The effect of oligomycin on net movements of sodium and potassium in mammalian cells in vitro

550

BIOCHIMICA ET BIOPHX~S1CA ACTA E~1¢A4I(i7

THE

EFFECT AND

OF OLIGOMYCIN POTASSIUM

ON

NET

MOVEMENTS

IN MAMMALIAN

CELLS

IN

OF SO1)IUM IITRO

G. 1). \'. VAN I(()SSUM* I.aboralory oj Physiological Chemi,stJ\v, University ~/ Amsterdam, Amste*'dam ('/'he Ne/herlaJid~)

(Received M~ty 8th, I0()3)

SUMMARY I. The effects af oligomycin on the net a c c u m u l a t i o n of p o t a s s n n n and net loss of sodium b y whole m a m m a l i a n cells have been studied. Three t y p e s of cell system, differing in the relationship of the cation m o v e m e n t s to the e n e r g y - p r n v i d i n g m e t a bolism, were used. e. In liver slices p r e p a r e d from a d u l t rats oligomycin inhibited sodium a n d potassium m o v e m e n t s b y a m a x i m u m of 50 0(,. H a l f - m a x i m a l inhibition of potassium u p t a k e was given b y oligomycin at a concentration of approx. 3 / , g / m l (o.e()/\g/rag liver-slice protein). The 5o % of the potassiuln u p t a k e which persisted in the presence of oligomycin was largely inhibited b y the further a d d i t i o n of c y a n i d e ()r e,4-dinitrophenol. 3. Oligomycin inhibited respiration of the a d u l t tissue by a m a x i m u m of eo %. h a l f - m a x i m a l inhibition being given b y 3 Ixg/ml oligomycin (o.2(~/\g/rag protein). The inhibition was released b y the further a d d i t i o n of diaitrophenoI. 4. Slices of liver p r e p a r e d from rat foetuses during the last 2 d a y s of gestation can a c c u m u l a t e a considerable a m o u n t of p o t a s s i u m when i n c u b a t e d in the presence of cyanide. Oligom.vcin gave a p p r o x . 5o % inhibition of this c y a n i d e - r e s i s t a n t accumulation of potassium, the h a l f - m a x i m a l effect being given b\' an oligomycin c~mcen t r a t i o n of a b o u t 3 / , g / m l (o.41/,g/nag liver-slice protein). In the absence of oligomycin, d i n i t r o p h e n o l ahnost c o m p l e t e l y inhibited c y a n i d e - r e s i s t a n t p o t a s s i u m accumulation. In the presence of oligomycin, p a r t of the p o t a s s i u m a c c u m u l a t i o n r e m a i n e d uniilhil)ited b y dinitrophenol. 5. Oligomycin did not inhibit anaerobic glycolysis in the slices of foetal liver. (). Oligomycin, at concentrations of IO a n d 2o/~g/ml, h a d no effect upon net sodium m o v e m e n t s in h u m a n e r y t h r o c y t e s . In contrast, s t r o p h a n t h i n (; inhibited the sodium movements. 7. Consideration of these results in relation to the known effects of oligornycin on e n z y m e activities in subcellular particles suggests t h a t oligomycin twobably i n h i b i t e d cation m o v c m e ~ t s in liver slices b y virtue of its i n h i b i t o r y effect upon o x i d a t i v e p h o s p h o r y l a t i o n , r a t h e r t h a n b y inhibition of a reaction directly involved in cation t r a n s p o r t . If this conclusion is correct, it follows t h a t p a r t of the energy required for cation t r a n s p o r t b y liver cells can be derived directly from an energy-rich i n t e r m e d i a t e of o x i d a t i v e phost)hnrylation. " Present address: J,dmson Research l:oundation, University of 1)ennsvlvania. Iq~ilatlelphia 4. Pa. (I'.S.A.). I~i~chi~J. 14iophy,~. ,tcla, ,s, (l(g~4) 7,3~ 57 j

E F F E C T OF O L I G O M Y C I N ON C A T I O N M O V E M E N T S

557

INTRODUCTION

There is evidence that the energy required for active movements of sodium and potassium by animal cells can be derived from ATP 1-n. According to most current hypotheses, the formation of ATP by oxidative phosphorylation proceeds via the formation of successive energy-rich intermediates 4-6, and SLATER7 has suggested that the energy of the intermediates m a y be utilized directly by energy-requiring processes, such as cation transport, without the intervention of ATP. The energy-rich intermediates m a y also be made available for processes requiring energy under anaerobic conditions if ATP synthesized by glycdysis, or introduced experimentally into the cell, can induce the reversal of some of the reactions of oxidative phosphorylation. The properties of oligomycin as an inhibitor of oxidative phosphorylation*, 9 suggest that this substance may be used as a tool for testing these suggestions. If the reactions of oxidative phosphorylation be schematically represented as, Respiration @ ~ 1~

~ 2 -% ~ P -% A T P ,

where ~ a, ~ 2, and ~ P represent energy-rich intermediates (see ref. 9), oligomycin inhibits the sequence in the region of the second intermediate ( ~ 3). Thus, in respiring mitochondria oligomycin inhibits the formation of ATP but allows the formation of the first intermediate and, conversely, in anaerobic mitochondria it inhibits the formation of ~1 from added ATP. The insensitivity to oligomycin of such energyrequiring reactions of isolated, aerobic mitochondria as the succinate-linked reduction of NAD 1°-14, and the accumulation of phosphate, manganese and magnesium15, TM, suggests that these reactions can utilize the energy of the first intermediate. Also, the inhibition by oligomycin of the energy-dependent reversal of electron transfer in anaerobic mitochondria in the presence of ATP suggests that this reaction, too, derives energy from the first intermediatO 7. Recently, evidence has been published which suggests that oligomycin m a y not act exclusively as an inhibitor of oxidative phosphorylation, but m a y also directly inhibit reactions which are intimately concerned in the mechanism of cation transport. Thus, oligomycin has been found to inhibit sodium- and potassium-stimulated adenosine triphosphatases (Na +- and K+-stimulated ATPases) of microsomal preparations of the electric organ of the eel Is, of mammalian brain microsomes TM,30and of erythrocyte membranes 3°. It has been suggested that such Na +- and K+-stimulated ATPases 3~,32 m a y form an integral part of the mechanism which brings about the active transport of sodium. GLYNNTM has also stated that the transport of sodium by human red blood cells, which is linked to glycolysis rather than to oxidative phosphorylation 23, is inhibited by oligomycin. The object of the work to be described was to examine the effect of oligomycin on cation movements in whole cells. In order to gain an insight into the relation of the effects of oligomycin on whole cells to its known effects on subcellular particles, three types of tissue system have been studied: (i) Liver slices from adult rats; the cation movements of these slices are almost entirely dependent upon respiration and oxidative phosphorylation 2~,25. (2) Liver slices prepared from rat foetuses at 21-22 days of gestation, and incubated in the presence of cyanide; the cation movements are here supported by anaerobic glycolysis24, yet the cells contain the enzymes necessary for oxidative phosphorylationa6, 3v. (3) H u m a n red blood cells, which derive energy Biochim. Biophys. Acta,

82 (1964) 5 5 6 - 5 7 1

558

G.D.V.

VAN ROSSUM

for cation movements exclusively from glycolysis 2a and do not appear to contain the enzymes necessary for oxidative phosphorylation ~*. While most of the results obtained were equally consistent with an action of oligomycin either as an inhibitor of a reaction directly concerned in cation transport or as an inhibitor of oxidative phosphorylation, some observations suggest that the latter type of action was the main cause of the effect of oligomycin on cation movements in whole cells. If the effects of oligomycin were, indeed, largely a result of an inhibition of oxidative phosphorylation, then the work provides evidence that part of the energy for cation movements in rat-liver slices was derived from all energyrich intermediate of oxidative phosphorylation. A preliminary account of some of this work has been published 29. METHODS

The general procedure adopted, both in experiments with liver slices and with red blood cells, was to disturb the normal cation content cf the cells by incubation at a low temperature; the recovery of the cell cation composition, and the rates of respiration or glycolysis, during subsequent incubation at 38° in the presence or absence of oligomycin, could then be studied. Since it was anticipated that oligomycin might penetrate intact cells only with difficulty, a period of pre-incubation with this substance was always included in the cold incubation. Slices of rat liver, prepared either from adult animals or from foetuses at 2~-22 days of gestation, were incubated for 9 ° min at I °, followed by 6o min at 38° (unless otherwise stated). Each incubation vessel contained lOO-2OO mg liver slices (wet weight) in 3 ml medium. The same 3-ml portion of medium was used for both the cold and w a r m incubations. Oligomycin, dissolved in 96 % ethanol, was added to appropriate incubation vessels before the start of the incubatioa at i°. The final concentration of ethanol in the medium varied from o.5 to 2.~, '/,o," the same concentration of ethanol was also added to the control (oligomycin-free) liver slices in each experiment. When required, potassium cyanide and 2,4-dinitrophenol were added to the incubation medium after tile cold incubation. Further details of the incubation procedure, together with the analytical methods used, have been described previously2< 27. Fresh, h u m a n erythrocytes were washed in isotonic saline a° and 2 ml of packed cells were then suspended in 2o ml of potassium-free medium for incubation at 4 '~ for 4-5 days (see ref. 31). The incubation medium contained (raM): Na+, I54.o; Mg 2+, L o ; Ca 2+, 1.3; CI , 137.o; SO~ 2-, i.o; phosphate, Io.o; glucose, Io.o, and had an initial p H of 7.8. After 48-72 h incubation at 4 °, the suspension was centrifuged and the packed cells were divided into three portions of approx. 0.6 ml each. Each portion of ceils was then resuspended in 3 ml of fresh medium; to one portion of the resuspended ceils was added oligomycin ; to the second, ethanol at the final concentration present in the medium containing oligomycin and to the third, this same concentration of ethanol plus strophanthin G. Incubation at 4 ° was then continued for a further 48 h. At the end of this period, each portion of cells was centrifuged clown and samples (o.I ml packed cells) were taken for analysis. The remaining o. 5 ml of each portion of ceils was suspended in 2.5 ml cf a medium having the same composition as t h a t used for the cold incubation, except that it contained I o mM K + and 13iochim. t3ioph, ys..-t c/a, 82 (t ()('4) 530 571

EFFECT OF OLIGOMYCIN ON CATION MOVEMENTS

559

only 144 mM Na +. Ethanol, oligomycin plus ethanol, or strophanthin G plus ethanol were added, as appropriate, to give the same concentrations as those used for the cold incubation. The cell suspensions were contained in 3o-ml erlenmeyer flasks and were incubated for up to 5 h in a shaking water bath maintained at 38°. Samples (0.5 ml) of the suspensions were removed at intervals by pipette and were centrifuged for IO min at 2000 × g in narrow centrifuge tubes (internal diameter 4.0 mm). The supernatant medium and the upper layers of cells were removed with a Pasteur pipette, and the remaining cells were transferred to hard-glass weighing bottles for analysis by methods identical with those used for analysis of liver slices. RESULTS

Experiments ~ith liver slices /tom adult rats Cation movements. Liver slices prepared from adult rats contained 84 ~ 2 (7) mmoles potassium and 639 + 24 (6) mmoles sodium per kg fat-free dry weight (mean ± standard error of mean, number of observations in parentheses) after incubation at I ° for 9 ° min in the absence of oligomycin; the sodium and potassium contents of slices incubated at I ° in the presence of oligomycin (0.5-20.0 vg/ml) were not significantly different from these values. During subsequent incubation for 60 min at 38° the potassium content of liver slices incubated without oligomycin increased '

12

&

o

~

O "O

E

0

0

'

3

.c_

gu - 2 0 i g g Z

,

,

i

i

o lO 20 Oligomycin concentration ()ug/ml)

Fig. i. Effect of oligomycin on respiration and net cation m o v e m e n t s of liver slices prepared from a d u l t rats. The slices were incubated for 90 rain at i ° followed b y 60 min at 38°, under conditions referred to in t h e text. Each point represents the mean of 2 - 7 observations ; vertical lines represent twice t h e s t a n d a r d error of the mean. A - - A , Qoz; @ - - @ , net change in potassium c o n t e n t during incubation at 38°; O - - O , net change in sodium c o n t e n t during incubation at 38°.

Biochim. Biophys. Acta, 82 (I964) 556-57 t

560

G.D.V. VAN ROSSUM

b y a m e a n of x72 ~_ 9 (7) in moles/kg fat-flee d r y weight. Fig. I shows t h a t the a c c u m u l a t i o n of potassium was partially." i n h i b i t e d b y oligomycin. H a l f - m a x i m a l inhibition was given b y oligomycin at a c o n c e n t r a t i o n of 3/~g/ml a n d the m a x i m a l inhibition (40-5 ° %) was given b y a c o n c e n t r a t i o n of Io/~g/mt. The p o t a s s i u m acc u m u l a t i o n in the presence of the l a t t e r c o n c e n t r a t i o n of oligomyein was 97 :[ 9 (6) m m o l e s / k g fat-free solids. However, the i n h i b i t o r y effects of oligomycin on isolated m i t o c h o n d r i a are more closely r e l a t e d to its c o n c e n t r a t i o n per unit of protein present in the reaction vessel t h a n to its c o n c e n t r a t i o n in the suspending nmdium (E. ('. SLATER, personal communication). F r o m the known wet weights of the liver slices used in the e x p e r i m e n t s i l l u s t r a t e d in Fig. i a n d from the protein content t)f liver given in ref. 27, the oligomycin c o n c e n t r a t i o n s which gave h a l f - m a x i m a l a n d m a x i m a l effects on p o t a s s i u m a c c u m u l a t i o n m a y be c a l c u l a t e d to be approx, o.20 a n d 0.74 /,g/ml liver-slice protein respectively. It should be p o i n t e d out t h a t a considerable fraction of the solid c o n t e n t of liver slices was lost to the m e d i u m during these experiments, m o s t l y during the cold i n c u b a t i o n 'a. Protein lost from the slices in this way will still bind oligomycin and, in order to allow for this, the calculations of oligoinycin c o n c e n t r a t i o n s p e r unit protein ha\'e been related to the initial protein content of the slices. The effect of oligomycin on tile loss of sodium from the liver slices during in. c u b a t i o n at 38o differed in some details from its effect on p o t a s s i u m accunmlation, I n particuhu-, concentrations of oligomycin below 4/zg/ml h a d no significant effect on the loss of sodium. However, as was the case with p o t a s s i u m a c c u m u l a t i o n , the inaximal inlfibitorv effect of oligomyciu was given b y a concentration of I o / ~ g / m l a n d a m o u n t e d to an approx. 5o % i n h i b i t i o n ; thus the sodium content of slices inc u b a t e d without oligomycin decreased b v 28i !- 30 (~) mmoles/kg fat-free d r y weight, while t h a t of slices i n c u b a t e d with ro/~g oligomycin/ml decreased b y onh, 134 i: 25 (5) mmoles/kg. The greater loss of sodium in the presence of 2o/~g o l i g o m y c i n / m l con> p a r e d to the loss in the presence of I o / , g / m l is not considered to be significant, since in the single e x p e r i m e n t in which the effect of 20/~g oligomycin/ml on sodium movem e n t s was examined, liver slices ineul)ated in tile presence of I o t~g/ml a n d 2o tzg/ml lost i d e n t i c a l a m o u n t s of sodium (210 m m o l e s / k g fat-free d r y weight). In general, it should be p o i n t e d out t h a t , because tile concentration of sodium in the extracellular phase of the slices is nnlch higher t h a n the p o t a s s i m n concentration, and beeatlse the vohnne of the extraceltular phase shows considerable changes during i n c u b a t i o n at 380 (ref. 25), changes in tile sodimn content of the whole slices are a less reliable indication of the i o n - t r a n s p o r t i n g a c t i v i t y of the cells t h a n are changes in the potassium content of the slices (see also ref. 24). The 6o-min period of i n c u b a t i o n at 38o used in the e x p e r i m e n t s of Fig. ~ was chosen because earlier e x p e r i m e n t s h a d shown t h a t the net u p t a k e ~,f p o t a s s i u m b y liver slices i n c u b a t e d w i t h o u t inhibitors was c o m p l e t e d within this time 2a. In order to ascertain t h a t 60 rain was also a suitable period of incubation for the comparison of the effects of different c o n c e n t r a t i o n s of oligomycin on cation m o v e n w n t s , the timecourse of p o t a s s i u l n u p t a k e in the presence of a c o n c e n t r a t i o n of oligomyein which gave m a x i m a l inhibition (IO/,g/Inl) was e x a m i n e d . Fig. 2 shows t h a t the accumulation of p o t a s s i u m in the presence of this concentration of oligomycin followed a similar course to t h a t in the absence of inhibitor. In each case, a relatively slow u p t a k e during the first lO-15 min i n c u b a t i o n at 380 was followed b y a more r a p i d u p t a k e Biochfm. Bioph~,s..tcla. 82 (19041 55° 571

EFFECT OF OLIGOMYCIN ON CATION MOVEMENTS

561

during the subsequent I5-2o min, and the net uptake of potassium in both the presence and absence of oligomycin was completed within 60 min. It has previously been shown that about 9 0 % of the net accumulation of potassium by liver slices prepared from adult rats is dependent upon respiration 24, and it therefore seemed probable that the oligomycin-resistant uptake of potassium observed in the present work was largely dependent upon respiration. However, in isolated mitochondria only respiration which is uncoupled from phosphorylating reactions can continue in the presence of oligomycin8,9, and it was therefore thought to be important to investigate directly the relationship between those fractions of the potassium accumulation and of the respiration of liver slices which persisted in the presence of maximally inhibiting concentrations of oligomycirI. In this experiment, slices incubated in the presence of IO/~g oligomycin/ml had a Qo2 of 9.8 /~l/mg fat-free dry weight/h and accumulated 12o mmoles potassium/kg fat-free dry weight, whereas slices incubated in the presence of the same concentration of oligomycin plus I mM KCN had a Qo2 of 1.4 and accumulated 5 mM potassium/kg fat-free solids. It is thus clear that the oligomycin-resistant potassium uptake was dependent upon the oligomycin-resistant part of the respiration.

200 . - -" C

W i t h o u t oligornycin

L

o

-5 k

E~7 u u c3

O

cJ

O

With oligomycin

100

._2 o

I tI

E

EE

13_

/zQ

o o

I

I

60 Time of incubation

I

I

120 at 38 ° (min)

Fig. 2. T i m e - c o u r s e of t h e n e t u p t a k e of p o t a s s i u m b y liver slices i n c u b a t e d in t h e presence of oligomycin. Liver slices p r e p a r e d from a d u l t r a t s were i n c u b a t e d for 9o rain at t°, followed b y i n c u b a t i o n a t 38o for t h e periods indicated. E x p e r i m e n t a l conditions are referred to in t h e text. E a c h p o i n t r e p r e s e n t s a single o b s e r v a t i o n of t h e n e t u p t a k e of p o t a s s i u m b y liver slices i n c u b a t e d in t h e presence of o l i g o m y c i n (~o/zg/ml). T h e b r o k e n line, which is d r a w n from d a t a given in ref. 25, r e p r e s e n t s t h e n e t u p t a k e of p o t a s s i u m b y liver slices i n c u b a t e d w i t h o u t inhibitors.

The finding that oligomycin gives a partial inhibition of net cation movements. in liver slices from adult rats can be explained by either of two hypotheses based on the observed effects of oligomycin on enzyme systems of sub-cellular particles: Hypothesis z: The whole of the mechanism responsible for cation transport derives energy directly from ATP, and oligomycin acts by partially inhibiting a reaction, e.g. a Na +- and K+-stimulated adenosine triphosphatase 18-2~, which is directly concerned in this mechanism. Biochim. Biophys. Acta, 82 (1964) 556-57I.

.562

(;.

1). V . V A N

ROSSUM

Hypothesis z : Only p a r t of the t r a n s p o r t i n g m e c h a n i s m is obligatorily d e p e n d e n t u p o n A T P . Oligomycin inhibits a reaction of o x i d a t i v e p h o s p h o r y l a t i o n which leads to the f o r m a t i o n of A T P and thus inhibits the A T P - d e p e n d e n t p a r t of the t r a n s p o r t i n g mechanism. The oligomycin-resistant p a r t of the t r a n s p o r t i n g mechanism, since it is d e p e n d e n t upon respiration (see above), m u s t derive its energy from the (rely energyrich c o m p o u n d which can be formed from r e s p i r a t o r y energy in the presence of oligomycin, n a m e l y the first ( ~ ~) i n t e r m e d i a t e of o x i d a t i v e p h o s p h o r y l a t i o n . According to both hypotheses, the oligomycin-resistant cation m o v e m e n t s should be i n h i b i t e d b y d i n i t r o p h e n o l a c c o r d i n g to H y p o t h e s i s 1, because d i n i t r o p h e n o l p r e v e n t s the formation of A T P b y o x i d a t i v e phost)horylation; according to H y p o thesis 2, because dinitrophenol is believed to p r o m o t e the hydrolysis, with consequent d i s s i p a t i o n of the energy, of the ~ 1 intermediatea2. Accordingly, the effect of dinitrophenol on the a c c u m u l a t i o n of p o t a s s i u m b y liver slices, i n c u b a t e d with and without oligomycin, was next examined. In these experiments, oligomycin ahme (lO/~g/ml) gave a 23 % inhibition of potassimn u p t a k e (Table I). The reason for the smaller i n h i b i t o r y effect of oligomycin in these e x p e r i m e n t s t h a n in the e x p e r i m e n t s of Fig. 1 is not clear. D i n i t r o p h e n o l ahme (5 ° / , M ) gave 18 %, inhibition of t)otassium u p t a k e , a n d the inhibitions caused b y this concentration of dinitrophenol a n d by oligomycin s e p a r a t e l y were a p p r o x i m a t e l y a d d i t i v e when the two compounds were a d d e d to t h e slices t o g e t h e r (4 .0 %). A t concentrations of i oo a n d 2oo/,M, d i n i t r o p h e n o l caused 83 % a n d 97 o inhibition of p o t a s s i u m u p t a k e in the absence ()f oligomycin and v e r y similar inhibitions in the presence of oligomycin. The ()ligomycin-resistant p o t a s s i u m a c c u n m l a t i o n was thus inhibited b y dinitrophenol. "I'AI~I.E EFFECTS

OF

2,4-DINI'FROPHGNOL

AND

F~Y L I V E R

1

OLIGOMYCIN

ON T H E

SLICES FROM

AI)ULT

ACCUMULATION

OF

PO'FANSIUM

RATS

Slices were incubated for 90 rain at I followed by t)o n[in at 38 . For details of incubation conditions see text. The values given for potassium accumulation represent the means L standard error of the mean (number of observations in parentheses) of the net increase in potassium content of the slices during incubation at 38'~. DiniHclphcnol FI~31)

Ohgomy~ in Qt~/ml)

Potassium accumulation (mm(des/kgfal-freedrywt.)

Inhihltio~ (",,~

o

o

t81

! 9 (to)

5°

o

149

~:4)([3)

18

too

o

3)

k: 3

(3)

s3

21>(~

o

5

! 4

(3)

97

o

)o

139

!

(8)

5o

Io

t°3

! 7 (lI)

ioo

Io

41

k 15 (3)

77

2oo

~o

13

~ .t

9z

1[

(4)

z3 4z

Respiration. I n the e x p e r i m e n t s i l l u s t r a t e d in Fig. I, liver slices i n c u b a t e d in the absence of oligomycin utilized oxygen at a r a t e of lO. 4 ± 0.4 (7) /,l/rag fat-free d r y weight/h. The rate of r e s p i r a t i o n was r e d u c e d in the presence of oligomycin, a m a x i m a l inhibition of a b o u t 25 % being given b y c o n c e n t r a t i o n s of IO i,g/ml (o.74 ~g/mg protein) or greater. H a l t - m a x i m a l inhibition was o b t a i n e d at an oligomycin concenBiochim. Biophys. ,qcla, 82 (1004) 554, 571

EFFECT OF OLIGOMYCIN ON CATION MOVEMENTS

563

tration of 3 ~g/ml (o.29 t~g/mg protein). The rate of respiration of the slices incubated in the presence of concentrations of oligomycin which gave maximal inhibition remained constant throughout the 5o-min period during which observations were made (the first IO min of the 6o-min incubation period at 38° were allowed for equilibration of the manometric apparatus), an observation which suggests that oligomycin had penetrated to the site of the cell at which it acted to inhibit respiration before the first readings were made. The finding that oligomycin inhibited cation movements by 40-50 % and respiration by 25 % is reminiscent of the observation that an appropriate concentration of a cardiac glycoside has similar relative effects on the two processes z~, and suggests that oligomycin, like cardiac glycosides (see ref. 33), m a y act on the liver cells primarily by causing a specific inhibition of cation movements (Hypothesis I). The partial inhibition of respiration could then arise, secondarily, if the rate of respiration is controlled by ADP formed during the hydrolysis of ATP by the transporting mechanism 34. According to Hypothesis 2, the inhibition of liver-slice respiration by oligomycin m a y be explained by the fact that inhibition of oxidative phosphorylation necessarily leads to the inhibition of respiration which is tightly coupled to the formation of ATP 8,9. According to either hypothesis, the inhibition of respiration by oligomycin should be released by agents which uncouple respiration from the phosphorylation reactions, and the effect of dinitrophenol on the respiration of liver slices incubated with and without oligomycin (IO/~g/ml) was therefore examined. The concentration of dinitrophenol used in these experiments (50 t,M) was that previously found to give maximal stimulation of the respiration of liver slices incubated without oligomycin 27. It can be seen from Table I I that dinitrophenol alone stimulated respiration b y 22 %, while oligomycin alone inhibited it by 23 %. In the presence of dinitrophenol and oligomycin together the slices respired at a rate which was not significantly different from that in the presence of dinitrophenol alone. Dinitrophenol thus released the respiratory inhibition caused by oligomycin. T A B L E II EFFECTS

OF D I N I T R O P H E N O L

AND

OF L I V E R

O L I G O M Y C I N ON T H E

SLICES FROM ADULT

RATE

OF R E S P I R A T I O N

RATS

F o r i n c u b a t i o n conditions, see text. Values given are m e a n s ~z s t a n d a r d error of t h e m e a n ( n u m b e r of o b s e r v a t i o n s in parentheses). Inhibitor

Rate of respiration (#l OJmg fat-free dry wt.[h)

None D i n i t r o p h e n o l (5o #M) Oligomycin (io/~g/ml) Oligomycin (i o/~g/ml) + dinitrophenol (5 ° #M)

11.8 ~ 0.5 (Iz) 14.4 ± 0. 5 ( 1 6 ) 9.1 :~z 0.5 (9) 13.7 ± 0.4 (7)

Experiments with liver slices from rat foetuses Liver slices prepared from rat foetuses at 21-22 days of gestation retained 186 -b IO (8) mmoles potassium/kg fat-free dry weight after incubation for 90 min at I o. During subsequent incubation for 6o min at 380 in the absence of inhibitors, these Biochim. Biophys. dcta, 82 (1964) 556-571

504

(;. l). v. VAN ROSSUM

slices showed a mean net u p t a k e of (;o ~ 9 (Io) mmoles potassium/kg fat-free solids, a n d respired at a rate of 7.6 ~ o.5 (9) /~l/mg fat-free solids/h; the corresponding values for slices i n c u b a t e d at 380 in the presence of i mM potassium cyanide were 5o := 7 (io_) mmoles potassium and i. 9 ~ o.3 (7) /~1 oxygen*. Evidence has previously been presented to suggest that the net u p t a k e of potassium in the presence of cyanide i~ d e p e n d e n t u p o n anaerobic glycolysis a n d not upon the cyanide-resistant respiration 'q. Fig. 3 shows the effect of increasing concentrations of oligomycin on the cyanideresistant a c c u m u l a t i o n of potassium 1)v these slices. Potassium a c c u m u l a t i o n was inhibited to a m a x i m u m of 5o-6o %, the m a x i m a l inhibition being given by concentrations of oligomycin greater t h a n IO/Lg/ml (I.3o/zg/mg protein). The half-maximal effect was given b y approx. 3 / , g / m l oligomycin (o.41 /zg/mg protein). Tal>le I I I shows t h a t the concentrations of oligomycin used had no effect on the rate of anaerobic glycolysis, as measured by lactate production, a n d oligomycin thus inhibited part of the cyanide-resistant potassium a c c u m u l a t i o n without limiting the supply of ATt'. Such an effect m a y be readily accounted for if oligom):cin partly inhibits a reaction which is specifically concerned in the mechanism of potassium transt;~wt (Hypothesis I). However, as suggested 1)\' SLATt';~:, A T P sx'nthesized during anaerobic glycolysis could lead to the formation of intermediates of oxidative phosphor3 latioll by a reversal of reactions of oxidative phosphorylatioa. In this case, any part of

the glyco]ysis-dependcnt potassium accumulation which is obligatorily linked to the first high-energy intermediate ( ~ ,) would be inhibited by oligoInycilLif this substance is able to act in the liver slices as an inhibitor of oxidative phosphorylation (Hypothesis e). The part of the potassium a c c u m u l a t i o n which is not inhibited by ~ligomycin u n d e r anaerobic conditions would then have to derive its energy directly from ATP,

t. o(

,

\

F_

oc

(5)

\\"~

2)0 l

r

I 7-T'

o E D o

)

m

o

2o

Oligomycbn concentration (}Jg/ml)

Fig. 3. Effect of oligomvcin on the cyanide-resistant net uptake of potassium l)y liver slices prepared from rat foetuses at z~ .e2 days of gestation. The slices were incubated for 9o min at ~', followed by 6o min at 38o in the presence of KCN (r raM). Experimental conditions are referred to in the text. Vertical lines represent twice the standard errors of the means; numbers of observations are given in parentheses. * It is of interest that the rate of cyanide-resistant respiration was unaffected by oligomycin at the concentrations used in this work.

13iochim. t3iophys..4 eta, ,";2 (I 904) 556--571

EFFECT

O F O L I G O M Y C I N ON C A T I O N M O V E M E N T S

565

or from an intermediate of oxidative phosphorylation lying between the reaction which is sensitive to oligomycin and the reaction which finally synthesizes ATP { e . g . ~ P). TABLE EFFECT

OF

III

OLIGOMYCIN ON THE FORMATION OF LACTATE BY LIVER SLICES PREPARED FROM FOETUSES AT 21--22 DAYS OF GESTATION

L i v e r slices w e r e i n c u b a t e d f o r 9 o m i n a t I °, f o l l o w e d b y 6 0 m i n a t 380 in t h e p r e s e n c e of c y a n i d e . V a l u e s g i v e n f o r l a c t a t e f o r m a t i o n r e p r e s e n t t h e m e a n ± s t a n d a r d e r r o r of t h e m e a n ( n u m b e r of o b s e r v a t i o n s in p a r e n t h e s e s ) of t h e d i f f e r e n c e b e t w e e n t h e l a c t a t e c o n t e n t of t i s s u e p l u s l a c t a t e c o n t e n t of m e d i u m b e f o r e a n d a f t e r i n c u b a t i o n a t 380 . Inhibitor

Lactate formed (mmoles/kg fat-free dry wt.)

Cyanide (i

raM) C y a n i d e (I raM) + o l i g o m y c i n ( 3 - 8 / z g / m l ) C y a n i d e (i m M ) + o l i g o m y c i n ( i o - 2 o # g / m l )

2 6 0 ± 3 ° (8) 261 ! 27 (4) 281 ± 36 (8)

A possible means of testing whether Hypothesis 2 is operative in the cyanideinhibited liver slices from the late foetus is offered by the fact that oligomycin inhibits the dinitrophenol-induced ATPase of isolated mitochondria 8& It can be seen from the upper portion of Table IV that, at a concentration of 200/~M, dinitrophenol almost completely inhibited the cyanide-resistant accumulation of potassium by the foetal slices. Since dinitrophenol does not inhibit anaerobic glycolysis, the most reasonable explanation of this result is that it stimulated the mitochondrial ATPase within the slices35 and thus removed the ATP (and/or other energy-rich compounds derived from it) which was formed by glycolysis and which was required for cation transport. The lower half of Table IV shows that, in these experiments, the addition of oligomycin (i0/zg/ml) alone to the slices led to a 40 % inhibition of glycolysisdependent potassium accumulation, and that the further addition of dinitrophenol gave no significant inhibition of potassium uptake beyond that due to oligomycin alone. TABLE

IV

EFFECTS OF OLIGOMYCIN AND DINITROPHENOL ON THE CYANIDE-RESISTANT ACCUMULATION OF POTASSIUM BY LIVER SLICES PREPARED FROM RAT FOETUSES AT 21--22 DAYS OF GESTATION T h e slices w e r e i n c u b a t e d f o r 9 0 r a i n a t I ° f o l l o w e d b y 60 r a i n a t 38°, u n d e r c o n d i t i o n s r e f e r r e d t o i n t h e t e x t . V a l u e s g i v e n f o r p o t a s s i u m a c c u m u l a t i o n r e p r e s e n t m e a n ± s t a n d a r d e r r o r of m e a n ( n u m b e r of o b s e r v a t i o n s in p a r e n t h e s e s ) of t h e n e t c h a n g e ill p o t a s s i u m c o n t e n t o f t h e slices d u r i n g i n c u b a t i o n a t 38o . Additions to incubation medium Cyanide (raM)

Dinitrophenol (pM)

Oligomycin (itg/ml)

Potassium accumulation (mmoles/kg fat-free dry wt.)

I

o

o

39 ±

I

50

o

21 ± 15 (5)

6 (12)

I

IOO

o

34 ±

I

200

o

I I I I

o 50 IOO 200

IO Io IO IO

7

(5)

4 ± 3

(6)

23 I2 22 22

± ± ± ±

7 (13) 5 (11) 7 (7) 6 (5)

B i o c h i m . B i o p h y s . A c t a , 82 (1964) 5 5 6 - 5 7 1

560

G. 1). V. \rAN ROSSUM

Tile uptake of potassium in the presence of oligomycin plus 200/~M dinitr()phenol was significantly greater than that found in the presence of this concentration of dinitrophenol alone ( P - o.oi). The results of this experiment tiros show that the part of tile cyanide-resistant potassium accumulation which was not inhibited by oligomycin was, in fact, protected by oligomycin from the inhibitory action of dinitro1)henol. This effect of oligomycin can be readily explained by its action as an i~lhibitor of the mitochondrial, dinitrophenol-induced ATPase, and the results thus provide evidence that oligomycin did act as an inhibitor of a reaction of oxidative phosphorylation in the liver slices. However, this by no means excludes the possibility that the inhibitory effect of oligomycin on tile cvalfide-resistant potassium accumulation in the absence of dinitrophenol resulted from inhibition of a reaction directly concerned in the transporting mechanism. Experiments with human ervthrocs, les

Mammalian red 1)lood cells do n o t appear to contain mitochondria '2s, and they therefore represent a system in which it is t/ossible to test whether ()ligomycin can inhibit ion movements linked to glycolysis independently of its effect on oxidative phosphorylation. After incubation for 4-5 days at 4 °, red blood cells contained 3I 9 _i: I6 (1i) mmoles sodium and 171 ~ 4 (8) mmoles potassium/kg dry weight; the presence of oligomycin (IO or 2o/~g/ml) or strophanthin (; (1o -4 M) in the medium during the last 48 h of incubation at 4 ° (see METHODS) did not significantly affect these values. During subsequent incubation at 38°, the control cells (i.e. those incubated without oligomycin and strophanthin (;) showed a net loss of 164 _1_ 25 (4) mmoles sodium/kg dry weight after 5 h (Table V). Oligomycin, at concentrations of IO and 2o/~g/ml, had no significant effect on the loss of sodium from the cells (the results obtained with both concentrations have therefore been pooled in Table V). In contrast, strophanthin (;, which is known to be a specific inhibitor of active cation movements aa, caused a considerable inhibition of the net loss of sodium. Neither control nor oligomycin-treated cells underwent a significant change in potassium content per unit dry weight during incubation at 38°, although in each case there appeared t~) be some tendency for the cells to accumulate potassium (Table V). These cells did show an increase in the concentration of t)otassimn in the cell water, the potassium concentration in control and oligomycin-treated cells increasing by i i _}- () (()) and 18 !: 7 (8) mmoles/kg water respectively after 5 h incubation. However, these, changes in potassium concentration were due to a loss of water from the cells rather than to an increase in potassium content. (;ells incubated in the presence of strophanthin (i, (m the other hand, lost some potassium per unit solids and maintained a constant potassium concentration in the (;ell water during incubation at 38°. After 5 h incubation at 38°, the potassium content of cells incubated in the presence of strophanthin G (x5o ,: 7 inmoles/kg dry weight, 6 observations) was significantly less than that of cells incubated with io-2o tzg oligomycin/ml (175 l- 8, 8 observations; P < o.o5). The potassium content of the control cells was I68 4 IO (6) mmoles/kg dry weight. Thus, although the control red cells showed no consistent net uptake of potassium during incubation at 38:', it seems clear that their potassium content was actively maintained by a transport mechanism which was inhibited by strophanthin (; but not bv oligomycin at the concentrations used Biochim. Biophys..4eta, S., (~9~4)55~' 571

567

EFFECT OF OLIGOMYCIN ON CATION MOVEMENTS TABLE V EFFECT OF OLIGOMYCINAND STROPHANTHING ON NET MOVEMENTS OF CATIONS IN HUMAN RED BLOOD CELLS

The cells were incubated for 4-5 days at 4 ° followed by incubation at 3 8° for the times stated. Incubation conditions are described in the text. The values given for loss of sodium and accumulation of potassium represent the means ± standard error of the mean (number of observations in parentheses) of the net changes in cation content of the cells during incubation at 38° in the presence of the inhibitors indicated. Time at 380 (h)

Control

Oligomycin (io-2o t*g/ml)

Strophanthin G (zo * M)

Sodium loss (mmoles/kg dry wt .)

0. 5 I 3 5

42 69 133 i 164 ±

(2) (2) 15 (4) 25 (4)

36 49 I15 ± 143 ±

(2) (2) 24 (6) 18 (6)

54 16 3 84

(2) (2) (2) (2)

Potassium accumulation (mmoles/kg dry wt. )

0. 5 I 3 5

--6 8 4 o

=c 2 ± 7 ± 7 ± 6

(4) (4) (6) (6)

o o 3 IO

± ~ ± ~

5 io 6 I2

(4) (4) (8) (8)

i II I3 --17

± ± ± ±

7 6 6 7

(3) (3) (6) (6)

DISCUSSION Of t h e two effects of o l i g o m y c i n t h a t h a v e b e e n d e m o n s t r a t e d w i t h s u b - c e l l u l a r particles, n a m e l y t h e i n h i b i t i o n of o x i d a t i v e p h o s p h o r y l a t i o n in m i t o c h o n d r i a a n d t h e p a r t i a l or c o m p l e t e i n h i b i t i o n of N a +- a n d K + - s t i m u l a t e d a d e n o s i n e t r i p h o s p h a t a s e s of microsomes, o n l y t h e f o r m e r has b e e n d e m o n s t r a t e d w i t h s u b - c e l l u l a r p r e p a r a t i o n s of liver s,~. T w o s u b - c e l l u l a r f r a c t i o n s of liver h a v e b e e n f o u n d to show N a + - a n d K + - s t i m u l a t e d A T P a s e a c t i v i t y - - a dense p a r t i c u l a t e f r a c t i o n c o n s i s t i n g of f r a g m e n t s of cell m e m b r a n e s zs a n d a m i c r o s o m a l fraction 37 (but c o n t r a s t ERNSTER AND JONESaS). I n n e i t h e r case was t h e p r e p a r a t i o n t e s t e d for its s e n s i t i v i t y to oligomycin, a l t h o u g h H. E . M. VAN GRONINGEN (quoted by HUIJING AND SEATER 9) a n d ERNSTER AND JONES ~ f o u n d t h a t t h e m a g n e s i u m - s t i m u l a t e d A T P a s e of r a t - l i v e r m i c r o s o m e s is n o t affected b y o l i g o m y c i n . As has b e e n p o i n t e d out, m o s t of t h e results o b t a i n e d w i t h liver slices in t h e p r e s e n t w o r k can, q u a l i t a t i v e l y , be e q u a l l y well e x p l a i n e d on t h e basis of a p a r t i a l i n h i b i t i o n b y o l i g o m y c i n of a r e a c t i o n which forms a n i n t e g r a l p a r t of t h e m e c h a n i s m of c a t i o n t r a n s p o r t e . g . a N a +- a n d K + - s t i m u l a t e d A T P a s e (see Fig. 4), or on t h e basis of a n i n h i b i t i o n of o x i d a t i v e p h o s p h o r y l a t i o n . I n t h e l a t t e r case p a r t of t h e e n e r g y for c a t i o n m o v e m e n t s w o u l d h a v e to be p r o v i d e d b y t h e first e n e r g y - r i c h i n t e r m e d i a t e of o x i d a t i v e p h o s p h o r y l a t i o n (Fig. 5). H o w e v e r , some a s p e c t s of t h e e x p e r i m e n t a l r e s u l t s a p p e a r to be m o r e decisive i n a c o n s i d e r a t i o n of t h e two h y p o t h e s e s : (I) T h e effect of o l i g o m y c i n i n p r o t e c t i n g p a r t of t h e g l y c o l y s i s - d e p e n d e n t p o t a s s i u m u p t a k e , b y slices of foetal liver, from i n h i b i t i o n b y d i n i t r o p h e n o l s t r o n g l y suggests t h a t o l i g o m y c i n p e n e t r a t e d to the m i t o c h o n d r i a w i t h i n t h e liver slices, a n d t h e r e i n h i b i t e d a r e a c t i o n of o x i d a t i v e p h o s p h o r y l a t i o n . (2) T h e c o n c e n t r a t i o n of o l i g o m y c i n per m l of i n c u b a t i o n m e d i u m w h i c h gave Biochim. Biophys. Acta,

82 (1964) 556-571

G. I). v . VAN R O S S U M

568

m a x i m a l inhibition of respiration and cation m o v e m e n t s in liver slices (about IO/,g/ml) was very similar to the concentration which was found by (ILVXX m and by J6BSIS AND VREMAN 19 to give m a x i m a l inhibition of Na ÷- and K+-stimulated adenosine triphosphatases. This concentration is, moreover, about ten times that required for m a x i m a l inhibition of oxidative phosphorylation in isolated mitochondria'~L However, Menlbranc

•

Respiration

~

~

a

~

2

~

I>

,VI't'

iIi.

I

{dinitrophcn()l)

i

,

(oligomycin I

Vl'l'as~

~

(cardiac glycoside)

,<

(;lycolysis

l:ig. 4. S c h e m a t i c r e p r e s e n t a t i o n of r e a c t i o n s l i n k i n g c a t i o n t r a n s p o r t a n d c n c r g y m e t a b ~ l i s m in r a t - l i v e r cells, o l i g o m y c i n b e i n g p r e s u m e d s p e c i f i c a l l y to i n h i b i t a N a ~ - a n d l ( ; - s t i m u l a t c d a d e n o s i n e t r i p h o s p h a t a s e r e a c t i o n c o n c e r l l e d in c a t i o n t r a n s p o r t . -- >, rc~tctiolls COllceriltd under aerobic conditions ; I , r e a c t i o n s c o n c e r n e d u n d e r a n a e r o b i c 17tlnditions : - ~. reactions promoted (directly or indirecth') by dinitrophcn,~l

Metal)nine

?

i l

Respiration

=" ~ 1

"

(cardiac glycosides)

I(

~ [ e n l t)l'aIl{!

( , ] i g o m y c i 11

Na ' .

~ u

~ I'

.VI'I' 5

Idinitrophenol) F i g . 5. S c h e i n a t i c r e p r e s e n t a t i o n of r e a c t i o n s in r a t - l i v e r cells, o l i g o m y c i n b e i n g p r e s u m e d The two "membranes" indicated mav or ma under aerobic conditions; ~:), reactions rcactions promoted (directly

I

~

A'l'l'ase

{cartliac g yt osi(lcsl

i

Glvcolvsis

l< "

linking cation transport and energy mctabolisnl specifically to inhibit oxidative phosphorylation. 5 n o t b e i d e n t i c a l . --., reactions c~mccrncd concerned under anaerobic conditions: ~ or indirectly) by dinitrophenol.

as m e n t i o n e d above, the q u a n t i t y of oligomycin per unit protein is probably a better measure of tile a c t i v i t y of this inhibitor. H a l f - m a x i m a l effects on the aerobic catio1: m o v e m e n t s of liver slices from adult rats, a n d on the cyanide-resistant p~tassiun: a c c u m u l a t i o n b\' the foetal tissue, were given bv oligoinycin concentrations of o.20 a n d o.4I /*g/rag liver-slice protein respectively. The results of HuUIXG AND SI.~vrH~'~' suggest t h a t the.' h a l f - m a x i m a l effect of oligomycin on oxidative tdlosphorylation in isolated m i t o c h o n d r i a is given by concentrations between o.2 and o. 5 /,g/rag mitochondrial protein. In contrast, .]0esIs AXD VREMANm found t h a t h a l f - m a x i m a l inhi bition of the N a ~- a n d K ~-stimulated ATPase of brain microsomes required approx. 3 / , g oligomycin/mg protein a n d VAx GRONINGEN ANI) SI.ATER2a found an even higher 141och~ln. I~iophys..Icla. >;2 (1~)04) 5.5t, 57~

EFFECT OF OLIGOMYCINON CATION MOVEMENTS

569

value (30/~g/mg). Moreover, VAN GRONINGENAND SLATER2° found that the Na +- and K+-stimulated ATPase of brain microsomes required approx. I00 times more oligomycin per unit protein for half-maximal inhibition than did the mitochondrial ATPase of the same tissue sample. Consideration of these results suggests that the concentration of oligomycin used in the present work was sufficient to give a large inhibition of oxidative phosphorylation in the mitochondria of the liver slices, but that it may have been too low for the inhibition of a Na +- and K+-stimulated ATPase caused by it to account for more than a small part of the observed inhibition of cation movements. However, this conclusion is based on the assumption that oligomycin becomes fairly evenly distributed over all tissue components in the incubated liver slices. If the ATPase system has a greater affinity for oligomycin than other tissue components, then the inhibition of this system by oligomycin m a y have been a significant factor in inhibiting cation movements in liver slices. (3) The failure to demonstrate an effect of oligomycin on cation movements in erythrocytes in the present work, even at a concentration of 2o ~g/ml medium, may also be taken to suggest that the observed action of oligomycin on cation movements in liver cells is mediated by its effect upon mitochondrial enzymes (mitochondria being apparently absent from mammalian erythrocytes2S). However, this finding is in contrast to that of GLYNNTM, who reported an inhibition of sodium transport in red blood cells by oligomycin at a concentration of IO/~g/ml. The reason for this difference in results is not clear, but the resolution of the discrepancy is clearly of importance in deciding whether the effect of oligomycin on net cation movements in liver cells can be entirely accounted for by an inhibition of oxidative phosphorylation, or whether another mode of action must also be invoked. A failure of oligomycin to inhibit the cation movements of erythrocytes would also be of interest in view of the finding by VAN GRONINGEN AND SEATER20 that it did inhibit the Na +- and K +stimulated ATPase of erythrocyte membranes. Whether or not part of the inhibition of cation movements in liver slices by oligomycin resulted from an inhibition of a reaction directly involved in the transporting mechanism, if oligomycin completely inhibited oxidative phosphorylation in the slices (and the results of Table IV suggest that it may have done so) then it must be concluded that the oligomycin-resistant part of the aerobic cation movements in adult liver slices can derive energy from the first energy-rich intermediate of oxidative phosphorylation (see Fig. 5). The inhibition by oligomycin of part of the cyanideresistant potassium uptake in the foetal tissue may also have been at least partly due to a requirement of the first intermediate of oxidative phosphorylation for the cation movements. This would be true even if part of the effect of oligomycin was due to an inhibition of an ATPase system directly concerned in the cyanide-resistant transport mechanism. The implication of a high-energy intermediate of oxidative phosphorylation as an energy donor for part of the cation transport of liver cells raises the question as to the site within the cell at which this fraction of the iontransporting mechanism might be situated. Since the energy-rich intermediates are presumably very labile, it seems probable that the mitochondria must be close to the site of transport. There is evidence that mitochondria lie very close to membranous portions of the cell structure sg. Alternatively, the mitochondria may themselves be the organelles which bring about part of the cation transport of the cells. The ability of isolated mitochondria to accumulate potassium by a mechanism which is dependent Biochim. Biophys. Acta, 82 (1964) 556-571

570

G. I). V. VAN R O S S U M

u p o n o x i d a t i v e p h o s p h o r y l a t i o n has been d e m o n s t r a t e d 4°, although the effect of oligomycin upon this s y s t e m does not a p p e a r to have been examined. The suggestion of CHANCE17 a n d of CHANCE ANI) HOLLVSGER4t t h a t the e n e r g y - d e p e n d e n t reduction of N A D in m i t o c h o n d r i a m a y be concerned in active t r a n s p o r t mechanisms is also of interest in this connection, p a r t i c u l a r l y since this reaction is resistant to oligomycin aerobically1°, 11, b u t sensitive to oligomycin anaerobieallvlL If cation t r a n s p o r t b y m i t o c h o n d r i a does p a r t l y account for the c a t i o n - t r a n s p o r t i n g a b i l i t y of the whole liver cells, t h e n the m i t o c h o n d r i a l t r a n s p o r t should be sensitive to cardiac glycosides, since cation m o v e m e n t s in liver slices are c o m p l e t e l y inhibited by s t r o p h a n t h i n K (ref. 25). A l t h o u g h SHARE a" has found t h a t p o t a s s i u m a c c u m u l a t i o n by isolated mitoc h o n d r i a is i n h i b i t e d b\" cardiac glycosides, the c~mcentrations of glycoside required were very much higher than those required for the inhibition of ion tr¢lnspoIt in whole cells. A further point of interest raised bv the effect of oligomycin on liver slices concerns t h e n a t u r e of the respiration of the slices. By virtue of its inhibition of a reaction of o x i d a t i v e p h o s p b o r y l a t i o n , oligomycin effectively inhibits that p a r t (and only t h a t part) of t h e respiration of isolated m i t o c h o n d r i a which is coupled to t>hosphory lotionS, 9. Thus, if oligomycin effectively ilthibits o x i d a t i v e p h o s p h o r y l a t i o n in liver slices from a d u l t rats, the i o % of the slice respiration which is inhibited b y oligomycin (see Fig. I) m u s t represent the p h o s p h o r y l a t i n g respiration of the slice, a n d the rem a i n i n g 8O°o m u s t b y uncoupled from p h o s p h o r y l a t i o n . Clearly, the utilization of the energy-rich b o n d of the first i n t e r m e d i a t e of o x i d a t i v e p l l o s p h o r y l a t i o n for such energy-utilizing reactions of the cell as cation t r a n s p o r t would effectively uncouple respiration from the oligomycin-sensitive step of o x i d a t i v e p h o s p h o r y l a t i o n (and hence from the p h o s p h o r y l a t i n g step) in a w a y which m a y be c o m p a r e d to the action of d i n i t r o p h e n o t (@ TAGER AND SLATER14). :\ further possibility arises from the e x p e r i m e n t a l procedure a d o p t e d in this work, since this required the pre-incub a t i o n of the liver slices at I °. This t r e a t m e n t ieads to considerable swelling of the liver cells4a a n d it seems not unlikely t h a t this will in t u r n lead to s~nne d a m a g e of the m i t o c h o n d r i a , with consequent i m p a i r m e n t of the coupling between respiration and phosptaorylating reactions. A('KNOWLEDGEMENTS

I wish to t h a n k Professor E. C. SLATER for his h o s p i t a l i t y and for suggesting this problem, Mr. F. H u I j I N 6 for help in the p r e p a r a t i o n of red blood cells and Miss M. VAN UrFELEN for technical assistance. The work was done during the tenure of a N A T O Research Fellowship. RFFERENCES 1 U. C. CALI)\VELL, \ .

l~. HODGKIN, 1~. l). I'(EYNES AND r .

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