Effect of K+ deficiency on oxidative metabolism in Ehrlich ascites tumor cells

Btoehtm:ca et Btophystca Acta, 325 ( 1973 ) 406-412 C, Elsevier Scientific P u b h s h m g C o m p a n y , A m s t e r d a m - Printed m The N e t h e r l a n d s BBA 46664

EFFECT OF K + D E F I C I E N C Y ON O X I D A T I V E METABOLISM IN E H R L I C H ASCITES T U M O R CELLS

GARY MOROFF*and

EDWIN E GORDON**

Department o f Medtcme, N e w YorL Unwersltv Med:cal Center. N e w YorL, N Y. 10016 (U S.A ) (Received June 21st, 1973)

SUMMARY

The effect of cell K + content, and cell-membrane K + transport on mltochondrlal ATP generation was studied in Ehrlich ascltes tumor cells. 1. Depletion of cell K + by repeated washings in cold K+-free medium results in an initial decline in ATP with concomitant increase in ADP and A M P content on incubation at 37 °C After prolonged incubation, ATP content falls to negligible levels and AMP degradation is evident as mamfested by a fall in total adenine nucleotides. The fall in ATP is a consequence of reduced electron transport through the mltochondrial respiratory chain. 2 ATP reduction in the K+-depleted cell can be prevented if the cells are incubated in a K+-containing medium. Addition of K + after prelncubation of K +depleted cells does not lead to restoration of adenine nucleotide levels. By contrast, addition of glutamme to K+-depleted cells, with or without premcubatlon, restores the ATP content. This effect of exogenous substrate, and the ummpalred conversion of [~4C]palmltate to ~4CO2 suggest that K+-depletion does not directly interfere with oxidative phosphorylation and that availability of endogenous substrates for mltochondrlal oxidation is hm~tmg in the K+-defic~ent state.

INTRODUCTION

Cell membrane K + transport has been implicated m the normal operation of both glycolysis and respiration in mammalian tissues. Thls energy-dependent, K + transport system serves as a ~pacemaker" for mitochondrial respiration ~ and d~rectly influences the rate of glycolysis in erythrocytes 2'3 and in Ehrlich ascites tumor cells 4' 5 probably at the phosphoglycerate klnase level 6'7. The lntracellular K + concentration has been assigned an important role in the regulation of the rate of glycolysis m Ehrlich asc~tes tumor cells 8'9, presumably through xts effect on pyruvate klnase 1°.

* Present address Mt Sinai Medical Center, 5th Avenue & 100th Street, N e w York, N Y. 10029, U.S A ** A d d r e s s reprint requests to: Dr Edwsn E G o r d o n , New Y o r k Umvers~ty Medical Center, 550 First Avenue, New York, N Y 10016, U S A

K + DEFICIENCY

407

A regulatory role for K + on mitochondrial metabohsm in intact cells has been difficult to assess. The observations of a high K + concentration in mitochondrm 1l, an effect of K + on the permeability of isolated mitochondria to substrate 12-~5 an effect on respiratory control ~6 and the P.O ratio ~7, and on adenine nucleotlde translocase ~8 has led to extrapolations bearing on the effects of K + deficiency on mitochondrml oxidative phosphorylahon in the intact cell. It has been argued that the mtracellular K + content is a more ~mportant determinant of oxidative metabolism than ~s cellular transport of K + (ref. 19). In the experiments presented here, the respwatory inhibition observed in K+-deficient cells could be overcome by addition of exogenous substrate suggesting that K + deficiency exerts its effect by reducing the ava~labdity of substrate for mltochondrml oxidation. In the absence of a functioning Embden-Meyerhof pathway, K + deficiency leads to a reduction m cell ATP and ultimately to an irreversible dechne in total cell adenine nucleotides. METHODS

Ehrlich ascltes tumor cells of the hyperdiplold strata were harvested from the peritoneal cavity of white mice, 6 to 8 days after inoculation. K+-depletion was accomplished by washing the cells repeatedly in ice-cold K+-free medium over a period of 3 h as previously described 2°. Non-depleted cells were similarly prepared except for the presence of 5 mM K + m all media. Incubations were carried out m a Dubnoff metabolic shaker in an mr atmosphere at 37 °C. The final cell protein concentration in the medium was approxmaately 4 mg/ml. At appropriate intervals aliquots of the cell suspension were removed and added to 6 o~, perchloric acid. The supernatant solution was neutralized to approximately pH 6.8 with 50/jo K O H containing 0.15 M glycylglycmezl. After removal of the potassium perchlorate precipitate by centrifugatlon, allquots of the supernatant solution were assayed for adenine nucleotldes 2z'23. The respwatory rates were determined w~th a Clark oxygen electrode at indicated intervals in a closed system at 37 °C. For K + determination, the cells were rapidly washed twice at room temperature with 0.154 M choline chloride, the washed cell pellet was digested over-night with 0.1 M HNO 3 and K + content was determined by flame photometry on the supernatant solution. Cell protein was determined by a biuret method with bovine serum albumin as the standard The [carboxy-14C]palmltate (New England Nuclear Corp. Boston, Mass.)-albumm complex was prepared by the method of Lorch and Gey 24 using fatty acid-free albumin (Pentex, Miles Labs, Kankakee, Ill.): the molar ratio of palm~tate to albumin was 4. The conversion of 14C-labeled palmltate to 14CO2 by Ehrhch ascites tumor cells was determined in stoppered Erlenmeyer flasks incubated at 37 cC. At the end of the incubation, perchlorlc acid was added and the flasks were shaken for an additional 30 min in order to insure entrapment of all 14CO 2 on K O H saturated filter paper m the center well. The filter paper and center well were washed with a large volume of water: carrier sodium bicarbonate was then added to the combined washings followed by barmm chloride leading to the precipitation of the radioactive CO2 as barium carbonate. The radioactivity of the plated barium carbonate was determined with a gas flow counter and the actiwty was corrected to mfimte thickness.

408

G MOROFF, E E GORDON

RESULTS

Adenme nucleotides and resptratton o/K+-depleted and non-depleted cells The collection and washing o f cells prior to i n c u b a t i o n necessitates t e m p e r a t u r e adjustments which result m a consistent dechne rn the A T P content o f cells d u r i n g the first 15 to 30 m m o f incubation at 37 ~C (Fig. 1 ) F o l l o w i n g this mitml dechne, the A T P content as well as the total adenine nucleotlde content of n o n - d e p l e t e d cells remain relatively constant. In contrast, there is a steady dechne o f A T P m K +-depleted cells (Fig. 2). Total adenine nucleotldes ( A T P ~ - A D P + A M P ) of K+-depleted cells r e m a i n relatively constant for 30 m m and subsequently exhibit a substantml dechne (Fig. 2). A n increase o f A M P and A D P c o u n t e r - b a l a n c e the decrease m A T P d u r i n g the first 30 m m o f incubation. Between 30 and 90 ram, A D P content decreases along with A T P levels while A M P increases shghtly indicating adenine nucleotide d e g r a d a t i o n . Incubation o f K + - d e p l e t e d cells m a m e d i u m containing 5 m M K + prevents the dechne in A T P : 5 m M Rb + or Cs + are s o m e w h a t less effectwe than K + m this regard.

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Fig 1 Ademne nucleotldes m non-depleted Ehrhch ascltes tumor cells. 5 mM K + was present m the collecting, washing, and incubation media. Fig. 2 Adenine nucleotldes m K+-depleted Ehrhch ascites tumor cells.

In the absence o f exogenous substrate, the oxygen u p t a k e o f depleted cells decreases significantly (Fig. 3); the early dechne m respiration c o r r e s p o n d s to the early fall in cell ATP. In K + - r e p l e t e d cells, the oxygen u p t a k e decreases m m l m a l l y during the first 30 m m after which t~me a relatively c o n s t a n t rate o f r e s p i r a u o n ~s maintained. Since A T P p r o d u c t i o n occurs only m m i t o c h o n d r i a m the absence o f exogenous substrate, the dechne o f oxygen u p t a k e in the K +-depleted cells c o r r e s p o n d s to a decline m A T P generation.

K + DEFICIENCY

409

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F~g 3 The resp irato ry rate of K+-repleted and K+-depleted cells m the presence and absence of 5 mM glu tamme. K+-rep leted cells were first depleted of K + by the s t a n d a r d procedure and then incubated m the presence of 5 m M K +. FLg 4 Effect o f e x o g e n o u s substrates on m a i n t a i n i n g the A T P c ont e nt o f K +-de pl e t e d cells The c o n c e n t r a t i o n o f substrates were g l u t a m m e 5 mM, s o d i u m g l u t a m a t e 10 mM. and s o d i u m succmate 10ram

Effect o / e x o g e n o u s substrate I n c u b a t i o n o f K + - d e p l e t e d cells in the presence of 5 m M glutamine, 10mM g l u t a m a t e o r l0 m M succinate prevents a dechne m A T P content; glutamine is m o r e effective than g l u t a m a t e o r succlnate (Fig. 4). This difference m a y be due to the relative rates o f p e n e t r a t i o n o f each o f these substrates across the cell z5 or mltochondr~al m e m b r a n e s 26. A d d i t i o n o f 5 m M g l u t a m m e to depleted cells p r e i n c u b a t e d for 30 mln at 37 c'C increases the A T P content from 4.8 nmoles o f A T P per m g protein to 14 nmoles o f A T P per m g protein, wlthm 5-10 m m The a d d i t i o n o f glutamlne to nondepleted cells a b o h s h e s the initial decrease m A T P that characteristically a p p e a r s d u r i n g the first 30 rain. A T P content o f n o n - d e p l e t e d cells incubated m the presence o f g l u t a m m e remains relatively constant at approxxmately 14 nmoles o f A T P per mg protein. The r e s p i r a t o r y rate o f depleted cells remains u n c h a n g e d when 5 m M glutamine is present m the i n c u b a t i o n mixtures (Fig. 3). W h e n K + is a d d e d to depleted cells after 30 m m o f incubation, no effect on the A T P content or r e s p l r a t l o n o f the cells is observed a n d the ceils d o not c o n c e n t r a t e K +. Conceivably, the A T P content o f the cell is t o o low to s u p p o r t the cell-membrane, energy-dependent, K + t r a n s p o r t system 2v'28 o r alternatively, substrate avaflabihty ~s limiting. K + transport A f t e r Ehrhch asc~tes t u m o r cells are removed and washed at 4 °C in a m e d i u m c o n t a l m n g 5 m M K +, the cell K + content is a p p r o x i m a t e l y 80 n e q u i v / m g cell protein; cells washed in the absence o f K + contain a p p r o x i m a t e l y 5-10 n e q m v / m g cell protein. I n c u b a t i o n o f either o f these cell p r e p a r a t i o n s at 37 °C in a m e d i u m c o n t a i n i n g 5 m M K + results in the a c c u m u l a t i o n o f m t r a c e l l u l a r K + ( a p p r o x i m a t e l y 300 n e q u i v / m g protein) against a c o n c e n t r a t i o n g r a d i e n t in agreement with earlier studies 29. Since u p t a k e o f K + against a c o n c e n t r a t i o n gradient requires energy, the decrease m

410

G MOROFF, E.E GORDON

A T P level d u r i n g the first 30 m i n of i n c u b a t i o n p r o b a b l y reflects the rapid utihzatlon of A T P for K + t r a n s p o r t into the cell. Other experiments indicate that changes in A T P c o n t e n t and m K + reaccumulat~on of non-depleted cells are i n d e p e n d e n t of the d u r a t i o n of the washing procedure.

Influence o)c ouabain Interference with the energy-dependent N a + - K + p u m p by addition of 1 m M o u a b a i n after lntracellular K + is partially restored, results m a prompt, progressive decline m K + (Fig. 5). A significant decrease in cell A T P c o n t e n t is also observed, but is not as precipitous as that seen with K+-depleted cells (Fig. 1). The largest fractional decrease in A T P c o n t e n t occurred d u r i n g the first 45 rain after addition of o u a b a m I n h i b i t i o n of the K + transport system itself could therefore not account for the rapid decline in A T P to negligible levels m K+-depleted cells. 4-

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Fig 5. The effect of ouabain on the K + and ATP content Non-depleted cells were incubated for 30 mln in order to increase the K + content; ouabaln was added to one vessel in a final concentration of 1 mM and medmm to the control vessel. The time of the additions is indicated by the vertical Interrupted line. Fig 6. The production of 14CO2 in K+-repleted and K+-depleted cells in the presence of 0 046 mM [14C]palmltate as the albumin complex.

Oxidation of fatty acids Exogenous fatty acids added to I n c u b a t i n g Ehrlich ascltes t u m o r cells as the alb u m i n complex are rapidly taken up a n d metabolized 3 o. I n order to determine whether K + deficiency influences fatty acid oxidation, Ehrlich asc~tes t u m o r cells were incubated with [l*C]palmitate a n d the f o r m a t i o n of 14CO2 was followed. The effect of K + - d e p l e t l o n on [14C]palmitate conversion t o 14C0 2 1s shown in Fig. 6. The rate of fatty acid oxidation in both repleted a n d depleted cells is linear with time a n d no dtfference is noted for the two types of preparations. These data suggest that neither

K + DEFICIENCY

41 1

fatty acid transport into mitochondria, fatty acid oxidation, or trlcarboxyhc acid cycle activity is affected by the K+-deficient state DISCUSSION

A high concentration gradient for K + between intracellular and extracellular fluid exists for most mammalian tissues. The intracellular K ÷ concentration is approxmlately 20-fold greater than that of the fluid bathing cells. A reduction in intracellular K + content is thought to influence energy-generating and biosynthetic pathways directly, or ln&rectly by restricting the cell membrane monovalent cation pump. In the present experiments K ÷ depletion induced by washing Ehrlich ascltes tumor cells at low temperature in K+-free medium 2° or by incubation in the presence of ouabain 31 leads to an imtial marked reduction in ATP without substantial decline in total adenine nucleottdes. The ATP dechne is a consequence of restriction of mitochondrlal energy generation as manifested by a decrease in cell respiration. Glycolysis plays no role in the ATP generation of Ehrlich ascites tumor cells unless glucose is added. It is unhkely that the reduced ATP generation in K+-depleted cells is primarily due to moperabdity of the cell membrane N a + - K + transport system. When pump activity is restricted by onmtlng K + from the incubation medium, ATP content and ATP generation can be maintained by addition of exogenous substrate, e.g. glutamine, succinate, or glutamate Further, inhibition of the ( N a + - K + ) A T P a s e with ouabain m non-depleted cells incubating in a K+-containmg medium does not affect energy generation 31. Maintenance and restoration of the respiratory rates and ATP in K+-depleted cells by exogenous substrate suggests that the intracellular K + concentration has little effect on the oxidation of substrate (glutamine, glutamate, succinate), the electron transport chain, or the conservation of energy. This conclusion is also supported by the almost identical conversion rates of [~4C]palmltate to ~4CO2 in control and K +deficient states. It is conceivable that the intramltochondrial K + concentration may be little affected despite reduction in overall cell K + content. The restoration of energy generation in K+-depleted cells by exogenous substrate speaks against an effect of high intracellular Na + on mitochondrial ATP production. It has been suggested that amino acids serve as the primary metabolic fuel for Ehrlich ascltes tumor cells 26'32'33 rather than carbohydrate 34 or lipid 33. If amino acids are an important metabohc fuel for Ehrhch ascltes tumor cells oxidizing endogenous substrates, then mtracellular K+-deficiency might affect the respiratory rate by limiting substrate availability It has been established 35'36 that the efflux of glycine from ascites tumor cells is stimulated by an mtracellular environment with high Na + concentration and low K + concentrations, a state that exists in the K+-depleted Ehrhch asotes tumor cell. If this same mechanism were operative for other amino acids, K+-depletion could lead to endogenous substrate depletion. The data presented in this report confirm previous observations suggesting that mtracellular K+-deficiency affects oxidative metabolism, The site affected by low K + in Ehrhch asotes tumor cells is proximal to the oxidative sequence of enzymes: the data point to a hmltatlon of substrate availability.

412

G. M O R O F F , E. E. G O R D O N

ACKNOWLEDGEMENTS This mvestlgatzon Grant AM-12404

was supported

from the National

by a grant from the New York of The Health

Research

Council

i n p a r t b y U S. P u b l i c H e a l t h S e r w c e R e s e a r c h

Institute of Arthritis and Metabohc

Heart

Association

Dr Gordon

of the City of New York

Diseases and

is a C a r e e r

under contract

Scientist 1-551.

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