Biochimica et Biophysica Acta, 389 (1975) 506-515

C) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Nctherlands BBA 76943

CaZ+-DEPENDENT TRANSPORT

INHIBITORY

IN S A R C O P L A S M I C

EFFECTS

OF

RETICULUM

Na + AND

K-

O N Ca -~+

VESICLES

CERL1 ROCHA GATTASS and LEOPOLDO DE MEIS lnstituto de Biofisica, Centro de Ci#ncias da Sat~de, Unit~ersidade Federal do Rio de Janeiro, Cidade Universit6ria, Ilha do Ftmdtio. Rio de Janeiro. GB (Brazil)

(Received October 25th, 1974)

SUMMARY Effects o f N a + a n d K + on C a 2 + t r a n s p o r t by s a r c o p l a s m i c reticulum vesicles were studied in a m e d i u m c o n t a i n i n g high M g 2+ a n d A T P ( 2 r a M ) and l o w Ca z+ (0.44/~M) concentrations. U n d e r these conditions, N a + a n d K + inhibit Ca 2 + uptake, A T P a s e activity a n d m e m b r a n e p h o s p h o r y l a t i o n by A T P . Since the c o n c e n t r a t i o n s o f A T P a n d C a 2 + used are consistent with relaxation in vivo, the results suggest that u n d e r physiological resting conditions the C a 2 + p u m p o f the s a r c o p l a s m i c reticulum o p e r a t e s b e l o w its m a x i m a l capacity.

INTRODUCTION A highly efficient A T P - d e p e n d e n t system for Ca z ÷ t r a n s p o r t has been described in s a r c o p l a s m i c reticulum vesicles isolated f r o m r a b b i t skeletal muscle h o m o g e n a t e s . This system plays a key role in the process o f e x c i t a t i o n - c o n t r a c t i o n coupling in muscle cells [ 1 4 ] . It has been shown t h a t N a + a n d K + activate the Ca 2 + p u m p in the presence o f 0.1 - 4.0 m M A T P a n d C a 2+ c o n c e n t r a t i o n s a b o v e 1 0 / t M [5-7]. This c o n c e n t r a t i o n o f C a 2 + is in the range o f that required to fully activate a muscle fiber [3, 8-9]. In this report, the effects o f alkali ions on Ca 2 + t r a n s p o r t were studied, using c o n c e n t r a t i o n s o f M g A T P a n d Ca 2 + in the range o f those expected to be f o u n d in a relaxed living fiber [10-11 ]. MATERIALS AND METHODS S a r c o p l a s m i c r e t i c u l u m vesicles

These were p r e p a r e d f r o m r a b b i t skeletal muscle as previously described [12]. Ca 2 + u p t a k e was determined, unless stated otherwise, using a m e d i u m consisting o f 10mM Tris/maleate buffer ( p H 6.85) 2 m M MgC12, 2 m M A T P , 2 m M Ethyleneglycolbis-(~-aminoethylether)-N,N'-tetraacetic acid ( E G T A ) , 4 m M p o t a s s i u m oxalate, Abbreviation: EGTA, ethyleneglycol-bis-(fl-aminoethylether)-N,N'-tetraacetic acid.

507 0.2 m M 45CAC 12, 0.25 mg sarcoplasmic reticulum vesicles protein/ml and the specified concentrations of NaC1 or K C I . The control medium, to which no monovalent cation was added, contained approximately 15 m M K + from the other reagents used. The reaction was started by the addition of sarcoplasmic reticulum vesicles and stopped after different incubation intervals at 37 °C by removing the vesicles with Millipore filters [13]. The concentrations of MgCl 2 and protein were chosen after 2 sets of preliminary experiments in which the Mg concentration was varied from 0.25 to 4.0 mM and sarcoplasmic reticulum vesicles protein varied from 0.05 to 0.4 mg/ml. The percentage of Ca 2 + removed from the medium after 12 rain was essentially constant in the range of 0.1-3.0 m M Mg and 0.2-0.4 mg sarcoplasmic reticulum vesicles protein/ ml, respectively. Different values for the C a - E G T A dissociation constant (Kd) have been reported [2, 12, 14-16]. In this paper, the value of 3.95 • 1 0 - 6 M was used [12, 16]. Thus the calculated free C a 2 ÷ concentration in the control medium was 0.44 uM. The apparent K d of the C a - E G T A complex in the presence of oxalate is not modified by Na +, K + or Li + [12].

Ca 2 ÷ release Ca 2 + release was determined after preloading the vesicles in a medium containing 10 m M Tris/maleate buffer (pH 6.85), 2 m M ATP, 2 m M MgC12, 4 m M potassium oxalate, 1.0 m M EGTA, 1.0 m M 4SCaC12 and 2 mg sarcoplasmic reticulum vesicles protein/ml. After a 3-min incubation, the vesicles had taken up more than 95 ~ of the Ca 2 ÷. Calcium release was initiated by diluting the loaded vesicles 20-fold in a medium similar to that described for loading but without Ca 2 ÷ or ATP. Ca 2 ÷ efflux was assayed by filtering aliquots of the reaction mixture at different times following dilution and measuring the radioactivity of the filtrate as in the determination of Ca 2 ÷ uptake. ATPase activity ATPase activity assayed as described previously [13] using the medium described for Ca 2 + uptake, after stopping the reaction by filtration through Millipore filters. The Ca 2 ÷-dependent ATPase was calculated as the difference between the activity of the (Ca 2 + + Mg 2 ÷)dependent ATPase (assayed in the presence of Ca 2 ÷ and Mg 2 ÷) .and the activity of the Mg 2 ÷_dependent ATPase (assayed in a medium with Mg 2 ÷ a n d 2 m M E G T A but without added Ca2+). E-P formation from [y-32p]ATP E-P formation from [y-32p]ATP was assayed as described by de Meis [17]. The reaction was started by the addition of sarcoplasmic reticulum vesicles protein and stopped after 20 s by adding perchloric or trichloroacetic acid. RESULTS The inhibitory effects of N a + and K + o n C a 2 + transport by sarcoplasmic reticulum vesicles in a medium containing low Ca 2 + and high A T P are shown in Fig. 1. In this experiment, the Ca 2 + uptake was measured after a prolonged incubation inter-

508

~.

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0 2O

I 0

I

I

I 100

I

I 20q

salt, mM Fig. I. Effect o f alkali ions a n d sucrose on the C a 2+ uptake at the steady state. The assay m e d i u m c o n t a i n e d 10 m M Tris/maleate buffer /pH 6.85), 2 m M MgC12. 2 m M ATP, 2 m M E G T A , 4 m M p o t a s s i u m oxalate, 0.2 m M "*SCaCIz, 0.25 m g sarcoplasmic reticulum vesicles protein/ml and the specified concentrations o f salts or sucrose. I n c u b a t i o n time was 12 rain. The wdues represent the average ~ S . E . o f 4 experiments. Ca 2 + incorporated in the control m e d i u m ,~as 0.43/maol/mg protein. ( Q ) control, control plus ( A ) Na +, (11) K -~, (Cl) Li + or (C') sucrose.

L

/"

1"

~ 80

~

40

2

minutes Fig. 2. Ca 2+ uptake as a function o f the initial free Ca z+ concentration in the assay medium. For the Ca 2+ concentrations o f (U) 0.981~M, ( 0 ) 0.44/~M and ( A ) 0.22/~M, the assay medium contained 0.2 m M 4~CaC]= and 1.0, 2.0 and 4.0 m M E G T A , respectively. Other conditions were as described for the control medium in Fig. 1. Each value represents the average :: S.E. o f 4 experiments.

509

val to ensure that maximal Ca 2 + accumulation was attained, i.e. at the steady state. N o inhibition was observed when LiC1 or sucrose was added to the assay medium. In the next experiments, we examined the parameters controlling the percentage o f Ca 2 ÷ removed from the medium by sarcoplasmic reticulum vesicles in the steady state. Fig. 2 is a control experiment which shows that the percentage of Ca 2 ÷ removed from the medium by sarcoplasmic reticulum vesicles in the steady state decreases with the initial free Ca 2+ concentration [3, 19, 20]. After 6 rain, maximal accumulation was obtained, implying a balance between the calcium influx due to active transport (ia) and the calcium efflux due to passive diffusion (ep). Since oxalate was included in the asssay medium, the free Ca 2 ÷ concentration inside the vesicles will be fixed by the calcium oxalate solubility product [4]. Table I shows that, independently o f the initial free Ca 2 ÷ concentration o f the medium, the equilibrium between ia and ep is reached at the same free Ca 2 ÷ concentration in the medium, i.e. 0.1 pM. Therefore, if the rate efflux depends only on the Ca 2 ÷ concentration gradient, it should be identical in the 3 steady-state conditions shown in Fig. 2. On the other hand, the rate of active transport decreases with a decrease in the Ca 2 ÷ o f the medium. Thus, as Ca 2 ÷ is removed by the vesicles, the rate of i a falls progressively until it reaches that o f ep [4, 20]. The inhibitory effects of N a ÷ and K ÷ shown in Fig. 1 could be related to a modification of the balance between ia and ep, so that the steady state is reached at higher external Ca 2+ concentrations. In fact, this appears to be the case, as increasing concentrations o f N a + or K ÷ in the assay medium lead to a progressively less effective removal o f Ca 2 ÷ from the medium in the steady state (Fig. 3). Fig. 4 shows that if N a ÷ or K ÷ is added to the control incubation medium after the equilibrium between ia and ep is attained, part of the Ca 2+ that was accumulated by sarcoplasmic reticulum vesicles is released, and subsequently a new steady state is reached as the rates o f i a and ep tend to equilibrate at a higher external Ca 2 ÷ concentration. In the experiments o f Figs 5 and 6, we attempted to distinguish an effect on ia f r o m an increase in the efflux. Since N a ÷ and K ÷ have no effect on the passive efflux (Fig. 5), but decrease the initial rate o f Ca 2 ÷ uptake (Fig. 6), we infer that the inhibitory effect o f these ions shown in Figs 1, 3 and 4 are due to a depression of the active Ca 2 ÷ influx. Active Ca 2+ transport is mediated by a m e m b r a n e - b o u n d ATPase which is highly sensitive to changes in the free Ca 2 ÷ concentration of the medium. In the process o f A T P hydrolysis, the 7-phosphate o f A T P is covalently bound to a m e m b r a n e TABLE I I O N I C Ca 2+ IN T H E A S S A Y M E D I U M A T T H E S T E A D Y STATE The experimental conditions were as described for Fig. 2. The incubation time was 6 min. Each value represents the average ± S . E . o f 8 experiments. Initial free Ca 2+ in the medium (/~M)

0.22

0.44

0.98

Ionic Ca 2 + at the steady state (/~M)

0.10~0.01

0.104-0.01

0.09dz0.02

510

protein. This phosphoprotein (E-P)represents an intermediate product in the sequence of reactions leading to Ca 2+ transport and Pi liberation [16, 19, 21]. If Na + and K + interfere with active Ca 2+ transport, they should also modify the Ca-dependent ATPase and E-P formation.

80

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16 i m m

25 (J

._c ¢o 34 .u

c::

o

6

9

12

0

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1

/

3

6

9

12

44

minutes

Fig. 3. Effects o f different N a + and K + concentrations on the time course o f Ca 2+ uptake. The assay m e d i u m as described in the legend to Fig. 1 contained in addition ( O ) none, ( ~ ) 50 m M , ( A ) 100 mM, ( ( 3 ) 2 0 0 m M NaCI ( l e f t ) o r KCI (right). The values represent the average ~ S . E . of 3 experiments.

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25

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3 20

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40

minutes

Fig. 4. Effects o f the addition o f N a + or K + to a system in which the Ca 2+ transport is in the steady state. After 10 min incubation in the control m e d i u m described in Fig. 1 ( O ) , 200 m M NaC1 ( A ) or KCI (11) was added to the medium. Each value represents the average ± S . E . of 5 experiments.

511

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40_

(J

20

~~J/

0

I 0

J

10

20

30

minutes

Fig. 5. Passive efflux of Ca 2+ in the presence of Na + and K +. Vesicles pre-loaded as described in Methods were diluted to a final concentration of 0.1 mg protein/ml in media containing 10 mM Tris/maleate buffer (pH 6.85), 2 mM MgCI2, 4 mM potassium oxalate, 1 mM EGTA, and ( 0 ) no added ions, (V1) 200 mM NaCI or (A) 200 mM KCl. Other experimental conditions were as described under Methods. Each value represents the average ±S.E. of 4 experiments.

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50

20

i 100 2o0

o loo salt,

J~

mM

Fig. 6. Effects of Na ÷ and K + on the initial rate of Ca 2+ uptake. NaCl (A) or KC1 ( A ) in the concentrations indicated were added to the control medium (111) described in Fig. 1. The reaction was started by the addition o f sarcoplasmic reticulum vesicles and stopped after a l-rain incubation by Millipore filtration. Each value represents the average 4-S.E. of 4 experiments.

512 Fig. 7 shows that the a d d i t i o n o f 200 m M Na + or K + to the assay m e d i u m results in inhibition o f the C a - d e p e n d e n t A T P a s e activity. The inhibition is evident in the first minutes o f the reaction. As the steady state is reached (after 6 min, cf. Figs 2 and 3), the effect o f the alkali ions virtually disappears. At this point, the higher ex-

0.6

j

¢.m

Jl'l

~. 0.4 E

h0.2

E

1

0

1

I

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--

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6

minutes

Fig. 7. Effects ofNa + and K + on the time course of the Ca2+-dependent ATPase. The Ca-dependent ATPase shown was the difference between the activities of the (Ca 2+ ÷ Mg2+)-dependent ATPase and Mg2+-dependent ATPase assayed as described in Methods. The values represent the average ± S.E. of 6 experiments. Control (O), control plus 200 mM NaCI (11) or KCI ( • ) .

c-

t~

E

O2o

13-

O

E

:3.

r 0

[

1

100

[

i

--

200

salt , mM

Fig. 8. Inhibition o f Ca2+-dependent ATPase activity by different Na + and K + concentrations. The experimental conditions were as described for Fig. 7 but with the indicated concentrations o f ( I ) NaCI or ( A ) KC1 added to the control ( 0 ) medium. Incubation time was 1 rain. Each value represents the average ± S . E . o f 4 experiments.

513

ternal Ca 2+ concentration in media containing N a + or K + is probably compensating for their inhibitory effects on the rate o f hydrolysis. The effect of a range of N a + and K + concentrations on the initial rate o f ATPase activity is shown in Fig. 8. T A B L E 1I E F F E C T S O F A L K A L I I O N S O N E-P F O R M A T I O N T h e assay m e d i a (see M e t h o d s ) contained 0.2 m M CaCl 2 plus 1 m M E G T A (free Ca 2+ = 0.98 t i M ) o r 0.1 m M CaCI2 a n d no E G T A . T h e i n c u b a t i o n time was 20 s. T h e values represent the average 4-S.E. o f the n u m b e r o f experiments indicated in parentheses. A d d i t i o n s to assay m e d i u m

flmol E-P/g protein 0.98 # M initial Ca 2+

100/*M initial Ca 2 +

none 50 m M NaC1 100mM NaCI 50 m M K C I 100 m M KCI

0.58-4-0.06 0.35 4-0.07 0.144-0.06 0.41 4-0.07 0.294-0.11

1.68±0.10 (5)

-~

(14) (9) (6) (7) (6)

1.864-0.12 (5) 1.814-0.09 (5)

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= o,~

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Fig. 9. Initial rate o f Ca 2+ u p t a k e as a function o f free Ca 2+ concentration; effect o f N a C l . (a) T h e i n c u b a t i o n m e d i u m described for Fig. 1 was altered by the addition o f different concentrations o f E G T A (3.0-0.38 m M ) to o b t a i n the indicated free Ca z+ concentrations in the presence o f (11) zero, ( [ ] ) 50 m M , ( A ) 100 m M or ( A ) 200 m M NaC1. T h e reaction was started by the addition o f 0.2 m g sarcoplasmic reticulum vesicles protein/ml for the two lowest Ca 2+ concentrations, 0.1 m g protein ml, for the next three Ca z + concentrations a n d 0.05 m g protein/ml for the higher Ca 2 + concentrations. T h e incubation time was 30 s. E a c h value represents the average ± S . E . o f 5 experiments. (b) T h e d a t a obtained for the initial rates o f Ca 2+ uptake in zero (11) or 200 m M NaCI ( A ) in the experiment o f Fig. 9 are normalized to the highest initial rate s h o w n for each condition (at 4 . 2 p M Ca2+).

514 N a + and K + also inhibit the E-P formation when the free Ca z + concentration of the medium is below 1.0/~M (Table lI). As has also been shown by de Meis [22]. this inhibition is no longer observed in high C a 2 + concentrations. The differences observed in high and low Ca 2+ in the effects of Na + and K + on i, (Figs 6-8 and Table ll) suggest that they act by altering the Ca 2~ sensitivit3 o f the pump. To test this possibility we measured the initial rate o f Ca z + accumulation over a range o f Ca 2 + concentrations on the presence of various concentrations o f one of the alkali ions. Three effects of NaC1 can be seen in Fig. 9a; a depression of the maximal rate of Ca 2 + uptake at 4.2 ttM Ca 2 +, a decrease in sensivitity to Ca 2 + and a change in the form o f the dependence on Ca z +. All three effects must contribute to the inhibition of C a 2 + uptake. At low Ca 2 + concentrations, however, the inhibition is proportionally greater: at 4.2 pM Ca z+, the rate in 200 m M N a C I is 45 o / o f that in the absence o f N a +, but at 0.4 t~M Ca 2+ this rate is only ,~. "/~,,o f the control. When the effect on the rate at 4.2 ltM is eliminated graphically by normalizing each curve to its maximum (Fig. 9b), it becomes obvious that the marked inhibitory effects observed at low C a 2 + can be attributed mainly to the increased sigmoidicity or decreased sensitivity to Ca 2 -.

DISCUSSION Previous reports have shown that in the presence of 0.1 - 4.0 m M A T P and Ca 2+ concentrations above 10 pM, Na + and K + do not inhibit Ca z+ transport but rather activate the C a 2 + p u m p [5-7]. Conversely, when the A T P concentration is between 2 and 10/tM, N a + and K + inhibit both C a / + uptake and ATPase activity [5, 17]. In this paper, it is shown that in low Ca / + concentrations, N a + and K + significantly inhibit Ca z + uptake (Figs 1 and 3), Ca-dependent ATPase (Figs 7 and 8) and E-P formation (Table II) when the A T P concentration in the medium is 2 raM. As was also observed for the high Ca z+ and low A T P condition [5], the inhibitory effects of N a + and K + appear to be specific and not related to the osmotic or ionic strength o f the medium (Fig. l). As shown in Fig. 9, these inhibitory effects are probably related to a decrease in the apparent Ca 2+ affinity of the pump. One result is a decrease in the ability of the sarcoplasmic reticulum vesicles to maintain a 1ow Ca z + concentration in the medium. The C a / + and A T P concentrations at which an inhibition by the alkali ions is observed are in the range o f those expected to be found in the sarcoplasm of a relaxed muscle fiber. Therefore in a resting muscle fiber, the Ca z+ p u m p of the sarcoplasmic reticulum m a y be operating below maximal capacity due to the K + concentration o f the sarcoplasm. A possible drawback to this hypothesis is the use in our reaction medium o f oxalate concentrations which are not found in psysiological conditions. Several reports have shown that oxalate increases the calcium storage capacity o f the vesicles, providing a sink for the entering calcium, without modifying the properties o f the p u m p [18, 23-25]. Oxalate merely serves to reduce the concentration o f the vesicles lequired to lower the Ca 2+ in a solution to the level found in the cytoplasm of a relaxed muscle cell. Relaxation o f myofibrils by sarcoplasmic reticulum vesicles has been obtained 'in vitro' both in the presence and in the absence of oxalate [2, 3, 23, 26, 27].

515 ACKNOWLEDGEMENTS T h i s i n v e s t i g a t i o n was s u p p o r t e d in p a r t by t h e C o n s e l h o N a c i o n a l de P e s q u i sas, Brasil, C o n s e l h o de E n s i n o p a r a G r a d u a d o s d a U . F . R . J . , Brasil a n d b y the B a n c o N a c i o n a l de D e s e n v o l v i m e n t o E c o n 6 m i c o ( F u n t e c 241). T h e a u t h o r s are g r a t e f u l to D r M a r t h a S o r e n s e o n f o r h e l p f u l d i s c u s s i o n o f the m a n u s c r i p t . C . R . G . is a r e c i p i e n t o f a F e l l o w s h i p f r o m the C o o r d e n a ~ o d o A p e r f e i q o a m e n t o de P e s s o a l de N i v e l Superior (Capes).

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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