooled (5 0C) Sephadex G-75 columns and separated as described by Till, Koehler & Loesche (1972). The effluent cells were collected for 35S activity determinations. Chromate influx was measured at an initial concentration of 0-05 mm. Samples of 0-5 ml. were added to 2 ml. ice-cold medium, centrifuged and aliquots of the supernatant were taken for 51Cr measurements. To determine sulphate efflux into sodium chloride media pre-equilibrated cells were first loaded with 35SO4. For this purpose erythrocytes were incubated at 50 % haematocrit in the original pre-equilibrating solution which contained 5S04 as well. The activity that remained outside at the end of the loading was removed by washing at 0 'C with the medium of the transport experiment. During the back exchange of ""SO, samples were processed as described for chromate uptake. If not stated otherwise, the indicated results are averages of triplicate measurements. Changes of water content and pH during sulphate-chloride exchange. The water content of the cells was measured before and after exchange of external sulphate for internal chloride. Slight shrinkage was observed but it never exceeded 5 %. These small differences were neglected and
ANION TRANSPORT OF THE RED CELL
503
the cell suspensions were characterized by the haematocrit values measured before the exchange. In unbuffered suspensions the medium became extensively acidified during exchange of external sulphate for internal chloride (Fig. 1). As sulphate transport has a strong pH-dependence (Passow, 1969; Schnell, 1972) addition of buffer was necessary. The incubating media contained small concentrations of buffer (generally 6-25 mM-NaPO4) to reduce the influence of a third anion. Mi8ceUaneou8. Chloride concentration of 20-fold diluted haemolysates and the efflux of chloride from cells was measured by chloride-sensitive electrode (Radelkis OP-Cl-711) connected to a voltmeter (Radelkis OP-203) and a recorder (Weinert eKN). pH was measured by combined glass electrode (Radelkis OP-8071-1/A). 85S activity was determined by an anticoincidence G.M. detector (Gamma NZ-305) and 51Cr was measured by means of an automatic gamma-counter (Gamma NZ-310).
I760 _
6-0
~~
~
5 0_
~
5*0
0
2
4
6
Time (min)
Fig. 1. Changes of the pH in the sodium sulphate medium after suspending red cells pre-equilibrated with sodium chloride. Temperature 25 'C; haematocrit 4-6 %; initial pH 7*4. Composition of medium: *, 100 mm sodium sulphate; 0, 95 mM sodium sulphate and 6-25 mm sodium phosphate buffer; A, 90 mmi sodium sulphate and 12 5 mm sodium phosphate buffer; A, 80 mm sodium sulphate and 25 mm sodium phosphate buffer.
[86S]sodium sulphate and [51Cr]sodium chromate were obtained from the Hungarian Isotope Institute (Budapest). SITS (4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid) was purchased from B.D.H. (Poole, England). Other chemicals were of reagent grade. Diffusion velocity constant, k, was derived from the initial rate of concentration change according to the equation AC/C = -k. At; and from the semilogarithmic plot of the fraction of the exchangeable amount of the anion, (a,0 - at)/aa, where a was determined by specific radioactivity or chloride concentration measurements. This representation gave straight lines when one type of anion exchange occurred whether it was a self exchange or a net one. The k values are considered suitable to compare the results obtained under the selected experimental conditions only, and not to calculate exact permeability constants.
0. ORMOS AND S. MANYAI
504
RESULTS
Inward and outward sulphate movement during sulphate-chloride exchange The efflux and influx of sulphate in exchange for chloride, at pH 6-4, are shown in Figs. 2 and 3 respectively. The sulphate diffusion rate was similar in the two directions at an initial concentration of 5 M (kout 0-0057 min-1, S.D. 000045; 1 0
08.
Time (min)
Fig. 2. Sulphate efflux from red cells into different media at pH 6-4. Temperature 20 0C; 6X25 mm phosphate buffer; haematocrit 82 %. Initial concentrations: *, inside: 4*8 mm sodium sulphate and 136 mm sodium chloride; outside: 143 mm sodium chloride; 0O inside: 95 mm sodium sulphate and 6 mm sodium chloride; outside: 143 mm sodium chloride; A, inside: 95 mm sodium sulphate and 6 mm sodium chloride; outside: 95 mM sodium sulphate. 1.0 8 -
8 1-
180 Time (min)
Fig. 3. Sulphate influx at pH 6-4 into red cells of different anion composition. Temperature 20 0C; 6-25 mm phosphate buffer; haematocrit 85 %. Internal anion composition was adjusted by pre-equilibrating the cells in solutions that beside phosphate contained: 0, 0, 143 mm sodium chloride; A, 95 mm sodium sulphate; A, 47.5 mM sodium sulphate and 71 mm sodium chloride. Composition of the extracellular medium: 0, 4-8 mmi sodium sulphate and 143 mm sodium chloride; 0, A, A, 95 mm sodium
sulphate.
ANION TRANSPORT OF THE RED CELL
F
002
I
0
a
I m.
a
N
I
100
50 Pre-equilibrating sulphate (mM)
Fig. 4. Efflux rate of sulphate from cells into sodium chloride medium at pH 7'8. Temperature 25 0C; 6-25 mM phosphate buffer; haematocrit 85%%. Velocity constants determined: *, from initial rate; 0, from semilogarithmic diagram.
0
006
I-
0*03
F
I
0
Fig.
50 Sulphate (mM)
I
iI 100
Dependence of the inward diffusion rate of sulphate on its initial concentration at pH 7-8. For experimental conditions and meaning of symbols see Fig. 4.
5.
505
G. ORMOS AND S. MANYAI kin = 0 0053 min-, S.D. 0.00030). Approximately a twofold increase of sulphate efflux rate was observed upon the elevation of the internal sulphate concentration to 95 mM (k0ut = 0-0096 min-, S.D. 0.00041). Sulphate self exchange proceeded at a similar rate (measured in both directions: kout= 00116 min-1, S.D. 0.00092; kin = 0-0102 min', S.D. 0.001 1). However, increasing the external sulphate concentration to 95 mm produced an asymmetrical, 15-fold increase of the velocity
506
0-40 r
x
v 0 20
0
0n
um
0
20
40
Time (min) Fig. 6. S04 efflux into sodium chloride medium at different intracellular sulphate level. Temperature 25 'C; 6-25 mm phosphate buffer pH 6-4; haematocrit, 8.9 %. Intracellular anion concentrations at the beginning of the diffusion measurement: *, 5-3 mm sulphate and 150 mm chloride; 0 91 mm sulphate and 11.8 mm chloride; A, 137 mm sulphate and 13-6 mm chloride. Ordinate: 35SO4 exit, fraction of the
equilibrium value.
constant of sulphate influx (kin = 00805 min-, S.D. 0.0065). These diffusion processes resulted in straight lines in the semilogarithmic representation. Only one experimental result deviated from linearity. In this case sulphate was the only external anion and the interior of the cells contained chloride and sulphate in comparable amounts. The exchange of chloride and sulphate was also measured at pH 7-8 at different initial sulphate concentrations (Figs. 4 and 5). In accordance with the findings at
ANION TRANSPORT OF THE RED CELL 507 pH 6-4 the elevation of intracellular sulphate produced a twofold increase of ko0t and the replacement of extracellular chloride by sulphate increased kin 15-fold. It should be noted that the concentrations of the pre-equilibrating solutions are indicated on Fig. 4. The actual intracellular sulphate concentration is smaller since at this pH the anion distribution ratio between the cells and suspending medium is less than unity (Funder & Wieth, 1966; Dalmark, 1975). Rate constants derived from both initial velocity and semilogarithmic representation were calculated to ascertain whether their values parallel the decrease of the initial sulphate concentration. Comparison of the data on Figs. 4 and 5 shows that 1.0
8
,
8
a
0.1 0
2
4
6
Time (min)
Fig. 7. Cl efflux into sodium sulphate media at pH 7-4. Temperature 30 'C; haematocrit 4*6 %. A, chloride efflux into a medium which contained 6-25 mm sodium phosphate buffer and 95 mm sodium sulphate; 0, chloride efflux, 0O sulphate influx, medium: 100 mm sodium sulphate. For chloride concentration calculation distinct points of the continuously monitored electrode-potential curves were taken.
it was the initial velocity that provided somewhat lower values. Similar findings were reported for anion transport under equilibrium conditions (Zaki, Fasold, Schumann & Passow, 1975), thus the deviations may rather reflect the time for perfect mixing and temperature equilibration than be a consequence of the non-
equilibrium exchange. The intracellular sulphate concentration was raised over 100 mm by means of ammonium chloride and ammonium sulphate (see Methods section). The results shown on Fig. 6 indicate that the elevation of sulphate concentration from 91 to 137 mm increased the velocity constant very little if at all. Efflux of chloride from red cells into sodium sulphate medium Fig. 7 shows the kinetics of the extracellular appearance of chloride at pH 7*4 determined by chloride-sensitive electrode. The diffusion of chloride depended
G. ORMOS AND S. MAN YAI 508 on the presence of buffer. A substantial part of chloride appeared at an enhanced rate when 6-25 mm phosphate buffer was added. In some experiments bicarbonate was excluded by using carbon-dioxide-free solutions and working under argon but the rapid efflux component was not diminished by this. When buffer was omitted chloride efflux followed first-order kinetics and its velocity constant agreed with that of sulphate transport in the opposite direction. It should be noted, however, that in the unbuffered medium the pH drops and therefore is quite different from that before the experiment. 10
8
T
Q
G
0.1 I
0
.
I
I
20
10
.
30
Time (min)
Fig. 8. Cl efflux into sodium sulphate medium at pH 6-4 and pH 7*0 and its inhibition by SITS. Temperature 26 0C; haematocrit 4.5 %; medium 95 mm sodium sulphate and 6-25 mm sodium phosphate. *, pH 6-4; 0, pH 7 0. Preincubation with SITS at 0 0C; pH 6-4 for 60 min: /, 0-25 ,umole/ml. cells; *, 2-5 /tmole/ml. cells.
The chloride efflux at pH 7-0 and at pH 6-4 in the presence of 6-25 mm phosphate buffer is shown on Fig. 8. The initial diffusion component was reduced at pH 7 0 and it disappeared at pH 6-4; thus chloride efflux could be characterized by a single velocity constant. At pH 6-4 0-25 molee SITS/ml. cells inhibited the chloride efflux by 92 %. Even a tenfold increase in the concentration of the reagent was unable to raise the level of inhibition. Effect on chromate influx of non-equilibrium distribution of anions The uptake of chromate was determined at chloride equilibrium and when sulphate was substituted for external chloride. Fig. 9 shows the results obtained at different temperatures. In parallel experiments sulphate influx was also determined. The velocity constant of sulphate transport together with the values obtained for chromate from the linear phases of the diagrams of Fig. 9 are listed in Table 1. The results show that at 30 00 sulphate influx was much faster than chromate diffusion whereas the situation was inverse at 16-5 and at 3 0C. Accordingly the original anion distribution changed significantly during the chromate uptake at high temperatures
ANION TRANSPORT OF THE RED CELL 100 80
0
Time (min) 60 0
30
30
60
60
120
509
60 40
20
.-o
0,0 0-0 0) .Y)
100 80 60
E
40
1655C
-
20
10 8 6 4
0
5
15 0 Time (min) Fig. 9. Comparison of the chromate uptake rate under 10
chloride/chloride and sulphate/ chloride distribution conditions at different temperatures. Haematocrit 20 %; pH 7-4; initial extracellular chromate concentration 0 050 mM. Cells were pre-equilibrated with 143 mm sodium chloride and 6-25 mm sodium phosphate. Media: 0O 143 mM sodium chloride and 6-25 mm sodium phosphate; *, 95 mm sodium sulphate and 6 25 mm sodium phosphate. TABLE 1. Comparison of chromate and sulphate transport into red cells at different temperatures Ion distribution between medium and cells n
eA
S04/Cl Temperature kso4*
(OC) 37 30 16-5 3
(min-') 0-257 0 014 0 00070
I
Cl/Cl kCrO*
kCrOn,j
(min-')
(min') 0-026 0-018 0 55 0-026
* Averages of duplicates. For details of the experimental conditions
0 025 0-017 0-0041 0 00049 see
Fig. 9.
510 5. ORMOS AND S. MA4NYAI and it was preserved at low temperatures. In line with this result at 30 and 37 TC only the initial section of chromate uptake was accelerated by the uneven anion distribution whereas at 16-5 and at 3 'C the uneven anion distribution greatly increased the entire process of chromate diffusion. It was attempted to remove the 51Cr-activity introduced into the cells at enhanced rate at 3 'C. 51Cr-tagged cells were suspended in 20 volumes of different washing solutions for 60 min. Washing at 0, 22, and 30 'C with isotonic saline resulted in 3-2, 3.9, and 4*1 % removal, whereas sodium sulphate medium produced 1 6, 2-4, and 4*1 % extracellular appearance of the incorporated 5lCr-activity. DISCUSSION
Characteristics of non-equilibrium sulphate transport The replacement of chloride by sulphate produced a large, asymmetric increase in the velocity constant of the transport of external sulphate into red cells compared to sulphate efflux. To interpret this finding we have to analyse first the possible experimental errors then to examine its relationship with the anion transport mechanism. Intracellular anion composition altered rapidly during anion translocations. This raised the possibility that an initial high velocity constant might have decreased during transport. However, the initial rate generally provided somewhat lower values for k than the long term exponential relationship. A rapid decrease of the initial sulphate level below a threshold concentration might also have produced asymmetric results. To examine this, sulphate efflux was measured also at 137 mm initial concentration. In this case more than 30 % of sulphate efflux occurred at sulphate concentrations above 95 mm without significant increase of Iout, so the differences between the two transport directions cannot be explained in this way. Transient pH changes occurred during sulphate movement due to the uneven anion distribution. The mobility of sulphate in the membrane is much less than that of chloride. Therefore, as an approximation, our selected experimental pH values were based on the pH sensitivity of sulphate self exchange. This is a bell-shaped curve with its maximum at pH 6-4 and flattening at more distant pH values (Schnell, 1972). Experiments were performed at pH 7-8 (limited pH sensitivity) and at pH 6-4. At the latter value any change could at most restrain the transport and so it could act against the increase of the velocity constant only. The asymmetry was observed at both of these pH values, suggesting its independence of the dissociation status of the intramembrane sites. Anion transport through the red cell membrane is strongly dependent on other anions present (Deuticke, 1967; Wieth, 1970). Schnell, Gerhardt & SchdppeFredenburg (1977) related the large increase of sulphate flux at increasing sulphate concentration to the release from a competitive inhibitory effect of chloride. The present findings suggest that external sulphate ions can more effectively compete for the anion transport sites than those present in the cell interior. There are also reports on the asymmetric inhibition of anion transport by some non-penetrating probe molecules from the external membrane side (Lepke & Passow, 1973; Schnell, Gerhardt, Lepke & Passow, 1973; Zaki et al. 1975). It is remarkable that an anion
ANION TRANSPORT OF THE RED CELL 511 for which the membrane is highly permeable also interacts with the transport sites asymmetrically. The unidirectional sulphate flux is the sum of different transport components. The effect of the membrane potential on them is not the same. The diffusion potential is determined by the permeability constants and the concentrations of ions on the two sides of the membrane (Goldman, 1943). In saline-washed red cells this potential is essentially equal to the chloride equilibrium potential. Sufficient changes in the concentrations and in the permeability of the individual ions, however, may alter the magnitude and the sign of the membrane potential. Changes due to increased cation permeability have been shown by micro-electrodes (Lassen, Pape & VestergaardBogind, 1976) and by fluorescent probes (Hoffman & Laris, 1974) and have been applied to load red cells with cations (Sarkadi, Szasz & Gardos, 1976). The potential is also influenced when the concentrations of permeating anions are altered on either side of the membrane. Since the exchange diffusion as defined by Ussing (1952) is electrically silent, the permeability constants of the chargetransferring diffusions are valid (Glynn & Warner, 1972). Accordingly, in a red cell suspension with unequally distributed chloride and sulphate the potential is maintained by that fraction of these anions that travel through ionic diffusion routes. Comparing the ionic movement and exchange rates of chloride (Hunter, 1971) and sulphate (Passow, 1974 a, b) one may assume that the ionic diffusions differ by a factor of 102-103 in favour of chloride. Therefore, the membrane potential is determined primarily by the uneven chloride distribution. The internal membrane side becomes positive when chloride, the anion of greater mobility, is in the cell interior. Chromate influx has been shown insensitive to the anion composition of the medium (Ormos & Manyai, 1974), hence changes in its uptake rate can be attributed to the alterations of the membrane potential. The enhanced diffusion rate of chromate when extracellular chloride is replaced by sulphate provides evidence that the membrane potential does change in the manner expected (inside more positive). In contrast to the electrically silent exchange the charge-transferring sulphate diffusion would be influenced by the membrane potential and it would contribute to the increase in the unidirectional flux. As the ionic sulphate diffusion is a small fraction of the total flux (Passow, 1974b) its contribution may be of minor importance. Although the main form of anion transport is generally attributed to potentialindependent exchange of anion-carrier complexes, it has frequently been put forward that even this main route may be under the influence of the membrane potential (Klocke, 1976; Passow, 1969, 1974b; Wieth, 1970). The results of our experiments do not exclude this possibility yet they do not offer a definitive answer to this question.
Chloride efflux into sodium sulphate medium Chloride efflux into media containing sodium sulphate and phosphate buffer consisted of two kinetic components at neutral and alkaline pH. The rapid component disappeared by lowering the pH or by omitting the buffer. Chloride self exchange is much faster than sulphate transport: therefore, at disequilibrium, chloride is likely to exchange with rapid penetrants if they are present. The rapid outflow of a portion of chloride may be the result of hydroxide influx/ chloride efflux. This supposition is consistent with the pH changes found as they also
512 5. ORMOS AND S. MANYAI involve the movement of hydroxyl ions as suggested by Scarpa, Cecchetto, & Azzone
(1970).
In the absence of chloride-hydroxyl exchange the velocity constants of chloride outflow and sulphate influx agreed. SITS, a specific inhibitor of anion transport (Knauf & Rothstein, 1971) inhibited this chloride movement. These observations support that the bulk of chloride efflux was a stoichiometric exchange against sulphate. There are reports on the determination of anion transport by the exchange of chloride against acetate, nitrate and halides (Gunn, Wieth & Tosteson, 1975), pyruvate (Motais, & Cousin, 1976) and bicarbonate (Klocke, 1976). The rate of exchange against monovalent inorganic anions is however too fast for convenient measurement at physiological temperatures whereas in the case of organic anions the contribution of non-ionic diffusion (Deuticke, 1973) may interfere with the exchange process. Measurements of sulphate-chloride exchange are not subject to these problems.
Rapid uptake of chromate at asymmetric anion distribution When sulphate was substituted for external chloride, chromate uptake proceeded 50 times (16.5 0C) or 100 times (3 0C) faster than in sulphate-free control. To obtain enhanced uptake rate it is important to keep the sulphate influx/ chloride efflux slower than chromate movement, otherwise the transient potential generated would act for only part of the period of chromate diffusion. Lowering the temperature has been shown to shift the relative transport velocities in favour of chromate diffusion as its activation energy is a third only of that of sulphate, 10 kcal/mole (Ormos & Manyai, 1974). The transient membrane potential could, accordingly, be applied to introduce chromate into the erythrocytes in a short time at low temperatures. 0 The skilful technical assistance of Miss Katalin Sarai is gratefully acknowledged. This work was supported in part by the Scientific Research Council, Ministry of Health, Hungary, no.
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