Passive Potassium Transport in LK Sheep Red Cells

Effects of Anti-L Antibody and Intracellular Potassium P H I L I P B. D U N H A M From the Department of Biology, Syracuse University, Syracuse, New York 13210

A B S T R A C T T h e passive K influx in low K (LK) red blood cells o f sheep saturates with increasing external K concentration, indicating that this mode o f transport is mediated by membrane-associated sites. T h e passive K influx, tML, is inhibited by external Na. I s o i m m u n e anti-L serum, known to stimulate active K transport in LK sheep red cells, inhibits IML about twofold, iML is affected by changes in intracellular K concentration, [K]~, in a complex fashion: increasing [K]~ from near zero stimulates ~Mk, while further increases in [K]l, above 3 mmol/liter ceils, inhibit ~Mk. T h e passive K influx is not mediated by K-K exchange diffusion. T h e effects o f anti-L antibody and [K]~ on passive cation transport are specific for K: neither factor affects passive Na transport. T h e c o m m o n characteristics o f passive and active K influx suggest that ~M k is mediated by inactive Na-K p u m p sites, and that the inability to translocate Na characterizes the inactive p u m p s . Anti-L antibody stimulates the K p u m p in reticulocytes o f LK sheep. However, anti-L has no effect on ~ML in these cells, apparently because reticulocytes do not have the inactive p u m p sites which, in mature LK cells, are a consequence o f the process o f maturation of circulating LK cells. T h e results also indicate that anti-L alters the m a x i m u m velocity o f both active and passive K fluxes by converting p u m p s sites from a form mediating passive K influx to an actively transporting form. I N T R O D U C T I O N

T h i s r e p o r t is c o n c e r n e d with t h e e f f e c t s o f i s o i m m u n e a n t i - L s e r u m a n d i n t r a c e l l u l a r p o t a s s i u m o n p a s s i v e p o t a s s i u m t r a n s p o r t in low K ( L K ) s h e e p r e d b l o o d cells. T h e r e s u l t s a r e c o n s i d e r e d in t e r m s o f t h e r e l a t i o n s h i p b e t w e e n t h e m e c h a n i s m s f o r active a n d p a s s i v e t r a n s p o r t o f K in t h e s e cells. S h e e p a r e d i m o r p h i c with r e s p e c t to t h e a b i l i t y o f t h e i r r e d cells to p u m p N a a n d K. R e d cells f r o m H K s h e e p , with h i g h K a n d low N a c o n c e n t r a t i o n s , h a v e h i g h e r p u m p f l u x e s t h a n L K cells ( h i g h N a a n d low K c o n c e n t r a t i o n s ) ( K e r r , 1937; E v a n s , 1954; T o s t e s o n a n d H o f f m a n , 1960). T h i s g e n e t i c a l l y d e t e r m i n e d d i m o r p h i s m ( E v a n s a n d K i n g , 1955; E v a n s e t a l . , 1956) is d u e in p a r t to a d i f f e r e n c e in n u m b e r o f a c t i v e t r a n s p o r t sites p e r cell ( D u n h a m a n d H o f f m a n , 1971a). T h e two cell t y p e s also d i f f e r in k i n e t i c p r o p e r t i e s o f t h e p u m p sites THE JOURNAL

OF GENERAL

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' VOLUME

68,

1976

• pages

567-581

567

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T H E ,JOURNAL OF GENERAL P H Y S I O L O G Y " V O L U M E 6 8 " 1 9 7 6

( H o f f m a n and Tosteson, 1971); in particular, intracellular K is a much m o r e effective inhibitor o f the p u m p in LK cells than in H K cells (Glynn and Ellory, 1972). T h e inhibition is a p p a r e n t l y by competition with Na for translocation loci o f the intracellular aspect o f the p u m p sites (Ellory et al., 1972; see also Hoffman, 1966; H o f f m a n and T o s t e s o n , 1971; k n i g h t and Welt, 1974). At very low concentrations o f intracellular K, [K],, raising [K]t stimulates the p u m p in H K cells (and also in LK cells p r e t r e a t e d with anti-L antibody; D u n h a m and Blostein, 1976). Only inhibition o f the p u m p with increasing [K]t is observed in u n t r e a t e d LK cells. Stimulation o f the p u m p by Kt had previously been r e p o r t e d in red cells o f goats (Ellory et al., 1972; Sachs et al., 1974b) and h u m a n s (Garay and G a r r a h a n , 1973). T h e r e are o t h e r types o f interactions o f K, with the Na-K p u m p in red cells: one for one exchange o f K (Glynn et al., 1970; Sachs et al., 1974 b) and complete reversal o f the p u m p (Lew et al., 1970), but these modes o f operation o f the p u m p have not been r e p o r t e d for sheep red ceils. In addition to lower p u m p fluxes, LK cells also have h i g h e r passive fluxes o f K and Na than do H K cells (Tosteson and H o f f m a n , 1960). Passive K influx in LK sheep cells a p p r o a c h e s saturation as the external K concentration, [K]o, is increased ( D u n h a m and H o f f m a n , 1971a), indicating that passive K t r a n s p o r t in these cells is not entirely by simple passive diffusion. T h e m u c h lower passive influx in H K cells showed no sign o f saturation as a function o f [K]o. (Passive transport is used h e r e to mean the flux m e a s u r e d in cells treated with ouabain.) T h e M-L blood g r o u p antigen system in sheep is associated with the H K - L K d i m o r p h i s m in that H K cells have only M antigen and the L antigen is f o u n d only on LK cells (Rasmusen and Hall, 1966). Anti-L antiserum, raised in H K sheep i m m u n i z e d with LK cells, stimulates active K t r a n s p o r t in LK cells fourfold or m o r e (Ellory and T u c k e r , 1969). Passive K t r a n s p o r t is r e d u c e d by some preparations o f anti-L serum (Ellory et al., 1972; D u n h a m , 1975, 1976) but not by others (Lauf et al., 1971; Lauf, 1974). Recently it was shown that anti-L serum can contain two populations o f antibodies with d i f f e r e n t effects on K t r a n s p o r t in LK sheep cells ( D u n h a m , 1976). One antibody, anti-Lp, stimulates the p u m p . T h e p r i m a r y action o f the o t h e r antibody, anti-Ll, is reduction o f passive K transport, t h o u g h it also stimulates the p u m p . T h e i m m u n o l o g y o f the M-L d i m o r p h i s m has recently been reviewed (Lauf, 1975). In the present work, this reduction o f passive K t r a n s p o r t by anti-L was characterized. In the course o f these e x p e r i m e n t s it was shown that intracellular K affects passive K t r a n s p o r t in LK cells in m u c h the same m a n n e r that it affects active t r a n s p o r t in H K cells: stimulation by low concentrations and m o d e r a t e inhibition at h i g h e r concentrations o f K~. T h e s e and o t h e r results suggest that passive K t r a n s p o r t in LK sheep cells is mediated by inactive p u m p sites, and that one o f the actions o f anti-L serum is a conversion o f inactive p u m p s to an active form. A preliminary" r e p o r t o f some o f these results has been p r e s e n t e d (Dunham, 1975). MATERIALS

AND

METHODS

Blood was drawn into heparin by venipuncture from adult male Dorset sheep, and was used on the day it was drawn. The red ceils were washed by centrifugation in an isotonic solution containing NaCI (150 mM), glucose (5 mM), Tris-Cl (10 mM) at pH 7.5.

DUNHAM PassiveK Transport in LK Sheep Red Cells

569

Unidirectional fluxes of Na or K were measured with 42K or ~4Na obtained as chlorides from International Chemical Corporation. Influxes and effluxes were measured and calculated as described by Sachs et al. (1974b) and D u n h a m and Hoffman (1971a), respectively. T h e passive influx of K and the passive efflux of Na were taken as the fluxes measured in cells pre-exposed to 10-3 M ouabain for 5 min at 37°C in K-free m e d i u m . During measurement of the fluxes, ouabain concentration was 10-4 M. Active, or p u m p fluxes were the ouabain-inhibited fluxes. Intracellular concentrations of Na and K and relative cell volumes were measured as described previously (Sachs and Welt, 1967; Sachs et al., 1974b). T o measure fluxes in Na-free m e d i u m , choline C1 was used as a substitute for NaCl. Purified choline chloride was obtained from Hoffman-Taff, Inc., Springfield, Mo. Intraceilular concentrations of Na and K were altered by reversibly increasing cation permeability with nystatin after the method of Cass and Dalmark (1973) as modified by D u n h a m and Blostein (1976). Reticulocyte-rich preparations were obtained as described previously ( D u n h a m and Blostein, 1976). Briefly, erythropoiesis was stimulated by phlebotomy and reticulocytes were concentrated by gradient centrifugation. Anti-L antiserum was raised in HK sheep by intramuscular injection of LK cells (Tucker and Ellory, 1970; D u n h a m , 1976). Packed LK ceils were mixed 1:1 with Freund's complete adjuvant, and injections of 5 ml each were made into the four u p p e r limbs of the animal. After 1 too, booster injections were made of cells mixed with incomplete adjuvant. Before use the serum was heated to 56°C for 20 rain to inactivate complement, and then was dialyzed at 4°C for 18 h against 50 vol 150 mM NaC1 or choline CI with 10 mM Tris-C1, pH 7.5. Before a flux was measured, cells were incubated with antiserum at 5-10% hematocrit at 32°C for 30 rain. Control aliquots of cells were incubated at the same time either in an isotonic NaCI solution or in heated, dialyzed serum from a n o n i m m u nized HK sheep. Affinity of p u m p sites for ouabain was investigated from the time course of the onset of inhibition of the p u m p d u r i n g exposure to ouabain (Sachs et al., 1974a). Aliquots of cells were incubated for various times in an isotonic solution containing 10-n M ouabain and 5 mM K, with Na the principal cation. Typically 8 ml of a 5% hematocrit suspension was used. Ouabain b i n d i n g was stopped by rapid chilling of the suspensions in an ice bath. After washing, fluxes were measured in: (a) these cells; (b) cells not exposed to ouabain (giving the uninhibited flux); and (c) cells pre-exposed to 10-3 M ouabain (giving the maximally inhibited flux). T h e logarithm of p u m p flux remaining was plotted against time of exposure to ouabain. This curve was taken as the time course of ouabain binding. A straight line would indicate a single rate constant of b i n d i n g and therefore homogeneous affinity of the p u m p s for ouabain, whereas a line of decreasing slope would indicate a heterogeneity of the affinity of the pumps for ouabain. RESULTS

T a b l e I shows p u m p a n d passive i n f l u x e s o f K in L K s h e e p cells, b o t h u n t r e a t e d a n d p r e - e x p o s e d to a n t i - L s e r u m . R e s u l t s are s h o w n f o r cells f r o m t h r e e LK s h e e p . A n t i - L s t i m u l a t e d t h e p u m p as r e p o r t e d p r e v i o u s l y (Ellory a n d T u c k e r , 1969; L a u f et al., 1970) a n d also c a u s e d sizable r e d u c t i o n s i n t h e passive K i n f l u x e s . T h i s was a c o n s i s t e n t f i n d i n g i n this s t u d y with p r e p a r a t i o n s o f a n t i - L s e r u m f r o m two i m m u n i z e d H K s h e e p .

Kinetics o f Passive K I n f l u x As m e n t i o n e d a b o v e , passive K i n f l u x in L K s h e e p ceils s a t u r a t e s as [K]o is

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i n c r e a s e d ( D u n h a m a n d H o f f m a n , 1971a). Fig. 1 s h o w s t h e kinetics o f p a s s i v e K i n f l u x e s , p r e s e n t e d as d o u b l e r e c i p r o c a l p l o t s , m e a s u r e d at v a r i o u s [K]o'S in a n t i L t r e a t e d cells a n d in c o n t r o l cells. T h e r e s u l t s fit s t r a i g h t lines f o r b o t h t y p e s o f cells, c o n s i s t e n t with s a t u r a t i o n k i n e t i c s f o r p a s s i v e K i n f l u x in a n t i - L t r e a t e d as well as c o n t r o l cells. T h e e x t r a p o l a t i o n s f o r t h e two t y p e s o f cells to t h e a b c i s s a w e r e n e a r l y t h e s a m e , g i v i n g a KaI2, t h e [K]0 at h a l f - m a x i m a l p a s s i v e i n f l u x , o f a b o u t 7 m M . T h i s Kt/z o f p a s s i v e i n f l u x c a n b e c o m p a r e d with Ktn's f o r active K i n f l u x o f 3 m M in N a - c o n t a i n i n g m e d i u m a n d 0.2 m M in N a - f r e e m e d i u m ( H o f f m a n a n d T o s t e s o n , 1971). T h u s , t h e m e c h a n i s m m e d i a t i n g p a s s i v e K i n f l u x has a l o w e r a p p a r e n t a f f i n i t y f o r K t h a n d o e s t h e active p u m p m e c h a n i s m . TABLE

I

T H E E F F E C T OF A N T I - L SERUM ON ACTIVE,iM~ , AND PASSIVE, tMk, K INFLUXES IN RED CELLS FROM T H R E E LK SHEEP Sheepno.

JM[

~Mk

control anti-L

0.15 0.75

0.67 0.18

control anti-L

0.16 0.24

0.61 0.22

control anti-L

0.10 0.18

0.45 0.26

1

Cells were pretreated with anti-L serum as described in the text. The same preparation of antiserum was used with all three aliquots of cells. Control cells were pretreated with isotonic saline. Fluxes (given as retool/liter cells × h), are means of two determinations, measured in a medium with 10 mM K and with Na as the principal cation. Similar results were obtained in at least three other experiments on cells from each sheep. On cells from sheep no. 1, similar results were also obtained with antiserum from a different immunized HK sheep. The above results show that anti-L reduces the maximal passive influx but not the apparent affinity for K of the system mediating passive influx. This effect of a n t i - L o n p a s s i v e K i n f l u x was r e p r o d u c i b l e q u a l i t a t i v e l y , b u t t h e Kv2's f o r b o t h c o n t r o l a n d a n t i - L t r e a t e d cells w e r e s o m e t i m e s two- to t h r e e f o l d h i g h e r . T h e r e a s o n f o r this v a r i a b i l i t y is n o t k n o w n .

Inhibition by External Na E x t e r n a l N a is a n i n h i b i t o r o f active K i n f l u x in s h e e p r e d cells ( H o f f m a n a n d T o s t e s o n , 1971). N a m a y act as a c o m p e t i t i v e i n h i b i t o r (Sachs et al., 1975) a n d in a m o r e c o m p l e x m a n n e r as well ( C a v i e r e s a n d E l l o r y , 1975; H o b b s a n d D u n h a m , 1976). Fig. 2 s h o w s t h e e f f e c t o f N a - f r e e m e d i u m c o m p a r e d to N a - m e d i u m o n p a s s i v e K i n f l u x in L K s h e e p cells. T h e f l u x was m e a s u r e d in u n t r e a t e d L K cells at v a r i o u s [K]o's in m e d i a with e i t h e r N a o r c h o l i n e as t h e p r i n c i p a l c a t i o n . I n t h e

"

571

DUNI~AM Passive K Transport in LK Sheep Red Cells

0

-_

.J. 0

0.5

1.0

1.5

I/ [K]o:(mM)-' FIGURE 1. Effect o f anti-L serum on passive K influx in LK sheep red cells with varying external K concentration, [K]o. Results are presented as double reciprocal plots. Pre-exposure to anti-L and m e a s u r e m e n t o f fluxes were as described in the text. Fluxes are in millimoles/(liter cells × hour) plotted as reciprocals. Points represent means of two determinations. T h e lines were fitted by eye. P u m p influxes measured at the same time at 10 mM [K]o were (mmol/liter × h): 0.09, control cells; 0.24, anti-L cells. In four other similar experiments the results were comparable in that the extrapolations to the abscissa were the same for control and anti-L cells. However in two of the experiments the extrapolation differed by a factor of 2-3.

~JEa=~O.lO E= "" 0.05

i chol

, 0

~

I 2

,

[ K ] . . ,,,M

I 4

F [ c u ~ 2. Passive K influx in LK sheep red cells with and without external Na. [K]~ = 28 retool/liter cells after nystatin treatment. Ouabain-insensitive K influxes, *ML, were m e a s u r e d in media with 1,2, or 4 mM K and with either Na or choline as the principal cation (146-149 mM). T h e points represent means o f two determinations; the curves were fitted by eye. T h e same results were obtained in four other experiments, both with cells pretreated with nystatin ([K], = 0.5-30 mmol/liter cells) and with fresh cells ([K], = 6-8 mmol/liter cells).

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T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 8 • 1 9 7 6

N a - f r e e m e d i u m , t h e i n f l u x e s w e r e a b o u t 25% h i g h e r t h a n i n N a m e d i u m . T h e r e f o r e , e x t e r n a l N a a p p e a r s to be a n i n h i b i t o r o f passive K i n f l u x .

K:K Exchange E x p e r i m e n t s were u n d e r t a k e n to d e t e r m i n e if a n t i - L r e d u c e s passive K i n f l u x by r e d u c i n g a o u a b a i n - i n s e n s i t i v e K : K e x c h a n g e o f the sort o b s e r v e d in goat r e d cells ( D u n h a m a n d Bleier, 1973; cf. Sachs et al., 1974b). T h e cells were first l o a d e d with 42K. W a s h e d a l i q u o t s o f t h e cells w e r e e x p o s e d to a n t i - L a n d t h e n w a s h e d a g a i n . K e f f l u x was m e a s u r e d in c o n t r o l a n d a n t i - L t r e a t e d cells in f o u r k i n d s o f m e d i a , with 20 m M K o r K - f r e e , a n d t h e s e two m e d i a each with 10 -3 M o u a b a i n . N a was t h e p r i n c i p a l c a t i o n . T h e r e s u l t s o f such a n e x p e r i m e n t , p r e s e n t e d in T a b l e I I , show first t h a t K e f f l u x was r e d u c e d n e a r l y t w o f o l d by TABLE

I I

EFFECT OF A N T I - L ON K EFFLUX IN LK SHEEP RED CELLS W I T H AND W I T H O U T EXTERNAL POTASSIUM AND O U A B A I N K efflux (mmol/liter cells x h) Control

anti-L

K-free

20 mM K

K-free

20 mM K

Control

1.38

1.23

0.70

0.78

Ouabain

1.41

1.28

0.72

0.73

Cells were loaded with 42K and treated with anti-L as described in the text. Fluxes were measured in either K-free medium or medium with 20 mM K, and in each of these media with or without ouabain (control). For measuring the efflux in ouabain-treated cells, the cells had been pre-exposed to 10-4 M ouabain at 37°C for 10 min in K-free medium. The principal cation in the media was Na (NaCI + KCI = 150 raM). The fluxes are means of determinations on two flasks of cell suspensions. The flux for each flask was in turn the mean calculated from samples taken at three consecutive 30-rain intervals. Intvacellular K concentration was 5.5 retool/liter cells. Similar results were obtained in two other experiments. Pump and leak influxes of K were measured simultaneously on aliquots of the same cells. Anti-L treatment caused a 1.85-fold increase in the pump and a 1.74-fold decrease in the passive influx. a n t i - L , as e x p e c t e d f r o m t h e r e s u l t s o n passive K i n f l u x . S e c o n d , o u a b a i n has n o effect o n K e f f l u x i n e i t h e r cell type i n t h e p r e s e n c e o r a b s e n c e o f Ko. F i n a l l y , r e m o v a l o f K0 h a d n o effect o n K e f f l u x in a n t i - L t r e a t e d cells. T h e r e is a s u g g e s t i o n o f a s m a l l i n c r e a s e in K e f f l u x u p o n r e m o v a l o f K0 in c o n t r o l cells, b u t K : K e x c h a n g e w o u l d be m a n i f e s t e d by a r e d u c e d K e f f l u x in K - f r e e m e d i u m , n o t a n i n c r e a s e . T h e r e f o r e , t h e r e is n o K : K e x c h a n g e , e i t h e r o u a b a i n - s e n s i t i v e o r o u a b a i n - i n s e n s i t i v e , i n L K s h e e p cells, a n d the effect o f a n t i - L c a n n o t be c o n s i d e r e d in t h e c o n t e x t o f s u c h a m e c h a n i s m .

Anti-L on Na Efflux T o d e t e r m i n e t h e specificity o f the a c t i o n o f a n t i - L o n c a t i o n t r a n s p o r t , t h e effects o f a n t i - L o n N a e f f l u x e s w e r e m e a s u r e d . T a b l e I I I shows e f f l u x e s o f N a i n t o c h o l i n e - m e d i u m for c o n t r o l a n d a n t i - L t r e a t e d cells. A n t i - L s t i m u l a t e d active N a e f f l u x f o u r f o l d , b u t t h e r e was n o effect o f the a n t i s e r u m o n passive N a

DUNHAM Passive K Transport in LK Sheep Red Cells

573

efflux. T h e r e f o r e the effect o f anti-L on passive cation efflux is specific for K, t h o u g h active t r a n s p o r t o f both K and Na are stimulated.

Passive K Influx and [K]~ T h e f o r e g o i n g results are consistent with mediation o f passive K t r a n s p o r t by a mechanism which has some p r o p e r t i e s in c o m m o n with the Na-K p u m p . Since Kt interacts with the p u m p , the effects o f varying [K]~ on passive influx in LK cells were d e t e r m i n e d ; the results are shown in Fig. 3. Fig. 3a shows the results o f one representative e x p e r i m e n t on u n t r e a t e d and anti-L-treated cells. It a p p e a r e d that, at low concentrations, increasing [K], stimulated passive K influx, while with a f u r t h e r increase in [K],, the influx was inhibited. T o substantiate that these effects are real, summaries o f a n u m b e r o f e x p e r i m e n t s are p r e s e n t e d in Fig. 3b and c. Fig. 3b shows the results o f 10 e x p e r i m e n t s with the fluxes TABLE EFFECT

OF ANT1-L SERUM

I I I

ON NA EFFLUXES

IN LK SHEEP

RED CELLS Na efflux (mmol/litercells x h) Total

OM~.

°M~.

Control

1.14-+0.022

0.96-0.019

0.19

Anti-L treated

1.72---0.031

0.96+0.008

0.76

T h e cells were loaded with 24Na and treated with anti-L as described in the text. Effluxes were m e a s u r e d in media containing (mM): choline Cl, 130; KCI, 20; NaCI, 1.0; glucose, 5; Tris-Cl, 10; p H 7.5. T h e passive efflux, °M~a, was that m e a s u r e d f r o m cells exposed, before the flux was m e a s u r e d , to 10-4 M ouabain for 10 min at 37°C in the choline m e d i u m without K. T h e p u m p efflux, °M~a , is the difference between total efflux and °M~a. T h e intracellular concentrations were (retool/liter cells): [K]l = 11.6 and [Na], = 82.4. Each value is the m e a n of fluxes determined on f o u r flasks o f cell suspensions. T h e flux for each flask was in t u r n a m e a n calculated f r o m samples taken at three consecutive 45-min intervals. T h e e r r o r s are SEM for n = 4.

normalized to 1.0 at the highest [K]~ in each e x p e r i m e n t (about 30 mmol/liter cells). T h e results are p r e s e n t e d in this fashion to emphasize the inhibitory effect obtained u p o n raising [Kit f r o m about 3 mmol/liter. T o emphasize the stimulation at low [K]~, the results o f f o u r additional e x p e r i m e n t s are shown in Fig. 3 c with the fluxes normalized to the lowest [Kit in each e x p e r i m e n t . T h e s e effects o f [K]t on passive K influx are c o m p a r a b l e to the effects o f varying [K]i on the p u m p flux in H K sheep cells ( D u n h a m and Blostein, 1976) and in goat cells (Ellory et al., 1972; Sachs et al., 1974b). As with the specificity o f the action o f anti-L on passive cation t r a n s p o r t , the specificity o f action o f [K], on passive cation influx was investigated. Na influx was m e a s u r e d at two external Na concentrations in cells with various [K],'s. As shown in Table IV, Na influx did not vary with [K],, and the effect o f [K]i on passive cation t r a n s p o r t , like the effect o f anti-L, is specific for K transport.

574

THE JOURNAL

or

GENERAL PHYSIOLOGY • VOLUME 68 •

0.2

1976

b • control o anti - L a~

E

0.1

anti - L

"5 1.2 E

E i

I 5 [K]i' m m o l / ~

~

J~

I I0

Lo o

5

[K]i:

cells

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cells

c

1.2

o

10

m mol/e

zo

N

I,I

2o

j ~ I.O

~

o

I

I

I

'-

l

I

2

[KJi, m m o l / Z c e l t s

FIGV~a~ 3. E f f e c t o f v a r y i n g i n t r a c e l l u l a r K c o n c e n t r a t i o n . [ K ] , o n passive K i n f l u x , ~M~, in LK s h e e p r e d cells. [K]~ was v a r i e d with n y s t a t i n . I n f l u x e s w e r e m e a s u r e d in m e d i a with a p p r o x i m a t e l y 4 m M K a n d with N a as t h e p r i n c i p a l cation, (a) Results o f o n e r e p r e s e n t a t i v e e x p e r i m e n t o n c o n t r o l cells, i.e. w i t h o u t p r e t r e a t merit, a n d o n cells p r e t r e a t e d with a n t i - L . T h e p o i n t s r e p r e s e n t m e a n s o f two d e t e r m i n a t i o n s . (b) Results o f 10 e x p e r i m e n t s with t h e fluxes n o r m a l i z e d to 1.0 at t h e h i g h e s t [K]t in e a c h e x p e r i m e n t (28-31 m m o l / l i t e r cells). Results a r e s h o w n f o r b o t h c o n t r o l a n d a n t i - L t r e a t e d cells. N u m b e r s n e x t to symbols a r e n u m b e r s o f d e t e r m i n a t i o n s f o r e a c h m e a n . E r r o r s a r e SEM. (c) Results o f f o u r e x p e r i m e n t s w i t h t h e f l u x e s n o r m a l i z e d to 1.0 at t h e lowest [K]~ in e a c h e x p e r i m e n t (0.2-0.4 m m o l / l i t e r cells). (b) a n d (c) c o n t a i n n o values in c o m m o n . TABLE

IV

EFFECT OF VARYING INTRACELLULAR K ON INFLUX OF N A (~Mr~a) I N LK S H E E P RED C E L L S IM~a(mmol/liter cells × h) [K]~ [Na],,

12 6 3 0.4

150 m M

15 ram

3.8 3.7 3.9 3.9

1.03 1.04 1.10 1.06

Methods for varying intracellular K concentration, [K], and for measuring Na influx are described in the text. T h e media contained no K. External Na concentration, [Na]o, was either 150 mM or 15 mM (+ 135 mM choline C1). T h e fluxes are means of 2 determinations. T h e same results were obtained in 2 other experiments.

DUNHAM PassiveK Transport in LK Sheep Red Cells

575

K I n f l u x in Reticulocytes R e t i c u l o c y t e s f r o m L K s h e e p d i f f e r f r o m m a t u r e L K cells in h a v i n g a c a t i o n c o m p o s i t i o n like t h a t o f H K cells, a p u m p f l u x m u c h h i g h e r t h a n in m a t u r e cells, a n d a m u c h h i g h e r p a s s i v e f l u x ( L e e e t al., 1966; T u c k e r a n d E l l o r y , 1971; D u n h a m a n d B l o s t e i n , 1976), a n d t h e m a t u r a t i o n o f L K cells is c h a r a c t e r i z e d b y l a r g e r e d u c t i o n s in b o t h active a n d p a s s i v e t r a n s p o r t o f K. T h o u g h t h e p u m p s o n L K r e t i c u l o c y t e s d i f f e r k i n e t i c a l l y f r o m p u m p s o n m a t u r e cells, a n t i - L s t i m u lates active K i n f l u x in r e t i c u l o c y t e s ( D u n h a m a n d B l o s t e i n , 1976). A c c o r d i n g l y it was o f i n t e r e s t to d e t e r m i n e i f p a s s i v e t r a n s p o r t in L K ceils is i n h i b i t e d b y a n t i - L b e f o r e t h e i n a c t i v a t i o n o f t h e b u l k o f t h e p u m p sites w h i c h o c c u r s d u r i n g m a t u r a t i o n . T a b l e V s h o w s b o t h active a n d p a s s i v e K i n f l u x e s in i m m a t u r e a n d TABLE

V

T H E E F F E C T OF A N T I - L SERUM ON K I N F L U X E S IN R E T I C U L O C Y T E S FROM AN LK SHEEP 'M[

~Mk retool~liter cells h

[K]~ ramol/liter cells

Reticulocytes control anti-L

3.04 4.04

2.82 2.97

4.46 3.10

Mature cells control anti-L

0.14 0.47

0.27 0.20

3.44 4.06

Active, tMt~, and passive, ~Mk, fluxes were measured. Reticulocyte-rich preparations (55% reticulocytes) were obtained from the top fraction after density gradient centrifugation as described in the text. Results are also presented for mature cells (bottom fraction) obtained at the same time from the same sheep. Intracellular K concentrations, [K]{s, were made the same in the young and mature cells by using nystatin. Cells were pretreated either with isotonic saline (control cells) or with antiserum as described in the text. Fluxes were measured in a medium with 4 mM K and with Na as the principal cation. The same results were obtained in one other experiment. m a t u r e L K cells, b o t h c o n t r o l a n d a n t i - L t r e a t e d . T h e [K]{s in t h e r e t i c u l o c y t e s a n d m a t u r e cells w e r e m a d e t h e s a m e with n y s t a t i n . B o t h active a n d p a s s i v e K f l u x e s w e r e m u c h h i g h e r in r e t i c u l o c y t e s . I n t h e m a t u r e cells, a n t i - L s t i m u l a t e d the pump more than threefold and reduced the leak somewhat. In reticulocytes t h e p u m p was s t i m u l a t e d to a l e s s e r r e l a t i v e e x t e n t t h a n in m a t u r e cells, b u t t h e a b s o l u t e s t i m u l a t i o n was n e a r l y 10-fold g r e a t e r . F i n a l l y , t h e p a s s i v e K i n f l u x in r e t i c u l o c y t e s was u n a f f e c t e d b y a n t i - L . I n this r e s p e c t t h e m e c h a n i s m s f o r p a s s i v e K t r a n s p o r t d i f f e r in m a t u r e a n d i m m a t u r e L K cells, a n d i n h i b i t i o n o f p a s s i v e t r a n s p o r t b y a n t i - L is p o s s i b l e o n l y a f t e r i n a c t i v a t i o n o f t h e p u m p s . Anti-L on Active K I n f l u x T o a i d in i n t e r p r e t i n g t h e m e c h a n i s m o f a c t i o n o n a n t i - L o n p a s s i v e K t r a n s p o r t , two k i n d s o f e x p e r i m e n t s w e r e p e r f o r m e d o n t h e e f f e c t o f a n t i - L o n active t r a n s p o r t . I n t h e first t y p e o f e x p e r i m e n t , t h e e f f e c t o f a n t i - L was d e t e r m i n e d as a f u n c t i o n o f [K]i. I n L K g o a t cells, t h e s t i m u l a t i o n o f t h e K p u m p by a n t i - L is

576

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y

•

VOLUME 68 •

1976

nearly abolished as [K]t is r e d u c e d below 1.0 mmol/liter o f cells, indicating that the p r i m a r y effect o f anti-L in LK goat cells is not on the m a x i m u m velocity o f the p u m p s , but is p r o b a b l y an alteration o f the affinity o f the p u m p s for K~ (Sachs et al., 1974b). Fig. 4 shows active K influx in LK s h e e p cells with varying [K]~. T h e relative m a g n i t u d e o f the effect o f anti-L was greatest at h i g h e r [K]~, u p to 15-fold stimulation at [K]~ o f 20 mmol/liter. Nevertheless the stimulation in cells nearly K-free ([K]t = 0.4 mmol/liter cells) was m o r e t h a n twofold. T h e r e fore, anti-L stimulates the m a x i m a l t u r n o v e r rate o f the p u m p in LK s h e e p cells, and the m e c h a n i s m o f action is not only t h r o u g h changes in affinity o f the p u m p for intracellular K. A l t h o u g h stimulation o f the p u m p with increasing [K], was not seen in these e x p e r i m e n t s (cf. D u n h a m and Blostein, 1976), the slope o f the

E x

0.2

N ~ control

0

•

I0 20 [ K ] ; ' m moll.e

30

cells

Fmum~ 4.

Effect of anti-L serum on active K influx, iMp, in LK sheep red cell~ with intracellutar K concentration, [K]t, varied by use of nystatin. [Na]t was between 75 and 90 mmol/liter cells. Fluxes are given for control cells, i.e. without pretreatment, and for anti-L treated cells. Points represent means of two determinations. The same results were obtained in three other experiments. curve is lowest at low [K]i, a finding consistent with the stimulation seen in o t h e r studies.

Anti-L on Rate of Ouabain Binding In LK goat cells the affinity o f the p u m p s for ouabain is h e t e r o g e n e o u s , as indicated by a n o n l i n e a r time course o f a logarithmic plot o f ouabain b i n d i n g (Sachs et al., 1974a). T h e rate o f o u a b a i n binding was increased by anti-L t r e a t m e n t in the goat cells a n d is also increased in LK s h e e p cells (Joiner a n d Lauf, 1975). In neither study was t h e r e an indication o f w h e t h e r or not anti-L also r e d u c e d the h e t e r o g e n e i t y o f the affinity o f the p u m p s for ouabain. T h e second type o f e x p e r i m e n t on stimulation o f the p u m p showed that this is the case in LK s h e e p cells (Fig. 5). Aliquots o f cells were first treated with anti-L, a n d

DUNHAM Passive K Transport in LK Sheep Red Cells

577

then the rate o f ouabain binding was m e a s u r e d in control a n d anti-L treated cells as described above. T h e time course for ouabain b i n d i n g to control cells indicates a heterogeneity o f the affinity o f the p u m p s for ouabain. Anti-L t r e a t m e n t increased the rate o f ouabain binding, and also r e d u c e d the heterogeneity, in such a way that only a b o u t 3% o f active t r a n s p o r t r e m a i n e d when the line fitting the time course o f binding d e p a r t e d f r o m linearity. T h e s e results suggest that anti-L increases the rate o f ouabain binding by increasing the ouabain affinity o f the low affinity sites. T h e results do not relate to possible effects o f anti-L on 1.0, c o E

z

.

0.5

0.2

o "2 o anti-L

o

0.05 c

o

i

i

io zo minutes, exposure toouabain

J

30

FIGURE 5. The effect of anti-L on rate of ouabain binding in LK sheep red cells. Rate of ouabain binding was determined, as described in the text, from the fraction of active K influx remaining (plotted on a logarithmic scale) after incubation for various times in media containing ouabain. The ouabain-incubation media contained 145 mM NaC1, 5 mM KCI, 5 mM glucose, 10 mM Tris-Cl (pH 7.5) and 10-6 M ouabain. The intracellular K concentration had been reduced with nystatin to (mmol/liter cells): 2.7, control cells; 3.3, anti-L ceils. After the cells had been incubated with ouabain and washed by centrifugation, K influxes were measured in media with 4 mM K and with Na as the principal cation. The points represent means of two determinations. The results are representative of four other experiments. m a x i m u m velocity o f the p u m p s since they are expresed in terms o f relative, not absolute fluxes. DISCUSSION

T h e results o f this study have bearing on the relationship between the mechanisms for passive and active K t r a n s p o r t , on the action o f anti-L antiserum on K transport, and on the processes o f m a t u r a t i o n o f LK and H K red cells. Passive K influx in LK sheep red cells is not by simple passive diffusion, but obeys saturation kinetics as a function o f [K]o as shown in Fig. 1. T h e r e are two i m p o r t a n t similarities between active K influx in H K cells and passive K influx in LK cells. (a) T h e kinetics o f the active a n d passive K influxes vary in the same

578

T H E J O U R N A L OF GENERAL P H Y S I O L O G Y ' VOLUME 6 8 "

1976

m a n n e r as a function o f intracellular K concentration: both types o f influx are stimulated and then inhibited as [K]t is increased f r o m n e a r zero. (b) Passive and active K influxes are both inhibited by external Na. T h e r e is in addition an i m p o r t a n t d i f f e r e n c e between the active and passive modes, indicated by the effects o f anti-L and Ki on K influx. Active K influx is coupled to Na efflux, but passive K influx is i n d e p e n d e n t o f Na transport. T h e evidence for this latter point is that both altering [K]f and t r e a t m e n t with anti-L affect passive transport o f K but not the passive flux o f Na (both o f these p r o c e d u r e s affect active transport o f Na as well as o f K). On the basis o f these points o f evidence, it is p r o p o s e d that passive K influx is mediated by inactive p u m p sites, sites which have become inactivated d u r i n g m a t u r a t i o n o f the i m m a t u r e LK cells. T h e specificity o f the passive t r a n s p o r t sites for K is a provocative finding, but no molecular basis for this characteristic suggests itself. T h e p u m p s operating in the passive m o d e are less sensitive to inhibition o f Ki than are the active p u m p s (compare curves for control cells in Figs. 3a and 4). I f this is so, then increased effectiveness o f Ki as an inhibitor is not associated with the process o f inactivation. T h e most striking difference between active and inactive p u m p s lies in their interaction with Nai: Na is translocated by active p u m p s but not by inactive ones. T h u s , u n c o u p l i n g o f Na and K fluxes may be a primary event associated with inactivation. (It should be emphasized that, alt h o u g h passive Na fluxes are not mediated by inactive p u m p sites, a large fraction o f passive Na t r a n s p o r t is by a ouabain-insensitive Na-Na exchange [Tosteson and H o f f m a n , 1960].) T h e stimulation o f passive K influx by Kt resembles the stimulation o f the active flux by K~ in H K cells and anti-L treated LK cells o f both sheep and goats (Sachs et al., 1974b; D u n h a m and Blostein, 1976; the present report); K-K exchange cannot account for the stimulation (Table II). T h e concomitant stimulation and inhibition o f K influxes by K~ suggest interaction o f K with two classes o f sites at the intracellular aspect o f the p u m p s . T h e s e two classes o f sites may be the "unloading" sites for K influx and the "loading" sites for Na efflux (with occupation by Na not resulting in translocation by inactive pumps). However, the data available do not allow the d e v e l o p m e n t o f a model which takes into account all o f the interactions o f K~ with the p u m p s (cf. Garay and G a r r a h a n , 1973; Sachs et al., 1974b). T h e results o f the present study are consistent with an earlier proposal that active and passive cation t r a n s p o r t sites are interconvertible ( D u n h a m and H o f f m a n , 1971a): p u m p s are inactivated d u r i n g maturation o f LK cells (Dunham and H o f f m a n , 1971b) and are activated by t r e a t m e n t with anti-L ( L a u f et al., 1970). It was shown in Table V that there is insignificant inhibition by anti-L of passive K influx in i m m a t u r e LK cells (and substantial inhibition in m a t u r e cells). This result indicates the absence in the i m m a t u r e cells o f sites (inactive pumps) which in m a t u r e LK cells mediate passive K influx and which are sensitive to anti-L. T h e s e inactive p u m p s arise d u r i n g maturation by conversion o f p u m p s f r o m an active to an inactive m o d e . Total passive K flux also declines d u r i n g maturation o f LK cells, but this change is probably associated with K permeability unrelated to the Na-K p u m p s .

DUNHAM Passive K Transport in LK Sheep Red Cells

579

T h e results in Fig. 1 p r o v i d e an indication o f the n a t u r e o f anti-L action on passive t r a n s p o r t . T h e m a x i m u m velocity o f passive K influx is r e d u c e d , but not the a p p a r e n t affinity f o r K o f the sites m e d i a t i n g the influx. This observation is consistent with anti-L simply r e d u c i n g the n u m b e r o f sites m e d i a t i n g the passive influx. It was recently s h o w n that anti-L], o n e o f the two p o p u l a t i o n s o f antibodies in anti-L s e r u m , is responsible for the r e d u c t i o n in passive K t r a n s p o r t ( D u n h a m , 1976). At the same time anti-L1 also increases the p u m p flux in LK s h e e p cells, as does anti-Lp, the o t h e r type o f antibody. In one possible scheme o f action o f antiL s e r u m , anti-Lt activates the inactive p u m p sites, sites which mediate only passive K t r a n s p o r t b e f o r e t r e a t m e n t . T h e sites' affinity f o r o u a b a i n m a y increase as a c o n s e q u e n c e o f the t r e a t m e n t with anti-L, p e r h a p s f r o m n e a r zero (cf. Sachs et al., 1974a). Anti-Lp would have no effect on inactive p u m p sites, but would increase the p u m p flux at sites active b e f o r e t r e a t m e n t by d e c r e a s i n g the inhibition o f the p u m p by K~. T h e results in Fig. 4 s u p p o r t these suggestions: the stimulation o f the p u m p in cells essentially K-free r e p r e s e n t s stimulation o f the m a x i m u m velocity o f the p u m p . T h i s would be an effect o f the anti-L~, an consequence o f the conversion o f sites f r o m a passive to an active m o d e . (In a related finding, Blostein et al. [1971] d e m o n s t r a t e d stimulation by anti-L o f NaA T P a s e activity in the absence o f K in m e m b r a n e s o f LK s h e e p cells.) T h e c h a n g e in the s h a p e o f the c u r v e in Fig. 4 by anti-L t r e a t m e n t , p r e s u m ably the effect o f anti-Lp, c o r r e s p o n d s to the effect seen previously with anti-L t r e a t m e n t o f LK goat cells (Sachs et al., 1974b) and i n t e r p r e t e d to indicate a r e d u c e d affinity for Kl at Na translocation sites. Related observations on the affect o f anti-L on LK goat cells are consistent with this scheme: (a) anti-Ll has no effect on the p u m p in LK goat cells ( D u n h a m , 1976); a n d (b) the p r i m a r y effect o f anti-L on the goat cells is on the relative affinities o f the p u m p s for Na~ a n d Ki since the a n t i s e r u m had little effect on cells with very low (K)~ (Sachs et al., 1974 b). T h e results on affinity for o u a b a i n (Fig. 5) are also consistent with the above scheme. U n t r e a t e d LK cells have p u m p s with h e t e r o g e n e o u s affinities for ouabain. T h e p u m p s with lower affinity m a y be partially inactivated sites with a h i g h e r relative affinity for Ki t h a n for Na~. T h e increased rate o f o u a b a i n b i n d i n g at these sites in anti-L treated cells would be associated with their altered affinity f o r intracellular cations. T h e h o m o g e n e i t y o f the p u m p s on anti-L t r e a t e d cells in their affinity for o u a b a i n is t h e n a consequence o f two effects o f c o m p o n e n t s o f anti-L s e r u m : activation o f passively t r a n s p o r t i n g sites, a n d alteration o f p u m p i n g sites sensitive to [K]~ a n d t h e r e f o r e partially inactivated b e f o r e interaction with a n t i s e r u m . T h e actions o f anti-Ll a n d anti-Lp differ in f o r m s o f the p u m p p r e f e r r e d , but m a y have in c o m m o n an effect on the association o f Na with the intracellular aspect o f the p u m p m e c h a n i s m . I am grateful to Mr. E. S. Hall for his competent assistance, and to Dr. R. Blostein for producing the reticulocytes. This work was supported by grant AM-16196 from the National Institutes of Health. Received for publication 10 May 1976.

580

THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 68 " 1976 REFERENCES

BLOSTEIN, R., P. K. LAUF, and D. C. TOSTESON. 1971. Characteristics of Na+-ATPase o f low K + sheep red cell membranes stimulated by blood g r o u p L antiserum. Biochim. Biophys. Acta. 249:623-627. CASS, A., and M. DALMARK. 1973. Equilibrium dialysis of ions in nystatin-treated red cells. Nat. New Biol. 244:47-49. CAVlERES, J. D., and J. C. ELLORY. 1975. Allosteric inhibition of the sodium p u m p by external sodium. Nature (Lond.). 255:338-340. DUNHAM, P. B. 1975. Passive K transport in LK sheep red cells: effect of anti-L antibody and intracellular potassium. Fed. Proc. $4:237. DUNHAM, P. B. 1976. Anti-L serum: two populations o f antibodies affecting cation transport in LK erythrocytes of sheep and goats. Biochim. Biophys. Acta. 445:219-226. DUNHAM, P. B., and J. S. BLEIER. 1973. Potassium effluxes in goat red blood cells. Physiologist. 16:301. DUNHAM, P. B., and R. BLOSTEIN. 1976. Active potassium transport in reticulocytes of HK and LK sheep. Biochira. Biophys. Acta. In press. DUNHAM, P. B.', and J. F. HOrrMAN. 1971 a. Active cation transport and ouabain binding in high potassium and low potassium r e d blood cells of sheep.J. Gen. Physiol. 58:94-116. DUNHA~t, P. B., and J. F. HOrrMAN. 1971b. T h e n u m b e r o f Na+:K + p u m p sites on red blood cells from HK and LK lambs. Biochim. Biophys. Acta. 241:399-402. ELLORY, J. C . , J . R. SACHS, P. B. DUNHAM, a n d J . F. HOFr~tAN. 1972. T h e L antibody and potassium fluxes in LK red cells o f sheep and goats. In Biomembranes. Vol. 3. Passive permeability of cell membranes. F. Kreuzer and J. F. G. Slegers, editors. Plenum Publishing Corp., New York. 237-245. ELLORY, J. C., and E. M. TUCKER. 1969. Stimulation of the potassium transport system in low potassium type sheep red cells by specific antigen antibody reaction. Nature (Lond.). 222:477-478. ELLORY, J. C., and E. M. TUCKER. 1970. Active potassium transport and the L and M antigens of sheep and goat red cells. Biochim. Biophys. Acta. 219:160-168. EVANS, J. V., 1954. Electrolyte concentrations in red blood cells of British breeds of sheep. Nature (Lond.). 174:931-932. EVANS, J. v . , and J. w . B. KING. 1955. Genetic control o f sodium and potassium concentrations in the red blood cells of sheep. Nature (Lond.). 176:171. EVANS, J. v . , J. w . B. KING, B. L. COHEN, H . HARRIS, and F. L. WARREN. 1956. Genetics o f hemoglobin and blood potassium differences in sheep. Nature (Lond.). 178:849-850. GARAY, R. P., and P. J. GARRAHAN, 1973. T h e interactions of sodium and potassium with the sodium p u m p in red cells. J. Physiol. (Lond.). 231:297-325. GLYNN, I. M., a n d J . C. ELLORY. 1972. Stimulation of a sodium p u m p by an antibody that increases the a p p a r e n t affinity for sodium ions of the sodium-loading sites. In Role of Membranes in Secretory Processes. L. Bolls, R. D. Keynes, and W. Wilbrandt, editors. American Elsevier Publishing Co., Inc., New York. 224-237. GLVNN, I. M., V. L. LEw, and U. L/0THI. 1970. Reversal of the potassium entry mechanism in red cells, with and without reversal o f the entire p u m p cycle.J. Physiol. (London). 207:371-391. HoBBs, A. S., and P. B. DUNHAM. 1976. Evidence for two sodium sites on the external aspect of Na-K p u m p in h u m a n erythrocytes. Nature (Lond.). 260:651-652. HOVrMAN, J. F. 1966. T h e red cell m e m b r a n e and the transport of sodium and potassium. Am. J. Med. 41:666-680.

DUNHAM PassiveK Transport in LK Sheep Red Cells

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