Biochem. J. (1978) 174, 801-8 10 801Printed in Great Britain

The Membrane Potential of Mouse Ascites-Tumour Cells Studied with theFluorescent Probe 3,3'-Dipropyloxadicarbocyanine

AMPLITUDE OF THE DEPOLARIZATION CAUSED BY AMINO ACIDS

By ROGER D. PHILO and A. ALAN EDDYDepartment oJ Biochemistry, University of Manchester Institute of Science and Technology, Manchester

M60 1QD, U.K.

(Received 30 January 1978)

1. The magnitude of the K+ gradient across the plasma membrane, which was in equili-brium with the membrane potential (E) of the tumour cells, was determined by the 'nullpoint' procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519-552] in whichthe fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. Amixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondriafrom interfering with the fluorescence signal. Transport functions at the plasmalemmawere maintained under these conditions in the presence of glucose. 3. Physiologicalcircumstances were found in which incubation with glycine or with glucose changedthe 'null point' value of Ewithin the range -2OmV to -100mV. The fluorescence intensityat the 'null point' was an approximately linear function of E over that range. The pro-cedure enabled E to be inferred from the fluorescence intensity in circumstances wheretitration to the 'null point' was not feasible. 4. The rapid depolarization caused by L-methionine or glycine was shown in this way to have a maximum amplitude of about6OmV. A mathematical model of this process was devised. 5. The electrogenic Na+ pumphyperpolarized the cells up to about -8OmV when the cellular and extracellular concen-trations of K+ were roughly equal. 6. The observations show that the factors generatingthe membrane potential represent a major source of energy available for the transportof amino acids in this system.

Work with vertebrate tissues, with derived prep-arations of plasmalemma vesicles and with isolatedcells (such as those of the mouse ascites tumour usedin the present investigation) has provided qualitativesupport for the view that the electrochemical gradientof Na+ acting across the plasmalemma serves as theenergy source driving the accumulation of the aminoacids and carbohydrates that these systems concen-trate (Eddy, 1977; Crane, 1977). The feasibility ofthe Na+-gradient hypothesis is thus not in doubt.However, lack of information about the quantitativerelations between the Na+ gradient (A,uNa) and theamino acid or carbohydrate gradient, in variousphysiological circumstances, has hindered acceptanceof the hypothesis (Blaustein & King, 1976; Christ-ensen, 1977; Schafer, 1977; Lever, 1977; Smith &Adams, 1977).The magnitude of the Na+ gradient is given by:

}AfNa =RT ln ([extracellular Na+]/[intracellular Na+]) - EF

(1)

Abbreviation used: Hepes, 4-(2-hydroxyethyl)-1-piper-azine-ethanesulphonic acid.

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where Eis the membrane potential and RTand Fhavetheir conventional significance. The ratio [extra-cellular Na+]/[intracellular Na+] = [Naj]/[Na1] isknown approximately and in the present investigationwe have developed a procedure (Hoffman & Laris,1974; Philo & Eddy, 1975; Eddy et al., 1977) forusing the fluorescent probe 3,3'-dipropyloxadicarbo-cyanine iodide [1-(3-propylbenzoxazolidin-2-ylidene)-5 -(3-propylbenzoxazol -2-ylonium)penta-1,3-dien-5-ylidene iodide] to assay the magnitude of Ein preparations of the mouse ascites-tumour cells.Work with two other carbocyanine dyes had a similarobjective (Laris et al., 1976; Burckhardt, 1977). Thedyes carry a delocalized positive charge, they pene-trate lipid membranes readily and are concentratedwithin the lipid boundary in response to the mem-brane potential. This process is amplified by thesubsequent binding of the dye on the membraneitself or on cellular proteins (Sims et al., 1974;Hladky & Rink, 1976; Kimmich et al., 1977).We studied the fluorescence of the cell suspensions

containing the dye under various conditions in whichthe glycine or methionine gradients formed in thepresence of selected concentrations of Na+ and K+have been assayed in a number of laboratories

26

R. D. PHILO AND A. A. EDDY

(Schafer & Heinz, 1971; Johnstone, 1972; Reid et al.,1974; Schafer, 1977). Gibb & Eddy (1972) suggestedthat the membrane potential of the mouse ascitestumour could become at least 40mV more hyper-polarized than is indicated by the typical values of upto -24mV that have been determined by impaling thecells with microelectrodes (Lassen et al., 1971; Smith& Levinson, 1975). The observations described belowsupport this supposition by showing that the mem-brane potential assayed with the fluorescent probevaries from about zero to about -lO0mV in variouscircumstances. This is apparently the result ofchangesin the activity of the electrogenic Na+ pump that aresuperimposed on a large potential generated by thepassive diffusion of Na+, K+ and Cl- across theplasmalemma. The absorption of the amino aciditself is shown markedly to lower the membranepotential.

Materials and Methods

Cell preparations

Mouse ascites-tumour-cell suspensions were pre-pared as described by Morville et al. (1973). Thus,after collection and washing, the tumour cells wereincubated for 30min at 37°C in the standard Ringersolution. The cells were then collected in the bottomof a graduated centrifuge tube of lOml capacity bycentrifugation at about 150Og for 1 min. The volumeof packed cells (1-2 ml) was noted. Tumour cells thatwere to be loaded with Na+ were subsequentlyincubated for a further 30min at 37°C in an Na+/Ringer solution from which K+ was omitted.

Solutions

The standard Ringer solution contained 155mm-Na+, 8mM-K+, 131mM-C1-, 16mM-orthophosphateand 1 .2mM-MgSO4. Na+/Ringer solution was ofsimi-lar composition, except that it contained 163 mM-Na+and no K+. K+/Ringer solution contained 163 mM-K+and no Na+. Some preliminary work was carried outin a solution containing 300mM-sucrose and 10mM-Hepes, adjusted to pH7.4 with Tris base.

Fluorimetry

The fluorescence of solutions of 3,3'-dipropyloxa-dicarbocyanine iodide was measured in an EEL-Corning 244 fluorimeter (Corning-Medical, Halstead,Essex C09 2DX, U.K.). Fluorescence was excitedwith a mercury lamp, the 577 nm and 579 nm linesbeing selectedwith an Ilford spectrum yellow filter (no.606). Emitted light was passed through an Ilford spec-

trum orange filter (no. 607) to the fluorimeter contin-uous wedge filter, which was set to 620nm (13 nmhalf-bandwidth). The stirred cuvette was thermostat-ically maintained at37°C. Assays wete carried out withcell suspensions at 1.3 mg dry wt. in4ml to which 4,ugof dye in 40,1 ofethanol had beenadded.To standard-ize the fluorescence scale before each series of experi-ments, the instrument reading at 620nm was adjustedto 100% (instrument sensitivity setting at 7) when thecuvette contained 2.5,g of the dye dissolved in 4mlof ethanol.

K+-titration 'null point' experiments

The intracellular K+ concentrations for thevalinomycin K+-titration experiments were deter-mined as follows. Packed cells (1 vol.) were suspendedin the standard Ringer solution (5 vol.) and thesuspension was split into 1 ml samples and stored onice. Each 1 ml sample, containing approx. 26mg drywt. of the tumour cells, was added to 80ml of Ringersolution of the required K+ concentration ([Na+] +[K+] was 163mm in all cases). The solution alsocontained 60,uM-2,4-dinitrophenol, 6ng of oligo-mycin/ml, 1 ug ofantimycin/ml and glucose (2mg/ml)as required. It was then incubated for an appropriateinterval (10 or 30min) at 37°C. A portion (80,ug) ofthe dye in 40,ul of ethanol was next added and 4mlof the mixed suspension placed in the fluorimetercuvette. About 70ml of the remaining suspension wascentrifuged at about lOOOg for 1 min and the pelletresuspended in the remaining 6ml. This suspensionwas added to a weighed tube containing 1 pCi of3H-labelled inulin and the cellular K+ concentrationdetermined as described by Morville et al. (1973). Theaddition of the valinomycin (lO,g) to the cellsuspension in the fluorimeter coincided with the endof the first centrifugation step of the K+ assay. Thiswas carried out 4-5min after the addition of thedye.

Cell respiration

The tumour cells were prepared in the Na+/Ringersolution and stored at a density of 1 ml of packedcells/2 ml of suspension at 0°C for up to 2 h before use.Assays were carried out in a Clark-type oxygenelectrode (Rank Brothers, Bottisham, Cambs., U.K.)at 37°C by addition of 0.2 ml of the stock cell suspen-sion to 4ml of the Na+/Ringer solution to which5mM-KCl had been added.

Chemicals

3,3'-Dipropyloxadicarbocyanine iodide was pre-pared as described by Sims et al. (1974), except that

1978

802

ELECTROGENIC AMINO ACID ABSORPTION

the initial crystallization by addition of hot KIsolution did not work in our hands. Instead thepyridine was removed from the reaction mixture byrepeatedly adding and distilling off ethanol. The dyewas crystallized from the resulting ethanolic solutionand recrystallized from ethanol. Oligomycin was ob-tained from Sigma Chemical Co., Kingston uponThames, Surrey KT2 7BH, U.K. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone and valinomycinwere obtained from Boehringer Corp. (London),Lewes, East Sussex BN7 1LG, U.K. 3H-labelled inulinand ['4C]methionine were obtained from The Radio-chemical Centre, Amersham, Bucks. HP7 OYB, U.K.

Mathematical Methods

Mathematical model of the Na+-dependent aminoacid-transport system

(1) Flux through the transport system. Considerthe scheme (Morville et al., 1973):

k+1A. + Na+ + CO x0 ANaCo (2)

k.1

k+2ANaCo0 ANaC, (3)

k-2

k+3ANaCi Ai+Nat +Ci (4)k_3

k+4Cis Co (5)

k_4

where the subscripts o and i refer to the extracellularand intracellular media respectively. Let k+2, kL2(eqn. 3) and k+4, k-4 (eqn. 5) be small compared withthe other rate constants. Further, let the system besymmetrical so that k+4 = k.4 = k4, k+1 = k.3, k-, =k+3 (eqns. 2 and 4) and let KA = 2kll/k+1 and the totalcarrier concentration be [C],. The net rate of aminoacid uptake is given by:

where D = [Na+]0[A]. I- k-RT(l eEF/RT)

+ [Na+]i[A]i - RT(eI T - 1))

- [Na+],[A],[Na+]i[A], 2 k (ERT (I eEFIRT)+ KA

In eqn. (7), E is the membrane potential, R is the gasconstant, F the Faraday, T the temperature, alsok+2 = k-2 = k2 when E= 0 and Vm= k2[C],. Eqn. (7) isbased on the constant-field assumption (Goldman,1943).Now there is also (Morville et al., 1973) a passive

amino acid flux:

(8)L = PA([A]O- [A]i)and in the steady state:

L+v=0 (9)Hence the steady-state value of [A], can be calculatedby substituting eqns. (7) and (8) into eqn. (9), pro-vided that the values of [Na+]0, [A]., [Na+]i, E, V.,KA and PA are known. We assumed for this purposethat k2= k4.

(2) Membrane potential in the presence of aminoacid. The passive fluxes of K+ (FRK), Na+ (FmNa) andCl- (Fcli) and the Na+ flux (iNa) through the aminoacid pump are related, in the absence of otherpumped fluxes, by the equation:

rNa+ mnK+ tNa- nci =0 (10)

tNa is given by eqn. (7) above and on the constant-fieldassumption:

, ([I]o - [I], eEFRT) nEFt7n, RT(1 - e EF/RT) (11)

where P, is the permeability coefficient of the ion I andn is the charge on the ion.

(6)

for an electroneutral carrier system.However, we shall suppose that the loaded form of

the carrier bears a net charge of + 1, whereas theunloaded form bears no net charge. Eqn. 6 then hasthe form (Morville et al., 1973):

= Vm r-[Na+]i[A]0 EF [Na+]i[A]i EFD (7)

LRT1 eEFIRT) + RT(e-EFIRT_ 1)J

Substitution of eqns. (7) and (11) into eqn. (10)gives:

PNa[Na ]O + PK[K+]O +E = RT In Pc,[Cl-]i + Vm[Na4]o[A]o/D

F PNa[Na+]i + PK[K+]i +Pc,[Cl-]0 + Vm[Na+]i[A]iID

(12)

Numerical solutions to eqn. (12) were obtained byusing the routine C05AAF. Given the values of[Na+],, [K+]0, [CI-]O, [A]0, [Na+]i, [K+]i, [CI-]i, PA,

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(k+2[Na+],[A]. - k-2[Na+]i,[A]i) [C],[Na]..V( k+2 [Na+]i[A]i(I+ k-)2+ 2[Na1]0[A]. [Na+],[A]i (k+2+ k-2..+KA

k4 ~~k4 KA +KA

803

R. D. PHILO AND A. A. EDDY

Vm, PNa, PCI and PK and that k2/k4 = 1, eqns. (9) and(12) can be solved to obtain the corresponding steady-state values of Al and E. Again this was carried outwith the routine CO5AAF.

(3) Amplitude of depolarization caused by theamino acid. The amino acid lowers the membranepotential from El to E2 where:

El = E2- In (1 + r[NaA

E2F+ RT(1l e E2FIRT)j) (13)

and a = Vm (PNa [Na+]o + PK [K+]O + PcI [C1]i)-'.We have supposed for this purpose that k2/k4 = 1.

Polynomialfitting

The routine EO2ABF was used where polynomialswere to be fitted through sets of points (NottinghamAlgorithms Group Subroutine Library, NAGCentral Office, 13 Banbury Road, Oxford OX2 6NN,U.K.). Routine CO5AAF was also from this source.

Results

Role of the mitochondria in the fluorescence signal

Cyanine dyes are known to be taken up by isolatedmitochondria, probably in response to the mitochon-drial membrane potential (Laris et al., 1975). Thispossibility complicates the interpretation of thefluorescence changes observed in intact cell prep-arations (Philo & Eddy, 1975; Eddy et al., 1977;Burckhardt, 1977). We therefore determined both (1)the minimum concentration of oligomycin andantimycin required for maximum inhibition ofrespiration of the intact tumour cells, and (2) theminimum concentration of 2,4-dinitrophenol re-quired for maximum stimulation of respiration afteroligomycin inhibition (Table 1). We suggest that thepresence of these concentrations of oligomycin,antimycin and 2,4-dinitrophenol is likely to prevent amitochondrial membrane potential being generatedby either the adenosine triphosphatase or by respir-ation. The fluorescence assays were therefore carriedout in the presence of 6ng of oligomycin, 1 pg ofantimycin and 60nmol of 2,4-dinitrophenol per mlof cell suspension containing 0.32mg dry wt. of thetumour cells and 1 ,ug of the dye.

Toxicity of the dye.The presence of the amount ofdye stipulated above, together with the mitochondrialinhibitors, had no significant effect on the amount ofmethionine the tumour cells accumulated during30min at 370C from the standard Ringer solutioncontaining glucose. The Na+ pumpwas not inhibited,as was shown by the maintenance of [Na+]i and

Table 1. Effect of graded amounts of antimycin, oligo-mycin and 2,4-dinitrophenol on the endogenous respiration

rateThe tumour cells were suspended in the standardNa+/Ringer solution (50mg dry wt. of cells/ml) andkept at 0°C for up to 2 h before use. For assay, 0.2mlof this suspension was added to 3.8 ml of Ringer solu-tion at 37°C containing 163mM-Na+, 5mm-K+ and4mg of glucose. The metabolic inhibitors were added(lO,pl) as solutions in ethanol, except for 2,4-dinitro-phenol, which was dissolved in water.

Successive additionsExpt. I Initial

Oligomycin (lOng)Oligomycin (lOng)Oligomycin (lOng)Oligomycin (lOng)Oligomycin (lOng)Oligomycin (lOng)

Expt. 2 InitialOligomycin (2,g)2,4-Dinitrophenol (50nmol)2,4-Dinitrophenol (SOnmol)Carbonyl cyanide p-trifluoro-

methoxyphenylhydrazone(SOOpmol)

Expt. 3 InitialAntimycin (51g)Antimycin (S5g)

Rate of02 uptake(nmol/minper mg dry

wt.)2.62.62.61.81.00.720.721.60.81.81.81.8

3.00.40.4

[K+]1 at about 50mM and 150mM respectively, valueslike those observed in controls without the metabolicinhibitors. Raising the dye concentration 3-foldlowered the ratio [cellular methionine]/[extracellularmethionine] by about 30%. Thus the preferred dyeconcentration appears to be just tolerated by thesystem provided glucose was added to maintain asupply of ATP. No evidence was obtained in thepresent investigation that this selected concentrationofcyanine dye altered the permeability characteristicsof the tumour cells towards methionine, Na+ or K+,although such effects have been observed in humanerythrocytes (Hoffman & Laris, 1974; Simons, 1976).

Relation between fluorescence and membranepotential (E). In the valinomycin 'null point' methodfor estimating membrane potential with cyanine dyes(Sims et al., 1974; Hoffman & Laris, 1974) a concen-tration ofK+ in the Ringer solution is found such thatthe addition of valinomycin causes no change influorescence. It is assumed that under these con-ditions the cellular concentration of K+ ([K+]i) andthe extracellular concentration of K+ ([K+]0) are in

1978

804

ELECTROGENIC AMINO ACID ABSORPTION

equilibrium with the membrane potential. ThusE= RT/F In ([K+]0/[K+]i). The method is valid only ifvalinomycin increases the permeability of the cellmembrane to K+ ions without affecting otherparameters governing the membrane potential. Theprocedure outlined below is based on the sameassumption.

In one such 'null point' assay (Fig. 1), the tumourcells were incubated for 10min with the metabolicinhibitors in various Ringer solutions containing[K+] in the range 1-150mM, and the dye was thenadded. The addition of valinomycin caused a gradedchange in the fluorescence of the various cell suspen-sions with a 'null point' corresponding to someintermediate value of [K+]. (Fig. la). Fig. 1(b) showshow the fluorescence observed before and after theaddition ofvalinomycin varied with log ([K+]L/[K+]i).The two solid lines were computed by fitting quadraticfunctions through the points. The intersection ofthese lines was taken to be the interpolated 'nullpoint'. Paradoxically, the use of this procedure doesnot imply that K+ ions were in reality strictly inequilibrium with the membrane potential at the 'nullpoint' for an extended period of time, for this wouldonly be the case if their absorption through the Na+pump had ceased. The validity of the method thusappears to depend on both the rapidity of the dyeresponse and on the ability of valinomycin rapidlyto increase the K+ permeability to a marked extent.A limitation of the above procedure is that the

membrane potential is eventually determined onlyunder the ionic conditions at the 'null point', thedye fluorescence serving, as it were, merely as atitration indicator. To circumvent this limitationEddy et al. (1977) suggested that a calibration curvemight be constructed showing the relation betweenthe fluorescence of the cell suspension at the 'nullpoint' and the membrane potential (E) when bothparameters varied with the physiological circum-stances. This raised the question whether there was aunique relation between the fluorescence and themagnitude ofE, or whether the effect ofthe membranepotential on the dye distribution would be obscuredby variations in the binding of the dye to the plasma-lemma under the different incubation conditions(Kimmich et al., 1977). The choice of conditions wasbased on qualitative evidence that the presence of anamino acid depolarizes the plasmalemma, whereasthe operation ofthe Na+ pump tends to hyperpolarizeit (Philo & Eddy, 1975; Heinz et al., 1975; Laris et al.,1976). A series of 31 assays like those illustrated inFig. 1 was therefore carried out with cell preparationsin which the magnitude ofEwas deliberately varied byincubating the tumour cells with methionine orglycine and glucose, for appropriate time intervals,as indicated in the legend to Fig. 2.

Fig. 2 shows that the fluorescence at the 'null point',

Vol. 174

ca

r.Ce*a

4)01-

C)

4)0rA

(a)J

HI~H

G ll-F

D E

5 mini-

Time (min)

70 [ (b)

60~

50 F

40 F

30

-2.0 -1.5 -1.0 -0.5 0

log ([K+lJ[K+],)Fig. 1. Observations made in the course of a single 'nullpoint' titration designed to determine the intensity offluorescence at the K+ equilibrium potential for that

particular cell preparation(a) Ten portions of a given preparation of the tumourcells were incubated for 10min without glucose inRinger solutions containing the mitochondrialinhibitors. [Na+] + [K+] was 163mM and [K+] was:A, 1.3mM; B, 1.8mM; C, 2.9mM; D, 4.5mM; E,8.1 mM; F, 14mM; G, 23mM; H, 42mM; 1, 71 mM;J, 144mm. The carbocyanine dye was then addedand a portion (4ml) of the cell suspension was put intothe fluorimeter cuvette (see the Materials andMethods section). These traces show how the fluore-scence intensity changed abruptly to a new value ineach case within about 1 min after the additionof lOpg of valinomycin to the system. (b) Transform-ation of the observations shown in (a). The fluore-scence intensity just before (e) and about I min after(o) the addition ofvalinomycin is shown as a functionof log (K+]h/[K+]J). The solid lines represent quad-ratic functions fitted to the observations.

assayed under the standard conditions, was roughlylinearly related to the corresponding membranepotential. A regression analysis based on E as thedependent variable showed that the slope was2.85 ± 0.17, with an intercept of -183.9 + 7.8(r = 0.951; P < 0.001). Although such linearityclearly cannot be maintained over an indefinitelyextended range of membrane potential, there aretheoretical grounds for expecting approximatelinearity over a restricted range of values of E

805

R. D. PHILO AND A. A. EDDY

0.-

cd

.

E

:-0

-20

-40

-60

-80

-100

(a)

(b)

S

30 35 40 45 50 55 60Fluorescence intensity (arbitrary units)

Fig. 2. Calibration curve showing the fluorescence intensityat the 'null point' as a function of the correspondingmembrane potential in 31 cell preparations in which it was

deliberately variedThree series of assays were carried with cell prepara-tions that initially contained at least 60mM-Na+.Each assay involved a titration like that illustratedin Fig. I (see the text). In six such assays [Na+] wasconstant at 30mM and choline chloride replacedKCI as [K+] was varied. The results fitted the sameregression line as the other 25 assays. The solid linesdepicted represent the regression ofEon the fluores-cence parameter together with the 95%/ confidencelimits. In the first series -E was varied from 22 to45mV. The Ringer solutions contained no glucoseand the cell preparations were incubated for 10min(four assays). Otherwise 1 mM-L-methionine was alsopresent (three assays). In the second series -E wasvaried from 33 to 52mV. The cells were incubatedwith glucose for 30min (three assays) and with 1 mm-methionine as well (three assays). In the third series-Evaried from 58 to I IOmV. The cells were incubatedwith glucose for 10min (five assays), with 2.5mM-glycine as well (seven assays), and with I mM-glycine(three assays) or with 1 mM-methionine as well (threeassays).

(Burckhardt, 1977; Kimmich et al., 1977). Weconclude that there was a systematic relation betweenthe fluorescence intensity assayed under the standardconditions and the magnitude of E. We have used thisrelation to estimate membrane potentials in con-ditions where the 'null point' method is inapplicablebecause K+ ions are not in equilibrium with themembrane potential. For instance, when the tumourcells were incubated for 30min in the standard Ringersolution containing glucose and mM-methionine,the fluorescence intensity (± S.E.M.) exhibited in thestandard assay was 45.0± 0.6 units (n = 3). Thiscorresponds to E= -56 ± 4mV. Similarly the meanof two determinations made in the absence of meth-ionine was -55 mV.

Time course of thefluorescence changesEffect of amino acid influx or efflux. The addition

of valinomycin caused a change in the fluorescence of

20

1 r (c)

(d)

5 min

Fig. 3. Increase in fluorescence intensity (depolarization)after the addition of glycine to a cell suspension in thestandard Ringer solution containing glucose, the mitochon-

drial inhibitors and the carbocyanine dye at 37°CThe fluorescence was assayed as described in theMaterials and Methods section. Traces (a) and (b)show how the fluorescence intensity varied immed-iately after the addition of 1 mM-glycine (trace a) or9.8mM-glycine (trace b). Controls to which a similarvolume of either water or the Ringer solution wasadded exhibited no significant changes in fluorescence(results not shown). Traces (c) and (d), which corres-pond to roughly the same initial fluorescence intensity,show what happened when the experiment wascontinued for 40min. For trace (c) the 2mM-glycinewas added at the start and the assay was continueduntil the fluorescence intensity was roughly constant.The control to which no addition of glycine was made

is represented by trace (d).

the dye in the standard assay system that was com-pleted in about 1 min (Fig. la). Likewise, the uptakeof glycine, provided the Ringer solution containedNa+ (Laris et al., 1976; Eddy et al., 1977), increasedthe fluorescence to a maximum value, reached inabout 0.5 min (Fig. 3, trace a). The fluorescenceintensity declined during the next 40min (Fig. 3,trace c). The fluorescence intensity was then slightlylarger than was observed in a parallel cell suspension(control) to which the amino acid was not added. Thesystem was intact at that time because the additionof glycine to the control caused a marked increase influorescence (results not shown). We attribute thebehaviour illustrated in Fig. 3(trace c) to the circum-stance that the uptake of glycine reached a steadystate by about 40min and that the influx and efflux ofglycine were both electrogenic. The inferred hyper-polarization accompanying the efflux of glycine wasdemonstrated more directly (Fig. 4). On being trans-ferred to a Ringer solution without glycine, cellpreparations that had previously accumulatedglycine exhibited a somewhat lower fluorescence

1978

806

ELECTROGENIC AMINO ACID ABSORPTION

(a) I (b)l (c) I (d) I

cn

c:3

?tm

.0-Fq

U)c2c

00cCD0(A01. 1 002 5 minU- -L-

Fig. 4. Lowering of the fluorescence intensity (hyper-polarization) associated with the efflux ofglycine into two

types of Ringer solutionsThe tumour cells were incubated for 15 min in the stan-dard Ringer solution. One portion was then incubatedfor 15min at 37°C in the standard Ringer solutionwith 30mM-glycine and 170mM-glucose to load thecells with glycine. A further portion was incubatedwith a similar solution without glycine (controls).Each cell preparation was then collected and washed.At the indicated time, a portion (50u1) was put in thefluorimeter cuvette containing either 4ml of thestandard Ringer solution or 4ml of the K+ Ringersolution lacking Na+, together with the mitochon-drial inhibitors and 4pg of the carbocyanine dye. Thefluorescence intensity was recorded for about 5minin each instance. Trace (a) is the control for trace (b)and trace (c) is the control for trace (d). For (b) and(d) the cell preparations were loaded with glycineand for (a) and (c) they were not. Traces (a) and (b)were obtained by using the standard Ringer solutionand traces (c) and (d) by using the K+/Ringer solution.

intensity than did those kept for a similar period inthe absence of glycine. The effect was observed whenthe fluorescence assay was carried out in the standardRinger solution or in one lacking Na+ ions (Fig. 4).

Role of the Na+ pump. Earlier work had shown (1)that the tumour-cell preparations that had accumu-lated Na+, at the expense of cellular K+, exhibited arelatively small fluorescence in the standard Ringersolution containing the cyanine dye, as though theplasmalemma was hyperpolarized. Since this behav-iour depended on the presence of both extracellularK+ and glucose, and since it was blocked by ouabain(Philo & Eddy, 1975; Laris et al., 1976; Eddy et al.,1977), hyperpolarization appeared to be due to theaccelerated working of the Na+ pump. (2) Gibb &Eddy (1972) and Heinz et al. (1975) suggested that thetumour cells loaded with Na+ were hyperpolarizedin Ringer solutions containing about 30mM- or

Vol. 174

(a)

t

(c)

(b)

._

ld) *"

t | ~~~~~~c2OJ

5min .1?tFig. 5. Effects of early or late additions of ouabain orvalinomycin on the fluorescence intensity exhibited by cellpreparations in a Ringer solution containing 80mM-Na+

and 150mM-K+The tumour cells were loaded with Na+ and trans-ferred to the hyperosmotic Ringer solution (Gibb &Eddy, 1972) in the cuvette at the time indicated bythe start (left-hand end) of the trace. The solution(4ml) also contained 2mg of glucose/ml, the mito-chondrial inhibitors and 44ug of the dye. For trace(a) 0.25mM-ouabain was added after about 5min.For trace (b), which refers to the same scale offluorescence intensity as trace (a), 0.25mM-ouabainwas added at 30min. For trace (c) 10,pgofvalinomycinwas added at 5 min. For trace (d), drawn on the samescale as trace (c), lOg of valinomycin was addedat 30min.

80mM-Na+ and 150mM-K+. The addition of either0.25 mM-ouabain or valinomycin (2.5,ug/ml) to suchpreparations, within 15min of the start of the incu-bation, caused a marked increase in fluorescence(Fig. 5). Our interpretation is that the Na+ pumpinitially hyperpolarized the cell preparations underthese particular ionic conditions and that the pumpaction was blocked by ouabain and short-circuited byvalinomycin. After 30min, the distribution of Na+and K+ would tend to reach a steady state in which thecontribution of the Na+ pump to the membranepotential would be small (Thomas, 1972).An artifact in incubations of 30min duration.

Preliminary work had shown that a fraction of theorder of 10% of the dye present in the cuvettecontaining the cell suspension progressively becameattached to the wall of the vessel during an assay of30min duration. The fluorescence reached at that timetherefore corresponds to a different membranepotential than would be predicted on the basis ofFig. 2. For this reason the observations shown inFig. 3 (traces c and d) and Fig. 5 provide only a roughguide to the magnitude of the membrane potential atany particular stage of the assay. Further quantitativework involved a standard incubation with the dye forabout 5min (Fig. 2). It was shown in that way that

807

R. D. PHILO AND A. A. EDDY

Table 2. Parameters deduced from the increase in fluor-escence (cellular depolarization) caused by glycine or

L-methionineIn both instances the observations obtained in threeindependent assays, like that illustrated in Fig. 6,were pooled. The maximum increase in fluorescenceand apparent Km were deduced by expressing thefluorescence increase as a hyperbolic function of theamino acid concentration.

0 1 2 3 4

[Methioninel (mM)Fig. 6. Eqn. (13) and the increase influorescence intensity

caused by selected concentrations Of L-methionineThe assays were carried out at 37°C in the standardRinger solution containing glucose. The increase influorescence intensity was assayed as illustrated intraces (a) and (b) of Fig. 3. The fluorescence changecaused by a given concentration of the amino acidwas extrapolated to the time the addition was made.The magnitude of the change determined in that wayis illustrated as a function of the methionine concen-tration. The solid line was computed from eqn. (13)with a = 33.6 and KA/[Na+JO = 2.74mM. The magni-tude ofEwas inferred from the fluorescence intensityby means of the calibration curve (Fig. 2).

Function(1) Apparent Km with respect

to amino acid concen-tration

(2) Maximum increase influorescence (arbitraryunits)

(3) Corresponding voltagechange inferred fromFig. 2

(4) KA/[NaJ of eqn. (13)(5) a of eqn. (13)(6) Km from velocity of amino

acid uptake (Eddyet al., 1967; Morvilleet al., 1973)

Glycine L-Methionine0.75mM 0.40mM

26 20

74mV

9mM484mM

the mean value ± S.E.M. of E after incubation of thetumour cells with glucose for 8min in a Ringersolution containing 80mM-Na+, 150mM-K+ and1 mM-L-methionine was -92 ± 6mV (5). Clearly thesecell preparations were hyperpolarized as comparedwith cells kept for 30min in the standard Ringersolution (see above).

Magnitude of the depolarization caused by amino acidabsorption

As was also observed with glycine (Fig. 3, trace a),the addition of methionine caused an increase in thefluorescence exhibited in the standard assay system.The magnitude of the change, determined by extra-polation to zero time, increased with the methionineconcentration up to a maximum value of about 25units of fluorescence intensity (Fig. 6), or about7OmV (Fig. 2). The observations roughly fitted arectangular hyperbola, with a Km of about 0.3mm formethionine. Similar behaviour was reported by Lariset al. (1976) for glycine. However, they observed anapparent Km of 2.5mM for that amino acid, which islarger than the value of about 0.75mM we have nowobtained (Table 2).

Laris et al. (1976) also reported rough quantitativeagreement between the apparent Km for glycine of1.5mm observed in transport studies and the abovevalue based on the fluorescence assay. Our obser-

vations, summarized in Table 2, indicate that theapparent Km values obtained by the two methods areprobably different. Indeed the fact that one assay isbased on changes in the membrane potential Ewhereas the other monitors the amino acid fluxmeans that the same value ofKm would not necessarilyapply in both instances.

Application of the mathematical model. The ampli-tude of the Na+-dependent depolarization caused bythese amino acids showed that a relatively largechange in Na+ permeability had taken place. More-over, the net uptake of Na+ with glycine was accom-panied by the expulsion of less than an equivalentamount of K+, possibly because Cl- was also ab-sorbed (Eddy, 1968). Thus the relative magnitudes ofthe flux of Na+ through the amino acid carrier andthe fluxes through the various channels conductingNa+, K+ and Cl would govern the membranepotential (eqns. 2-13).

In practice the values of a and KA/[Na]. shown inTable 2 were obtained by fitting the observationsillustrated in Fig. 6 to eqn. (13). The position of thesolid line depicted in Fig. 6 shows that a satisfactoryfit was obtained. This procedure thus leads (1) to avalue of the dissociation constant KA of the ternarycomplex ANaC. and (2) to a value of the complexparameter a, defined as the ratio of Vm for the aminoacid carrier to a second function based on the perme-ability coefficients for Na+, K+ and Cl-. The furtherdevelopment of the mathematical model is describedby Philo & Eddy (1978).

1978

I-8 208cnow,

15o Ca

o 5

56mV

3.1mM230.96mnm

808

ELECTROGENIC AMINO ACID ABSORPTION

Discussion

Although the membrane potential of the strain oftumour cells used in the present investigation has notbeen studied by conventional electrophysiologicaltechniques, work with the related mouse Ehrlichascites tumour has given results either in the vicinityof -llmV (Hempling, 1962; Aull, 1967; Smith &Levinson, 1975) or in the range from -2OmV to-4OmV (Johnstone, 1959; Lassen et al., 1971).Lassen et al. (1971) found that, after insertion of theglass electrode, the apparent membrane potentialdecayed rapidly with time, from a value in theforementioned upper range to one in the lower range.This behaviour was attributed to the failure of theplasmalemma to form an insulated seal around theinserted electrode. Moreover, work with HeLa cells,with fibroblasts and with excitable cultured cells ofneural or myocytic origin has led to estimates of themembrane potential varying from about -4OmV to-6OmV (Sachs & McDonald, 1972; Okada et al.,1973). Thus the conclusion reached by Williams(1970), on the basis of earlier literature, that the'resting' membrane potential in non-excitable cells ismuch lower than in nerve and muscle does not holdin general and may not apply to the mouse ascites-tumour cells.The fluorescence intensity exhibited by the tumour

cells incubated for 30min in the standard Ringersolution with glucose corresponded to a membranepotential of about -55mV. Comparable assays withanother carbocyanine dye and the Ehrlich ascitestumour in the absence of glucose gave values near-4OmV, in a system that was calibrated on the basisof the assumption that the membrane potential wasequal to the K+ equilibrium potential in the presenceof 18.6pM-valinomycin (Burckhardt, 1977). SimilarlyLaris et al. (1976) inferred values ranging from-18mV to -42mV by using a different carbocyaninedye as an indicator in a 'null point' titration withvalinomycin. Both of the above estimates depend onthe further assumption that the addition of valino-mycin caused a change in fluorescence intensity thatwas not due to the redistribution of the dye betweenthe mitochondria and the cytosol. Bearing in mindthe very different procedures used by Burckhardt(1977) and Laris et al. (1976) their observations aresimilar to the upper range of estimates of the mem-brane potential obtained by inserting microelectrodesinto the Ehrlich cells.The markedhyperpolarizationuptoabout-100mV

that took place when the Na+ pump was expellingNa+ rapidly from the tumour cells (Figs. 2 and 5)corresponds to values much larger than the K+equilibrium potential. Similar phenomena have beenobserved by implanting glass electrodes in othermammalian cells (see Sachs & McDonald, 1972). Onthe other hand, the magnitude of the depolarization

Vol. 174

of up to 7OmV caused by the addition of the aminoacids (Fig. 6) is larger than has been detected bymicroelectrode techniques in either the kidney or theintestine. This is possibly because the methods usedonly detected the voltage changes in these last-namedsystems in an attenuated form, owing to the presenceof a low-resistance shunt (Maruyama & Hoshi, 1972;Rose & Schultz, 1971). It is noteworthy in thisconnection that the proton-dependent absorption ofglucose by Neurospora depolarized the hyphae by upto 1OOmV (Slayman & Slayman, 1974).

Smith & Levinson (1975) have concluded that theEhrlich ascites-tumour-cell membrane is atypical inbeing unable to support valinomycin-mediated K+transport. These workers nevertheless observed thatvalinomycin caused a greater loss of cellular K+ andinflux of cellular Na+ than the accompanyingdepletion of the cellular ATP content was itselfexpected to cause. Reid et al. (1974) attributed thesimilar behaviour they observed, with the strain oftumour cells used in the present investigation, to theincrease in K+ permeability (PK) caused by valino-mycin. This increase would be expected to raise themembrane potential (E). However, Smith & Levinson(1975) found that the membrane potential of-12mVrecorded by microelectrode impalement ofthe Ehrlichcells was scarcely affected by the presence of valino-mycin. We suggest that the changes in the ioniccomposition of the tumour cells observed by Smith& Levinson (1975) are consistent with the hypothesisof Reid et al. (1974) and that the method the formerworkers used for assaying the membrane potential,which involves the introduction of La3+ into thesystem, may not be sound (see Laris et al., 1976). Thisview that valinomycin increases PK and therebychangesE is indeed consistent with other phenomena.(1) It explains the effect of valinomycin and of otherionophores on the accumulation of amino acids incell preparations depleted ofATP (Reid et al., 1974).(2) It accounts for the ability of valinomycin to affectthe cellular accumulation of the carbocyanine dyes(Fig. 2; Laris etal., 1975; Burckhardt, 1977) and otherlipophilic cations (Cespedes & Christensen, 1974).(3) It explains why a mixture of nigericin and aproton conductor mimicked the effect ofvalinomycinon the dye fluorescence exhibited by suspensions ofthe tumour cells (R. Philo & A. A. Eddy, unpub-lished work; cf. Kimmich et al., 1977).

It is useful to enumerate the assumptions involvedin using Fig. 2 to assay the membrane potential.First, there is the supposition that the changes in thefluorescence signal, measured under the standardconditions, varied in a systematic way with themagnitude ofE as inferred from the K+ equilibriumpotential at the relevant 'null point'. The latterinference in turn depends ontwofurther assumptions.(1) Valinomycin affected only PK among the factors

809

810 R. D. PHILO AND A. A. EDDY

governing the membrane potential. (2) Changes inthe distribution of the dye and hence the fluorescenceof the cell suspension were governed by only oneindependent variable, namely the magnitude of E.The very fact that the change in K+ equilibriumpotential correlated with a major fraction of thevariance associated with the fluorescence intensity(Fig. 2) encourages us to believe that the aboveassumptions are valid. The effect of various iono-phores on the dye distribution shows, moreover, thatit is the membrane potential and not the ratio[K]I/[K]0 that is the significant variable (Fig. la;Laris et al., 1976; Burckhardt, 1977; Kimmichet al., 1977).

The above work was supported by grant no. B/RG/42357 from the Science Research Council, U.K.

References

Aull, F. (1967) J. Cell. Physiol. 69, 21-32Blaustein, M. P. & King, A. C. (1976) J. Membr. Biol. 30,

153-173Burckhardt, G. (1977) Biochim. Biophys. Acta 468,227-237

Cespedes, C. & Christensen, H. N. (1974) Biochim.Biophys. Acta 339, 139-145

Christensen, H. N. (1977) FEBS Symp. 42, 222-235Crane, R. K. (1977) Rev. Physiol. Biochem. Pharmacol. 78,

99-159Eddy, A. A. (1968) Biochem. J. 108, 489-498Eddy, A. A. (1977) in Intestinal Permeation (Kramer, M. &

Lauterbach, F., eds.), pp. 332-349, Excerpta Medica,Amsterdam and Oxford

Eddy, A.A., Mulcahy, M.F. & Thompson, P.F. (1967)Biochem. J. 103, 863-876

Eddy, A. A., Philo, R., Earnshaw, P. & Brocklehurst, R.(1977) FEBS Symp. 42, 250-260

Gibb, L. E. & Eddy, A. A. (1972) Biochem. J. 129,979-981Goldman, D. E. (1943) J. Gen. Physiol. 27, 37-60Heinz, E., Geck, P. & Pietrzyk, C. (1975) Ann. N. Y. Acad.

Sci. 264, 428-441Hempling, H. G. (1962)J. Cell. Comp. Physiol. 60,181-198

Hladky, S. B. & Rink, T. J. (1976) J. Physiol. (London) 263,287-319

Hoffman, J. F. & Laris, P. C. (1974) J. Physiol. (London)239, 519-552

Johnstone, B. M. (1959) Nature (London) 183, 411Johnstone, R. M. (1972) Biochim. Biophys. Acta 282,

366-373Kimmich, G. A., Philo, R. D. & Eddy, A. A. (1977)

Biochem. J. 168, 81-90Laris, P. C., Bahr, D. P. & Chaffee, R. R. J. (1975)

Biochim. Biophys. Acta 376, 415-425Laris, P. C. Pershadsingh, H. A. & Johnstone, R. M.

(1976) Biochim. Biophys. Acta 436, 475-488Lassen, U. V., Nielsen, A. M. T., Pape, L. & Simonsen,

L. O. (1971) J. Membr. Biol. 6, 269-288Lever, J. E. (1977) Biochemistry 16, 4328-4334Maruyama, T. & Hoshi, T. (1972) Biochim. Biophys. Acta282,214-225

Morville, M., Reid, M. & Eddy, A. A. (1973) Biochem. J.134, 11-26

Okada, Y., Ogawa, M., Aoki, N. & Izutsu, K. (1973)Biochim. Biophys. Acta 291, 116-126

Philo, R. & Eddy, A. A. (1975) Biochem. Soc. Trans. 3,904-906

Philo, R. & Eddy, A. A. (1978) Biochenm. J. 174, 811-817Reid, M., Gibb, L. E. & Eddy, A. A. (1974) Biochem. J.

140, 383-393Rose, R. C. & Schultz, S. G. (1971) J. Gen. Physiol. 57,

639-663Sachs, H. G. & McDonald, T. F. (1972) J. Cell. Physiol.

80, 347-358Schafer, J. A. (1977) J. Gen. Physiol. 69, 681-704Schafer, J. A. & Heinz, E. (1971) Biochim. Biophys. Acta

249, 15-33Simons, T. J. B. (1976) Nature (London) 264, 467-469Sims, P. J., Waggoner, A. S., Wang, C. H. & Hoffman,

J. F. (1974) Biochemistry 13, 3315-3330Slayman, C. L. & Slayman, C. W. (1974) Proc. Natl.

Acad. Sci. U.S.A. 71, 1935-1939Smith, T. C. & Adams, R. (1977) J. Membr. Biol. 35, 57-74Smith, T. C. & Levinson, C. (1975) J. Men1br. Biol. 23,

349-365Thomas, R. C. (1972) Physiol. Rev. 52, 563-594Williams, J. A. (1970) J. Theor. Biol. 28, 287-296

1978