BIOCHIMIE, 1979, 61, 1185-1192.

Ionophore-mediated cation translocation in artificial systems. I. A23187-mediated

calcium

translocation.

W. J. MALAISS,E~, I. VALVERDE, G. SO'MERS and E. COUTUR~IER.

G. D E ¥ I S ,

Laboratory o[ Experimental Medicine, Brussels University, Brussels, Belgium.

(22-3-1979),

R6sum6.

Summary.

L ' i o n o p h o r e A23187 p r o v o q u e l a t r a n s l o c a t i o n de calcium d'un tampon Hepes en milieu aqueux dans une phase orqanique immiscible. Aux concentrations basses de calcium darts la p h a s e a q u e u s e i n i t i a l e (0,01 et 1,0 raM), l e s mol6cules d'ionophores semblent aqir en coop6rativit6 positive dans le ph6nom~ne de transloc a t i o n . En effet, l e r a p p o r t e n t r e l e s c o n s t a n t e s d e v i t e s s e d ' a s s o c i a t i o n et d e d i s s o c i a t i o n d u calcium et de l'ionophore auqmente avec les concentrations croissantes d'ionophore. De ce raft, l a q u a n t i t 6 d e c a l c i u m a p p a r a i s s a n t d a n s la phase orqanique est proportionnelle & une puissance de la concentration en ionophore. Compte tenu de ce ph6nom6ne, la relation entre l a t r a n s l o c a t i o n d e c a l c i u m et s a c o n c e n t r a t i o n e s t r6cjie p a r l e s r 6 q l e s c o u r a n t e s c o n c e r n a n t l a cin6tique des liaisons substrat-r6cepteur.

T h e i o n o p h o r e A23187 s t i m u l a t e s t h e t r a n s l o c a t i o n of c a l c i u m f r o m a n a q u e o u s H e p e s b u f fer into a n o r q a n i c i m m i s c i b l e p h a s e . A t s a t u r a t i n q c a l c i u m c o n c e n t r a t i o n s , 2 m o l e c u l e s of i o n o p h o r e s e e m to c o m p l e x e a c h a t o m of c a l c i u m . Consistent with such a stoichiometric behaviour, t h e a p p a r e n t r a t i o of c a l c i u m - i o n o p h o r e a s s o c i a t i o n to d i s s o c i a t i o n r a t e c o n s t a n t s i n c r e a s e s a s t h e c o n c e n t r a t i o n of i o n o p h o r e is r a i s e d . A s a result, at low calcium concentrations, the a m o u n t of t r a n s l o c a t e d c a l c i u m i n c r e a s e s a s a p o w e r f u n c t i o n of A23187 c o n c e n t r a t i o n . W h e n a l l o w a n c e is m a d e for s u c h a p h e n o m e n o n , t h e r e l a t i o n b e t w e e n c a l c i u m t r a n s l o c a t i o n a n d conc e n t r a t i o n is c h a r a c t e r i z e d b y u s u a l s u b s t r a t e receptor bindinq kinetics.

Mots-el4s : Ianophore (A23187), calcium, transport.

Key-words : Ionophore A23187 - - calcium- transport.

Introduction.

Material

The a n t i b i o t i c s A23187 a n d X53qA are, at the p r e s e n t time, the c a l c i u m - i o n o p h o r e s most curr e n t l y used in e x p e r i m e n t a l b i o l o g y [1, 2]. The p r e s e n t series of investigations deals w i t h the influence of cations, b i o l o g i c a l f a c t o r s a n d p h a r m a c o l o g i c a l agents u p o n i o n o p h o r e - m e d i a t e d calc i u m t r a n s t o c a t i o n in artificial models. In this r e p o r t , w e have c h a r a c t e r i z e d the m o d a l i t y of calcium-A2r3187 i n t e r a c t i o n in a t~vo-phase m o d e l system.

Unless otherwise stated, a small volume (0.2 ml) of a Hepes buffer (25 mM ; pH 7.0) containing Na + 123, K÷ 5 and C1- 120 mM and 45calcium (10 ~tC/m]) xvas placed in a glass tube (51 mm height, 5 mm inner diameter) and vigorously mixed for 1 win at room temperature with an equal volume of a mixture of toluene/butanol (7/3, v/v), containing, when required, the ionophore A23187 (Eli Lilly Research Laboratories, Indianapolis, USA). An aliquot (0.1 ml) of the supernatant immiscible phase was then examined for its radioactive content by liquid scintillation. The amount of calcium present in the final organic phase is expressed in terms of 45calcium radioactivity (cpm/0.1 ml) or as a molar concentration. In some experiments, the radioactivity of the final aqueous phase was also measured. For this purpose, an aliquot (0.1 ml) of such a phase was removed with the aid of a fine needle mounted on a syringe. 81

To whom all correspondence should be addressed.

and Methods.

1186

W. J. Malaisse and coll. (100.0 ~M), t h e c o n c e n t r a t i o n of c a l c i u m i n t h e f i n a l o r g a n i c p h a s e a m o u n t e d to 2.16 _ 0.0~5 ~M.

Results. Influence o[ the mixing procedure. W h e n t h e i n i t i a l a q u e o u s p h a s e c o n t a i n e d a t r a c e a m o u n t of r a d i o a c t i v e c a l c i u m (11.1 ~ M ; ~3.62 × 106 c p m / 0.2 m l ) a n d w h e n i n c r e a s i n g c o n c e n t r a t i o n s of A23187 (2.0, 10.0 a n d 100.0 ttM) w e r e p r e s e n t i n t h e i n i t i a l o r g a n i c p h a s e , t h e r a d i o a c t i v i t y of t h e f i n a l o r g a n i c p h a s e a v e r a g e d 3~1,1 _ 11 ( n = 6), 6,270 _ 157 (n = 8) a n d 3,51,30'6 _ 8,206 c p m / 0.1 m l ( n = 9), r e s p e c t i v e l y , a s d i s t i n c t f r o m a c o n t r o l v a l u e ( i n t h e a b s e n c e of A23,187) of 3,5 _ 6 c p m / 0 . 1 ml. F r o m t h e s e d a t a , it c a n b e c a l c u l a t e d t h a t , at t h e h i g h e s t l e v e l of i o n o p h o r e

T h e d u r a t i o n of t h e m i x i n g p e r i o d h a d l i t t l e e f f e c t u p o n t h e a m o u n t of r a d i o a c t i v e m a t e r i a l recovered in the final organic phase. At the most, t h e r e w a s a t r e n d f o r t h e a m o u n t of c a l c i u m t r a n s l o c a t i o n to b e s l i g h t l y l o w e r a f t e r p r o l o n g e d (10 r a i n ) t h a n a f t e r s h o r t (1 to 5 m i n ) m i x i n g periods. Thus, pooling the data obtained in the a b o v e - m e n t i o n e d e x p e r i m e n t at t h e t h r e e c o n c e n t r a t i o n s of A23~287 (2.0, 10.0 a n d 100.0 ~M), t h e r e a d i n g s , r e l a t i v e to t h e o v e r a l l m e a n v a l u e o b t a i n e d at e a c h i o n o p h o r e level, a m o u n t e d t o 100.7 _

TABLE I.

E[[ect of temperature upon the appearance o[ ~Scalcium in the final organic phase. A23187 (~M)

Temperature

M i l i n g time (rain)

(0-~oC)

(Ca. 20~C)

(37"C)

Nil

1 10

0.3 -{- 0.1 0 . 5 -~- 0.1

0.1 --F- 0.1 0 . 2 -t- 0.1

0 . 2 ___ 0.1 0 . 2 -4- 0.1

10

1 5 10

2 1 . 0 -I- 0 . 3 22.1 ___~0.5 20.9 -t- 0 . 5

14.4 -F- 0 . 2 15.0 -~- 0 . 4 11.7 -t- 0 . 4

13.3 -4- 0 . 3 14.3 ___~0.1 12.0 ~ - 0 . 2

Mean values ( _ SEM) refer to 3 i n d i p e n d e n t m e a s u r e m e n t s , a n d are expressed as nM, after correction for t h e b a c k g r o u n d r a d i o a c t i v i t y . The initial c o n c e n t r a t i o n of t a l c u m in the aqueous p h a s e a m o u n t e d to 11.1 ~tM.

TABLE II.

lonophore-mediated calcium translocalion in relation to the volume of the initial aqueous and organic phases. Line

Aqueous (ml)

Organic (ml)

~.~Ca translocation (per cent}

Aqueous (nil}

Organic (ml)

4~Ca translocation (per cent}

0.20 0.20

0.20 0.30

100.0 ___ 1 . 4 ( 6 ) 98.3 -}- 5 . 4 (3)

0.20 0.20 0.20 0.20

0.40 0.60 0.80 1.00

100.0 94.2 88.6 81.0

Small tubes 1 2 3 4

0.10 0.20 0.30 0.40

0.20 0.20 0.20 0.20

87.7 100.0 110.4 110.3

5 6 7 8

0.50 0.65 0.80 0.95

0.20 0.20 0.20 0.20

100.0 100.8 98.6 98.2

-I-~-t-~-

1.4 (6) 2.3(9) 1.9(5) 5.8(3)

Large tubes + 1.4(6) ~__ 5 . 9 ( 8 ) ~__ 0 . 8 (3) -~- 4 . 4 ( 6 )

-~-~-~___

1.6(6) 3.2(6) 2 . 9 (3) 2.2(3)

Mean values ( _ SEM) for the radioactive c o n t e n t of the final organic p h a s e (cpm/0.1 ml) are expressed relative to the control values s h o w n in line 2 (small tubes) a n d line 5 (large tubes), respectively, a n d are followed b y the n u m b e r of i n d i v i d u a l o b s e r v a t i o n s (in parentheses). Data o b t a i n e d w i t h s m a l l a n d large t u b e s are presented separately because, in the large tubes, the recovery of 45 calcium was i n v a r i a b l y lower t h a n t h a t f o u n d in the s m a l l tubes. All e x p e r i m e n t s were carried out at a low calcium c o n c e n t r a t i o n in t h e i n i t i a l aqueous p h a s e (11.1 or 22.2 ttM) a n d at a A23187 level of 10.0 ~tM in the initial organic phase. Under the e x p e r i m e n t a l conditions given in line 2, w h i c h are identical to those c u r r e n t l y used in the p r e s e n t study, the c o n c e n t r a t i o n of calcium in the final organic p h a s e averaged 28.9 a n d 52.5 nM a t calcium 11.1 and 22.2 ~tM, respectively.

BIOCHIMIE, 1979, 61, n ° 10.

A 2 3 1 8 7 - m e d i a t e d calcium translocalion. 2.4 (n = 11), 102.3 __ 2.9 (n = 6) and 96.3 __ 1.1 p er cent (n = 6'). after a m i x i n g p e r i o d of 1, 5 and 10 min, r es p e c t iv e l y .

Influence of temperature. T h e amount of radioa c t i v i t y r e c o v e r e d in the fi,nal o r g a n i c phase was h i g h e r at l o w t e m p e r a t u r e than at r o o m temper a l u r e (table I). W e have not e x p l o r e d the factor(s) responsible for this p h e n o m e n o n (e.g. greater increase in the rate constant for c a l c i u m - i o n o p h o r e dissociation t h a n in the rate constant for association, at the h i g h e r t e m p e r a t u r e ; see ref. 3). Th e

1187

Influence of changes in the volume of the aqueous and organic phases. Th e r e c o v e r y of 45calcium in the final o r g a n i c p h ase was slightly but significantly affected by the r e s p e c t i v e volumes of the initial aqueous and o r g a n i c phases (table II). W h e n the v o l u m e of the o r g a n i c phase was maint a i n e d constant (0.2 ml), the a m o u n t of 45calcium t r a n s l o c a t e d (cpm/0.1 ml) i n c r e a s e d as the volume of the aqueous phase was raised f r o m 0.1 to 0.3 ml ,oo

50

//-'/.~;

50

/|~|

/

40

/ 10

%

/

~2 ~1

20 ~L 10

50

1 2 3 4 5

i 10

' 15

.

"o

40

30

m

.

~5.0

z 2.5

.

.

.

.

.

io" A23187

a

°IZ,I)

.1 .0~ / ( .02

"=~-" 5

10

15

0

1

0.50 I

o.25 I

.-="

I ,x" 0 b" , , = i 0 1 2 3 4

,4 23107

i 5 CALCIUM

I

pM

¢ i 10 15 CONCENTRATION (rnM)

i 20

FIG. 1. Effect of increasing calcium concentration in the initial Hepes buffer (abcissa) upon the calcium concentration of the final organic phase (ordinates), at three concentrations of A23187 in the initial organic phase. Mean values (___ SEM, which are only indicated when their size exceeded that of the mean value) refer to 6 (top panel) or 3 individual measurements. In the insert to the right, the values for calcium translocation at calcium 1.0 mM are expressed in per cent of the saturation value found at high calcium concentration (15 to 20. mM), in the presence of A23187 100 ~M (closed circles, solid line), 10 ttM (open circles, dashed line) and 1 IxM (crosses, dotted line), rcspectively. -

-

a m o u n t of 4~calcium t r a n s l o c a l e d in lhe final o r g a n i c phase w a s not vastIy different at 37°C and r o o m t e m p e r a t u r e . All f u r t h e r m e a s u r e m e n t s w e r e p e r f o r m e d at r o o m t e m p e r a t u r e and after a s h o r t (1 to 2 min) m i x i n g period.

BIOCHIMIE, 1979, 61, n ° 10.

v

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