Effect of anions on potassium self-exchange in ascites tumor cells

292 Biochimica et Biophysica Acta, 513 (1978) 292--295 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 71360

E F F E C T O F ANIONS ON POTASSIUM SELF-EXCHANGE IN ASCITES T U M O R CELLS

TILLY BAKKER-GRUNWALD Department of Physiology, University of Colorado Medical Center, 4200 E. Ninth Avenue, Denver, Colo. 80262 (U.S.A.) (Received May 24th, 1978)

Summary Potassium self-exchange in ascites cells was partially suppressed when chloride in the incubation medium was replaced b y bromide, and completely abolished in iodide-, nitrate- or sulfate-containing saline. Thus, it appears that potassium self-exchange activity is anion-dependent.

One of the modes by which monovalent cations are transferred across cell membranes is self-exchange [1]. In this communication we present evidence that K ÷ self-exchange in ascites cells is dependent on the anion composition of the medium. Fig. 1A shows the effect of ouabain and furosemide on the influx of 86Rb into ascites cells; this isotope serves as a tracer for K ÷ in this system (e.g., ref. 2 ). In the absence of furosemide ouabain had a small inhibitory effect. Furosemide alone decreased the rate of 86Rb influx by 50%. Both inhibitors together brought about a marked reduction to approximately 8% of the control value. These findings are similar to those of Tupper [2]. Ouabain caused a net loss of K ÷ (Table I); we show elsewhere (unpublished) that the concentration of ouabain employed is sufficient to totally inhibit the (Na + + K + ) ATPase. Furosemide did not influence the cellular K ÷ content (Table I). In another experiment (Table II) the furosemide-sensitive c o m p o n e n t of 86Rb influx was shown to be equal in absolute magnitude to that of efflux, both in the absence and in the presence of ouabain; in the presence of ouabain the furosemide-sensitive c o m p o n e n t was markedly stimulated, as in the experiment of Fig. 1. Fig. 1B shows the results of a similar experiment carried o u t in a saline solution in which 100 mM NaNO3 was substituted for NaC1. In this system furosemide had no effect. Moreover, all fluxes were reduced to the rates observed in the presence of furosemide in C1- saline (1.9 and 0.3 ~mol K +. g-1

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time (min} Fig. I . E f f e c t o f i n h i b i t o r s o n SSRb i n f l u x i n t o ascites cells in Cl---saline a n d NO~--saline. A s c i t e s cells w e r e g r o w n in t h e a b d o m i n a l cavities o f Swiss W e b s t e r m i c e , a n d h a r v e s t e d a f t e r 6 - - 1 2 d a y s . T h e y w e r e w a s h e d in a saline s o l u t i o n c o n t a i n i n g : 50 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid ( H E P E S ) a n i o n , n e u t r a l i z e d w i t h 2 0 m M N a O H a n d 4.4 m M K O H , 1 m M CaCI 2 , 1 m M MgSO 4, 20 m M glucose, a n d e i t h e r 1 0 0 m M NaCI o r 1 0 0 m M N a N O 3 . T h e final p H w a s 7.5. T h e y w e r e t h e n s u s p e n d e d at a conc e n t r a t i o n o f 1.5% in t h e r e s p e c t i v e saline plus o r m i n u s f u r o s e m i d e (1.2 m M ) , a n d i n c u b a t e d f o r 9 0 rain at 3 7 ° C to a l l o w t h e i o n c o n t e n t t o stabilize. A t t i m e z e r o SCRb (20 n C i / m l ) w a s a d d e d a l o n e or t o g e t h e r w i t h o u a b a l n (1.2 raM). A t t h e i n d i c a t e d t i m e s , d u p l i c a t e 3 m l s a m p l e s o f s u s p e n s i o n w e r e s p u n d o w n at 1 5 0 0 r e v . / m i n , t h e s u p e r n a t a n t d e c a n t e d a n d s a v e d , a n d t h e cells e x t r a c t e d w i t h 5% t r i c h l o r o a c e t i c acid. R a d i o a c t i v i t y o f cell e x t r a c t s a n d s u p e r n a t a n t s was c o u n t e d b y liquid scintillation c o u n t i n g , a n d K + cont e n t d e t e r m i n e d b y f l a m e p h o t o m e t r y (see T a b l e I). E x t r a c e l l u l a r space c o r r e c t i o n w a s m a d e b y e x t r a p o l a t i n g t h e u p t a k e c u r v e s t o z e r o t i m e . Points s h o w n are a v e r a g e o f d u p l i c a t e s ; d u p l i c a t e s w e r e e q u a l w i t h i n 5%. A, Cl---saline; B, NO~--saline. o (C), c o n t r o l ; • (O), o u a b a i n ; ~ (F), f u r o s e m i d e ; • (O + F), o u a b a l n plus f u r o s e m i d e .

dry w t . . m i n -1 , in the absence and presence of ouabain, respectively). K ÷ contents were comparable to those in the C1- system (Table I). From these data it appeared that either removal of C1- or addition of NO~ abolished the furosemide-sensitive components of SSRb influx. In order to further examine this point we investigated the effect of several other anions in the presence of ouabain, where the size of the furosemide-inhibitable component was larger. Fig. 2 shows the results of these experiments. The dotted line gives the level of influx in the presence of furosemide and ouabain, i.e. the non-mediated influx. This level was the same in all saline systems tested (0.37 + 0.02 pmol K+.g -l dry w t . . m i n - ' ). As C1- was gradually replaced by I-, NO~ and SO~-, the furosemide-sensitive component of the flux was reduced; at 2 mM Cl-it was completely abolished. In contrast, Br- at 90 mM suppressed TABLE I E F F E C T O F I N H I B I T O R S A N D N I T R A T E O N K+ C O N T E N T S a m p l e s w e r e o b t a i n e d f r o m t h e e x p e r i m e n t s h o w n in Fig. 1, 24 rain a f t e r a d d i t i o n o f SSRb plus o r m i n u s o u a b a i n . V a l u e s given are t h e a v e r a g e o f d u p l i c a t e s ; d u p l i c a t e s w e r e e q u a l w i t h i n 5%. Major a n i o n

K + content (mmol. g-I dry wt.)

NO;

c1 -

- Furosemide + Furosemide

-- o u a b a i n

+ ouabain

- ouabain

+ ouabain

0.31 0.30

0.25 0.26

0.32 0.32

0.27 0.27

294

the furosemide-sensitive component by only 35%. Cellular K + content and the net loss induced by ouabain (not shown) were equal for all anions tested except SO~-, and comparable to those in Table I. In SO~- saline, values were approximately 24% lower on a dry weight-basis; this is probably due to shrinkage of the cells [3]. Tupper [2] interpreted his data on the effect of furosemide to mean that this compound inhibited K + self-exchange. For the experiments presented here this interpretation is supported by the observation that the furosemideinhibitable fractions of influx and efflux were equal in absolute magnitude (Table II), as well as the fact that furosemide did not alter K + content (Table I). It thus seems justified to interpret the furosemide-inhibitable 86Rb flux components as arising from K + self-exchange. Our data show that this self-exchange is influenced by the anionic composition of the medium. The concentration dependence of this effect was the same for I-, NO~, and SO~- (Fig. 2). This suggests that C1- is required for K + self-exchange with a rather high apparent Kin. Br- appears partially able to substitute for C1- in sustaining K + selfexchange. Effects of anions on unidirectional cation fluxes comparable to those described here have been reported by Funder and Wieth [4] for human red blood cells.

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Fig. 2. E f f e c t s o f d i f f e r e n t a n i o n s o n t h e o u a b a i n - i n h i b i t e d i n f l u x o f S6Rb. Saline s o l u t i o n s w e r e similar t o t h a t d e s c r i b e d in the l e g e n d t o Fig. 1.with either NaBr, NaI, NaNO s (100 mM) or Na 2 SO 4 (50 raM) s u b s t i t u t e d f o r NaCI. Cl--saline w a s m i x e d w i t h t h e o t h e r salines t o give t h e i n d i c a t e d CI'- c o n c e n t r a t i o n s . T h e e x p e r i m e n t w a s p e r f o r m e d as d e s c r i b e d for Fig. 1. R a t e s w e r e c a l c u l a t e d f r o m t h e d i f f e r e n c e in celinlar r a d i o a c t i v i t y b e t w e e n 3 0 rain and 1 0 rain after t h e a d d i t i o n o f SSRb (20 nCi/ml) p l u s o u a b a i n (1.2 mM). T h e d o t t e d line gives t h e i n f l u x rate for t h e five u n m i x e d saline s o l u t i o n s in t h e p r e s e n c e o f f u r o s e m i d e (1.2 mM). I - , w h i c h w a s f o u n d t o b e a s t r o n g q u e n c h e r in our s c i n t i l l a t i o n s y s t e m , w a s p r e c i p i t a t e d w i t h A g N O s prior t o c o u n t i n g o f r a d i o a c t i v i t y , o, Br-; o, I ' ; ~, NOs;V, SO~-.

295 T A B L E II E F F E C T O F I N H I B I T O R S O N S6Rb I N F L U X A N D E F F L U X IN C l - S A L I N E F o r t h e r a t e o f S~Rb e f f l u x , a s c i t e s cells w e r e e q u i l i b r a t e d f o r 9 0 m i n i n t h e p r e s e n c e o f t h e i s o t o p e ( 1 0 n C i / m l ) . A t t h e e n d o f t h i s p e r i o d cells w e r e s p u n d o w n f o r 1 r a i n a t 1 0 0 0 r e v . / m i n , a n d a t t i m e z e r o t h e p e l l e t w a s r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 1 . 5 % in C l - s a l l n e in t h e p r e s e n c e o r a b s e n c e o f i n h i b i t o r s ( c o n c e n t r a t i o n , 1 , 2 r a M ) . R a d i o a c t i v i t y r e m a i n i n g in t h e cells a t a g i v e n t i m e w a s d e t e r m i n e d as d e s c r i b e d in t h e l e g e n d t o Fig. 1. E f f l u x r a t e c o n s t a n t s ( k e ) w e r e o b t a i n e d b y l i n e a r r e g r e s s i o n from semilogarithmie plots of the decrease in cellular radioactivity with time. Correlation coefficients (r) e x c e e d e d 0 . 9 4 in all cases. T o t a l e f f l u x w a s c a l c u l a t e d f r o m t h e p r o d u c t o f k e a n d t h e a v e r a g e o f t h e c e l l u l a r K + c o n t e n t s a t z e r o t i m e a n d 2 4 m i n . I n f l u x w a s d e t e r m i n e d in t h e s a m e e x p e r i m e n t b y m e t h o d s d e s c r i b e d i n t h e l e g e n d t o Fig. 1; t h e e x t e r n a l K + c o n c e n t r a t i o n w a s 2 . 5 m M . T h e v a l u e s f o r the furosemide-sensitive components were obtained by subtraction from the values given for the unidirectional fluxes; the error in these derived values can be estimated at about 10%. Efflux rate constant k e (rain -t )

-+ Furosemide + O uabain + Furosemide + ouabain

Unidirectional fluxes (~umol K + . g - I d r y w t . . m i n - I )

Furosemide-sensitive components O~mol K +. g - t d r y w t . . m i n - t )

Influx

Efflux

Influx

Efflux

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7.1 3.2 7.1

6.8 2.8 10.8

3.9

4.0

6.8

8.2

0.008

0.3

2.6

The nature o f the anion effect on K + self-exchange is not dear. Apparently the passive permeability of the anion is not involved since C1- and NO~- have comparable passive permeabilities (unpublished data), whereas that of SO24- has been reported to be considerably lower [3]. A clue to the mechanism o f K + self-exchange and its susceptibility to the anion composition of the medium may possibly be found in the observation of Heinz et ah [5] that K + and C1- are co-transported under certain conditions. The fact that K + self-exchange was stimulated in the presence of ouabain suggests a close relation between pumping and exchange activities. This relation (often of a complementary nature) has been observed in a number of systems [6]. This research was supported by NIH grant AM 15807 to Dr. Margaret C. Neville, whom I would like to thank for her invaluable input. References 1 2 3 4 5

G l y n n , I.M. a n d K a r l i s h , S . J . D . ( 1 9 7 5 ) A n n u . R e v . P h y s i o L 3 7 , 1 3 - - 5 5 Tupper, J.T. (1975) Biochim. Biophys. Acta 394, 586--596 L e v i n s o n , C. a n d Villeral, M.I~ ( 1 9 7 4 ) J. Cell. P h y s i o L 8 5 , 1 - - 1 4 Funder, J. and Wieth, J.O. ( 1 9 6 7 ) A c t a P h y s i o L S c a n d . 71, 1 6 8 - - 1 8 5 H e i n z , E., G e c k , P., P i e t r z y k , C., B u r c k h a r d t , G. a n d Pfeiffer0 B. ( 1 9 7 7 ) J. S u p r a m o L S t r u e t . 6, 125--133 6 Mills, B. a n d T u p p e r , J . T . ( 1 9 7 6 ) J. C e i l P h y s i o l . 8 9 , 1 2 3 - - 1 3 2