Biochem. J. (1965) 95, 378

Calcium Ion Accumulation and Volume Changes ofIsolated Liver MitochondriaCALCIUM ION-INDUCED SWELLING

By J. B. CHAPPELL* AND A. R. CROFTS*Department of Biochemi8try, Univer8ity of Cambridge

(Received 27 July 1964)

1. Liver mitochondria suspended in an iso-osmotic buffered potassium chloridemedium containing an oxidizable substrate and phosphate accumulated added Ca2±.During this process H+ appeared in the medium and the mitochondrial suspensionshowed increased light-scattering. Respiration was markedly stimulated. 2. Theaddition of excess of Ca2+, respiratory inhibitors or uncoupling agents causedextensive mitochondrial swelling associated with release ofCa2+ into the suspendingmedium. When the suspension became anaerobic extensive swelling also occurred.Only under conditions when the addition ofuncoupling agents wouldhave producedhigh rates of electron transport, e.g. in the presence of succinate, was the structuralintegrity of the mitochondrion maintained after Ca2+ accumulation. 3. Condi-tions that prevented respiration-dependent Ca2+ accumulation also preventedCa2+-induced swelling. Bovine plasma albumin was without effect, indicatingthat U-factor was not involved. Oligomycin together with ADP or ATP partiallystabilized the mitochondria against Ca2+-induced swelling. 4. It is suggested that a'high-energy' intermediate generated by coupled electron transport is required toprevent the mitochondrial swelling that results as a consequence of Ca2+ accumula-tion.

It has been known for a number of years thatCa2+ causes a swelling of large magnitude ofisolatedmitochondria (Raaflaub, 1953; Slater & Cleland,1953; Hunter & Ford, 1955; Tapley, 1956). It hasbeen claimed that this swelling action of Ca2+ ismore immediately due to the enzymically catalysedrelease of U-factor from an endogenous precursor,a process activated by Ca2+. It is characteristic ofswelling of this type, including that producedby Ca2+, that it is prevented by low concentrationsof serum albumin, probably because of the bindingofU-factor (Lehninger & Remmert, 1959; Wojtczak& Lehninger, 1961). Like other forms of large-mag-nitude swelling, e.g. that produced by phosphate.or thyroxine, Ca2+-induced swelling is abolished bythe addition of respiratory inhibitors and appearsto be dependent on electron transport through evena restricted portion of the respiratory chain (seeChappell & Greville, 1963a). Use of selectiveinhibitors of phosphorylation (2,4-dinitrophenol,oligomycin) rather than of electron transport hasindicated that this type of swelling is perhaps

* Present address: Department of Biochemistry, MedicalSchool, University of Bristol.

directly dependent on electron transport (Chappell& Greville, 1959, 1960, 1961, 1963a).Recent work from a number of Laboratories has

revealed that mitochondria isolated from a varietyof tissues are able to accumulate Ca2+, Mg2+,Mn2+ and Sr2+ with the simultaneous uptake ofphosphate in either a respiration-dependent processor one that requires the presence of ATP (e.g.Brierley, Bachmann & Green, 1962; Brierley,Murer, Bachmann & Green, 1963; Chappell, Cohn& Greville, 1963; Chappell & Greville, 1963b;Chappell, Greville, & Bicknell, 1962; Lehninger,Rossi & Greenawalt, 1963a,b; Saris, 1959, 1963a,b).During the investigation of this process in thisLaboratory it was discovered that Ca2+ causes aswelling of mitochondria that appears to beintimately related to the accumulation process(Chappell et al. 1963). In the present paper theresults of this investigation are presented. It hasbeen shown that U-factor is probably not involvedin this type of swelling, but rather that the accumu-lation of Ca2+ by the mitochondria is the causalfactor. Under conditions where accumulation is pre-vented swelling does not occur. In an accompany-

378

CALCIUM-INDUCED SWELLINGing paper (Crofts & Chappell, 1965) the reversal ofthis type of swelling is described.

METHODS AND MATERIALS

Rat-liver mitochondria were isolated in a freshly preparedmedium consisting of 0-25M-sucrose in 5mM-tris-ehloridebuffer, pH7-2, essentially by the method of Hogeboom,Schneider & Pallade (1948). The mitochondria were

washed twice and stored for use at a protein concentrationof 60-80mg. of protein/ml. at 00 for not more than 4hr.Oxygen consumption, changes in H+ concentration and

light-scattering were measured simultaneously in thereaction vessel shown diagrammatically in Fig. 1. Thetotal volume of the reaction vessel, which was thermostatic-ally controlled at 300, was 6-Oml. The reaction mediumconsisted of 80mM-KCl in 20mm-tris-chloride buffercontaining the various additions indicated in the text andlegends to Figures. The initial pH was 7-20-7-25 in eachcase.

Oxygen consumption. This was followed by using theClark oxygen electrode (Yellow Springs Instrument Co.,Ohio, U.S.A.), as described by Chappell (1964a). In someexperiments the rate of respiration was recorded directlyby differentiating the amplified signal from the oxygen

electrode (see Longmuir, 1957) by means of the circuitshown in Fig. 2. The 'noise' of the differentiated signal was5-10% of the ADP-stimulated suceinate rate (0-2,ug.atomof 0/min.), the small fluctuations in the electrode currentdue to imperfect stirring of the medium contributing mostof this 'noise'. In the Figures shown in the present paperthe traces of the recorded rate of oxygen consumption havebeen smoothed; the other traces have been presented as

they were recorded.pH changes. Changes in H+ concentration were followed

by using a Radiometer (Copenhagen, Denmark) concentric

glass electrode (model GK2026C) and radiometer pH-meter(model PHM22r) adapted for recording. The saturatedKCI solution of the concentric glass electrode was replacedby 0-1M-KCI. It was found that this diminished consider-ably the 'noise' due to stirring of the medium, presumablybecause of the decrease in the fluctuations of the junctionpotential at the porous plug separating the 0 1 M-KCI andthe reaction medium. The use of 0.1 m-KCI instead ofsaturated KCI had no effect on the behaviour of the pH-meter. The output from the pH-meter was used to drive a

Honeywell-Brown (Motherwell, Scotland) recorder (lmvfull-scale deflection, 1 see. response time). A suitableresistance network was used to match the pH-meter andrecorder, and a suitable backing-off circuit was incorporatedto enable a pH change of 0-1-0-2 unit to give a full-scaledeflection on the recorder, starting at any initial pH value.

Light-cattering. This was measured by using a 2-2vpre-focus torch bulb (Ever Ready 2225) supplied from a

stabilized 2v source (model 115D spectrophotometer powersupply; Labgear Ltd., Cambridge) and measurement of thelight-intensity at 1800 to the incident beam by using a

red-sensitive vacuum photocell (Cintell VS50S) coveredwith a gelatin filter with maximum transmission at 750mU.The current from the photocell was amplified with a PyeInstrument Co. (Cambridge) d.c. amplifier (model 11370)and recorded on a Control Instruments Ltd. (Birkenhead)potentiometric recorder (1 mv full-scale deflection, 0-3sec.response time).

There was no detectable interaction between the oxygenand glass electrode systems if care was taken to see thatthere was no electrical leak between the Clark electrode andthe reaction vessel.

Reagent&. Most organic chemicals used in the presentinvestigation were obtained from the California Corp. forBiochemical Research, Los Angeles, Calif., U.S.A. Sucroseused in the preparation of mitochondria and inorganic

Fig. 1. Reaction vessel for the simultaneous recording ofoxygen consumption, changes in H+ concentration andchanges in light-scattering at 180°. A, Photocell; B, filter;C, concentric glass and calomel electrode; D, oxygen

electrode; E, reaction vessel; F, lens-ended bulb; G, waterjacket; H, stirrer. The apparatus was constructed ofPerspex, the cell from clear Perspex, and the base, waterjacketing, electrode support and photocell hood from blackPerspex. Details of components are given in the Methodsand Materials section.

I -Sv ~~~~~A/iRI2

AMPI AMP2Pt c

Ref VRI Recorder 2R3 Al R

Recorder I R4

Fig. 2. Circuit for recording oxygen uptake together withthe rate of oxygen uptake. Components were as follows:VR1, lkQ; R1, 1-5kQ; R2, 1-0kQ; R3, 100 kQ; R4,50Q;R5, 5Q; C1, 8 pF, 750v d.c., paper; A/1, A1, AssociatedElectrical Industries Ltd. synchronous chopper (CK 3);AMP1, Pye d.c. amplifier (model 11370); AMP2, Prince-town AppliedResearchlock-in amplifier (modelJB4) ;Recor-ders 1 and 2, Control Instruments Ltd. recorders (0-5mvfull-scale deflection, 1-3sec. response time). The frequencyof chopping was determined by a tuning circuit incorpor-ated into the lock-in amplifier, which then filtered out allsignals except those at the set frequency, thus eliminating'noise' from the signal.

Vol. 95 379

J. B. CHAPPELL AND A. R. CROFTSchemicals were of A.R. grade. EGTA* was obtained fromL. Light and Co. Ltd., Colnbrook, Bucks. Oligomycin was

a gift from Charles Pfizer Inc., Groton, Conn., U.S.A.,and antimycin was purchased from the Wisconsin AlumniFoundation, Madison, Wis., U.S.A. Atractyloside was

a kind gift from Dr E. C. Slater and rotenone from Dr R. W.Estabrook. Carbonyleyanide-p-trifluoromethoxyphenyl-hydrazone was provided generously by Dr P. G. Heytler ofE. I. du Pont de Nemoirs and Co. Inc., Wilmington, Del.,U.S.A.

RESULTS

Some bivalent metal ions (Ca2+, Mg2+, Mn2+,Sr2+) are accumulated by mitochondria suspendedin the presence of phosphate and an oxidizablesubstrate by a process that has the followingcharacteristics.

(1) There is an increased rate of respiration(Chance, 1959) that decreases when the added ionhas been accumulated by the mitochondria(Chappell et al. 1962). The rates of electron trans-port produced by Ca2+ are two- to four-fold greaterthan those that occur during ADP-stimulatedrespiration (Chance, 1959, 1963; Chappell et al.1963; Gutfreund & Jones, 1964); in this respectCa2+ resembles 2,4-dinitrophenol, which can alsoinduce rates of electron transport well in excess ofthose produced by ADP together with phosphate(Chappell, 1962, 1964b). It is emphasized that thishigh rate of electron transport is not necessarilyassociated with any increase in the permeability ofthe mitochondrial membrane.

(2) Phosphate is accumulated at the same timeas the bivalent metal ion. Arsenate is able toreplace phosphate (Chappell et al. 1963). In thepresence of phosphate, analysis shows that it isprobable that the product accumulated has thecomposition M3(PO4)2 for Mg2+ (Brierley et at.

1962) and Mn2+ (Chappell et al. 1963) and somethingvery close to this for Ca2+ (Rossi & Lehninger,1963). This accumulation process accounts ade-quately for the appearance of H+ in the medium:

3M2++ 2HPO42---M3(PO4)2 + 2H+ (1)

3M2++ 2H2PO4-- M3(PO4)2 + 4H+ (2)

At pH 7-2, which is close to the second aciddissociation constant of phosphate, it would beexpected that approx. 1H+ would appear in themedium for each metal ion accumulated by themitochondria. This has been observed for Mn2+(Chappell & Greville, 1963b; Chappell et al. 1963).

(3) The accumulation of bivalent metal ions andphosphate is prevented by respiratory inhibitors(hydrogen cyanide, Amytal, rotenone, antimycin)appropriate to the oxidizable substrates presentand by a variety of uncoupling agents. It is not

* Abbreviation: EGTA, ethylene glycol bis(aminoethyl)-tetra-acetate.

affected by oligomycin (Chappell & Greville,1963b; Chappell et al. 1963; Rossi & Lehninger,1963) or by concentrations of atractyloside thatcompletely block ADP-stimulated respiration andATP synthesis (J. B. Chappell, unpublished work).The ATP-dependent accumulation of bivalentmetal ions is inhibited by oligomycin, but is un-affected by respiratory inhibitors (Rossi & Leh-ninger, 1963).

Light-scattering measurements have revealedthat during the accumulation of Mn2+ and Ca2+mitochondria undergo an apparent contraction(Chappell et al. 1963; and Fig. 3). This increasedlight-scattering by the mitochondrial suspensionmay well have been due to an increased opacity ofthe mitochondria as a result of the accumulatedmetal phosphate, rather than the type of effect thatis observed on addition ofADP (see Packer, 1960),although this latter alternative cannot be ruled out.

, F> 1.0 _

0 0-5

5 c

00020 _2O _

0~

C.

c0

&b

I min.0=

Fig. 3. Ca2+ accumulation and Ca2+-induced swelling ofmitochondria. The upper broken trace is a recording of thelight-scattering at 1800, a downward deflection representingmitochondrial swelling. The lower broken trace is a record-ing of the change in oxygen tension in the suspendingmedium, a downward deflection representing oxygenuptake by the mitochondria. The upper continuous traceis a recording of the differential of the change in oxygenconcentration, an upward deflection representing an

increase in the rate of oxygen uptake, and the height of thetrace giving the rate of oxygen uptake, which can be readoff on the scale. The lower continuous line is a recording ofthe change in H+ concentration, an upward deflectionrepresenting a production of H+. 8 mM-Succinate, 2-1 mM -

phosphate and 017,uM-rotenone (to prevent oxalo-acetate accumulation; see the text), were present initially,and additions were made where indicated as follows:A, mitochondria (9-7mg. of protein); B, 075,umole ofCaCl2; C, 0 5,umole of CaC12 D, 0 5,umole of CaCl2.

380 1965

CALCIUM-INDUCED SWELLING3The ADP-induced contraction is smaller than thatproduced by Ca2+ (Fig. 3). Successive additionsof Mn2+ or Sr2+ led to further increases in light-scattering synchronous with the accumulationprocess, but with Ca2+ at some stage extensivemitochondrial swelling occurred and Ca2+ wasreleased again (Fig. 3).

Inhibition of Ca2+-induced swelling. Inhibitorsthat prevented the accumulation of Ca2+ bymitochondria, as revealed by H+ production andincreased respiration, also prevented the swellingaction. Thus if 1001M-hydrogen cyanide or0- 1 ,tg. ofantimycin/ml. was present when succinate,or glutamate together with malate, served assubstrate then there was no pH change on addingCa2+, i.e. there was no accumulation, and themitochondrial volume remained unchanged, i.e.neither swelling nor contraction occurred. How-ever, if ascorbate (2mM) plus tetramethyl-p-phenylenediamine (0-2mM) were added whenswelling had been inhibited by antimycin,then Ca2+ was accumulated and extensiveswelling occurred (Fig. 4). Ascorbate plus tetra-methyl-p-phenylenediamine had no action whenrespiration had been blocked by hydrogen cyanide.With this latter inhibitor and with succinate assubstrate, neither accumulation of Ca2+ normitochondrial swelling occurred, but the furtheraddition of ferricyanide (2mM) as electron acceptorallowed both Ca2+ accumulation and extensive

B D E G

(a) (b)|j S 1

A C F

I min.

Fig. 4. Swelling supported by electron transport through a

restricted portion of the respiratory chain. In each of theseexperiments, 8 mM-succinate, 2-ImM-phosphate and 0-17 uM-rotenone, together with mitochondria (9-3mg. of protein),were present before the recording was started. The tracesare of light-scattering at 1800, downward deflectionrepresenting swelling. Oxygen uptake and ferricyanidereduction were followed by recording changes in oxygentension and H+ concentration respectively. These traceshave been omitted for clarity. Additions were made whereindicated as follows: (a) A, 1 5jmoles of CaCl2. (b) B,0 4,g. of antimycin; C, 1l5 jtmoles of CaCI2; D, ascorbate(20mm) with tetramethyl-p-phenylenediamine (0.2mM).(c) E, HCN (100PiM); F, 1 51tmoles of CaCl2; G, 5O0,umolesof K3Fe(CN)6.

swelling. These and other similar results indicatethat Ca2+ accumulation and Ca2+-induced swellingcan both be supported by electron transportthrough even a restricted portion of the respiratorychain, e.g. from succinate to ferricyanide (cyto-chrome b to cytochrome c) and from tetramethyl-p-phenylenediamine to oxygen (cytochrome c tooxygen).The addition of dinitrophenol (100,UzM) or car-

bonylcyanide-p -trifluoromethoxyphenylhydrazone(0.1 ,UM), a potent uncoupler of respiratory.chain phosphorylation (Heytler & Prichard, 1962),before the addition of Ca2+ prevented the pHchange associated with the accumulation process,any further increase in respiration beyond that dueto the uncoupling agent alone, and swelling.EDTA (5mM) or EGTA (0.5mM), a potent

chelating agent for Ca2+, Sr2+ and Mn2+ (Schmid &Reilly, 1957), completely inhibited the accumula-tion process and swelling. However, when Ca2+was added in excess over the EGTA present thenboth accumulation and swelling occurred.The ease with which Ca2+ produced swelling of

mitochondria was found to depend on a variety offactors, many of which are discussed below.Freshly prepared mitochondria could accumulatemuch larger quantities of Ca2+ (up to 300,uequiv.of Ca2+/g. of protein) without swelling occurringthan mitochondria that had been stored for 3-6hr.at 0°. The presence of lmg. of defatted bovineplasma albumin/ml. had no effect on the rate orextent of Ca2+-induced swelling.

Relationship between rate of respiration andCa2+-induced swelling. Inhibition of respiration(anaerobic conditions, respiratory inhibitors) pre-vented both Ca2+ accumulation and Ca2+-inducedswelling. In contrast, in the absence of respiratoryinhibitors substrates capable of producing poten-tially low rates of electron transport, e.g. endogen-ous substrates or DL-.-hydroxybutyrate, muchmore readily supported Ca2+-induced swellingthan those substrates, e.g. succinate or glutamatetogether with malate, that were capable of sup-porting relatively high rates of electron transport(Chappell, 1962, 1964b). Some explanation of theseresults is provided by the following experiments.The addition of either respiratory inhibitors or

uncoupling agents to mitochondria that hadalready accumulated Ca2+ resulted in a rapidswelling and a release of the accumulated Ca2+, asrevealed by a reversal of the pH change thatoccurred during the accumulation process. InFig. 5 the results of an experiment of this type areshown. In this case oligomycin was present beforethe mitochondria were added. The addition ofADP led to no increase in respiration and only asmall pH change, very much smaller than thatwhich would have occurred as a result of the

Vol. 95 381

J. B. CHAPPELL AND A. R. CROFTS

A B C D

0~~~~~~~~~~~~~~

I-o- ~~~~~~~~~~~0

Fi.5Cwlinafmtconrata a acmlted

0-5+\ b -0 0

0

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Imin. 02

Fig. 5. Swelling of mitochondria that had accumulatedCa2+, initiated by uncoupling agents. The convention ofthe traces is the same as that given in Fig. 3. 8mM-Suc-cinate, 2 lmM-phosphate, 017,um-rotenone and 033,ug. ofoligomycin/ml. were present initially. Additions weremade where indicated as follows: A, mitochondria (9.7mg.of protein); B, 05,umole of ADP; C, 2 O,moles of CaCl2;D, dinitrophenol (133,UM).

I.C1-0 _0

o0.° 0.5

S0,

CZ:: :U 0 L

-0-02

< O

1-I._C)

I-

4-

to

4-,

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1 min.

Fig. 6. Swelling of mitochondria that had accumulatedCa2+, initiated by respiratory inhibitors. The conventionof the traces is the same as that given in Fig. 3. 8 mM-Suc-cinate, 2 1mM-phosphate, 017,uM-rotenone and 033,ug. ofoligomycin/ml. were present initially. Additions weremade where indicated as follows: A, mitochondria (9.7mg.of protein); B, 05bjumole of ADP; C, 2-Oymoles of CaCl2;D, 04,ug. of antimycin.

phosphorylation of ADP. The addition of Ca2+led to a marked increase in the rates ofH+ produc-tion and respiration. After a new steady statehad been reached the addition of dinitrophenol ledto increased respiration, swelling of the mito-chondria and Ca2+ release. In Fig. 6 a similar experi-ment is shown; in this case antimycin was addedinstead of dinitrophenol.

In brief, it appears that if the potential rate ofaccumulation of Ca2+ exceeds the rate at whichelectron transport is able to supply the energy

necessary for this accumulation then swellingresults. This action of respiratory inhibitors anduncoupling agents in inducing mitochondrialswelling is to be contrasted with their inhibitoryeffect when they are added before the Ca2+, i.e. at atime when they prevent the accumulation of thebivalent metal ion.The hypothesis that an inadequate rate of elec-

tron transport is responsible for Ca2+-inducedswelling once some measure of accumulation hasoccurred is supported by results obtained whensuccinate served as substrate. As mentioned aboveCa2+, like dinitrophenol, is able to induce rates ofelectron transport in excess of those produced byADP together with phosphate, and it may bepredicted therefore that Ca2+ ought to lead tonon-linear rates of succinate oxidation as a result of

the accumulation of oxaloacetate and the conse-quent inhibition of succinate dehydrogenase(Pardee & Potter, 1948; Chappell, 1961, 1963,1964b; Gutfreund & Jones, 1964) and that thiswould in turn lead to Ca2+-induced swelling as aresult of failure of respiration. If this were so,then rotenone or Amytal, both of which inhibit theoxidation of those substrates with NAD-linkeddehydrogenases, including malate dehydrogenase(Ernster, Jalling, Low & Lindberg, 1955; Fukami& Tomizawa, 1956; Lindahl & Oberg, 1961),should diminish to some extent the onset of Ca2+_induced swelling in the presence of succinate. Thisin fact proved to be the case. Glutamate (5mm),which also prevents the inhibition of succinateoxidation by high concentrations of dinitrophenol(greater than 20LM) (Chappell, 1964b), alsostabilized the mitochondria against Ca2+-inducedswelling when succinate served as substrate.However, even in the presence of rotenone, the

addition of malonate (0*5-1.OmM) to mitochondriathat had accumulated Ca2+ in the presence ofsuccinate (4mM) led to both inhibition of respira-tion and to swelling. This action of malonateresembled that ofantimycin and uncoupling agents.It appears therefore that once accumulation ofCa2+ has occurred a high concentration of some' energy-rich' intermediate is required by themitochondria for the maintenance of their mor-phological integrity. Anaerobic conditions, or

382 1965

CALCIUM-INDUCED SWELLING3the addition of a respiratory inhibitor or un-coupling agent, leads to immediate and extensiveswelling.

Effects of oligomycin, adenine nucleotides andarsenate. Oligomycin (up to 0-8,ug./ml.) waswithout effect on the respiration-dependent accum-ulation of Ca2+ by liver mitochondria and had onlya small and variable inhibitory effect on Ca2+_induced swelling. However, in the presence of200 ,LM-ADP and oligomycin the mitochondriawere able to accumulate very much greaterquantities of Ca2+ than in the absence of thesecompounds. ATP was as effective as ADP in thepresence of oligomycin. This stabilizing action ofadenine nucleotides provides an explanation of theobservation that ATP is required for extensiveaccumulation of Ca2+ and phosphate by mito-chondria (Brierley et al. 1962; Lehninger et al.1963a).Arsenate could replace phosphate at the same

concentration in both the accumulation andswelling processes. Oligomycin completely inhibitsarsenate-stimulated respiration (Estabrook, 1961),but had no effect on the rate of accumulation ofCa2+, Mn2+ or Sr2+ in the presence of arsenate.However, under these circumstances it was foundthat mitochondria were more prone to the swellingaction of Ca2+ in the presence of arsenate as com-pared with phosphate. With Mn2+ or Sr2+ nodifference in behaviour with regard to H+ produc-tion or oxygen consumption was observed. Evenin the presence of arsenate neither Mn2+ nor Sr2+caused mitochondrial swelling.

Changes in rate of respiration during Ca2+_induced swelling. The addition of Ca2+ to mito-chondria suspended in the presence of phosphateled to an increased rate of respiration, H+ produc-tion and an increased light-scattering. With lowconcentrations of Ca2+ (approx. 1 ,mole of calciumchloride/6ml.), once Ca2+ accumulation was com-pleted then the rate ofrespiration fell to a low value,characteristic of the resting state, H+ productionceased and the mitochondrial volume, as reflectedby light-scattering, remained unaltered. Withhigher concentrations of Ca2+, after a period ofaccumulation, characterized by a rapid rate of H+production and respiration, extensive swellingoccurred. This led to an inhibition of respiration anda reversal of the pH change, owing to release ofCa2+. When succinate was the substrate it could beshown that this inhibition of respiration probablyderived from two causes, loss of cytochrome cand accumulation of oxaloacetate, since theaddition of cytochrome c (2-5 uM) and rotenonevery largely prevented the decrease in rate ofrespiration (cf. Gutfreund & Jones, 1964).More informative results were obtained when

intermediate concentrations of Ca2+, which caused

some measure of, but incomplete, swelling of themitochondria, were used (Fig. 7). Here four phasesof behaviour in response to the addition of Ca2+could be characterized.Phase 1 occurred immediately after the addition

of Ca2+. This phase was characterized by highrates of respiration and H+ production and anincreased light-scattering of the mitochondria;Phase 2 started when Ca2+ accumulation was

complete. It was possible to show this by makinguse of the fact that, where EGTA is added to asolution containing free Ca2+, 2 H+/metal ion areproduced as a result of chelation. As mentionedabove, when Ca2+ is accumulated by mitochondriaonly 1 H+/metal ion is produced (Chappell et al.1963). It is possible to assess therefore the extent towhich Ca2+ is free at any time and it is also possibleto determine the rate at which bound Ca2+ leavesthe mitochondria, once accumulation has occurred.Thus the addition of EGTA in this phase led to noimmediate H+ production (Fig. 7b), which it wouldhave done had any significant quantity of Ca2+been free (see Fig. 7a).Phase 3 occurred some 10-20 sec. after phase 2

and was characterized by a relatively slow swellingof the mitochondria to a new steady state and anincreased rate of respiration (Fig. 7c). During thisphase relatively little Ca2+ was available to addedEGTA, either at the start of this phase (Fig. 7b) orlater (Fig. 7a), since no rapid change ofpH occurredon the addition ofEGTA. It seems that under theseconditions Ca2+ was being lost and taken up againby the mitochondria, which would account for theincreased rate of respiration. However, EGTA didnot prevent the swelling. The addition of cyto-chrome c (2-5(LM) during this phase considerablyincreased the rate of respiration, indicating thatthe mitochondria had lost much of their cytochromec, yet were able to retain the Ca2+ that they hadaccumulated.Phase 4 occurred when the suspension became

anaerobic. In this phase, all the accumulated Ca2+was rapidly lost (since added Triton X-100 causedno further Ca2+ release; Fig. 7a) and extensivemitochondrial swelling occurred, even in thepresence of EGTA (Figs. 7b and 7c).The most reasonable interpretation of these

results may be made in terms of the hypothesisdeveloped above. The behaviour in phase 3 isperhaps the most interesting. Here it is apparentthat the mitochondria, probably as a result of theirpartially swollen condition, are losing Ca2+ andaccumulating it at the same time. This processrequires energy and this must be provided by anincreased rate of respiration. In this respectphase 3 resembles the state that occurs when livermitochondria have been treated with Mn2+ in theabsence of added phosphate (see Fig. 2 of Chappell

Vol. 95 383

384

1*0

O

V

o.d

J. B. CHAPPELL AND A. R. CROFTS

1..f02O0

B (c) c

1 min. 0. _0

Fig. 7. Experiments showing the sequence of events leading to complete mitochondrial swelling induced byaccumulation of Ca2+. The convention of the traces is the same as that given in Fig. 3. In each experiment8mM-succinate, 2 1mM-phosphate and 0-17,uM-rotenone were present initially. Additions were made whereindicated as follows: (a) A, mitochondria (14.7mg. of protein); B, 1.25btmoles of CaC12; C, 4 Oumoles of EGTA;D, 5Opl. of 5% Triton X-100. (b) and (c) Additions were the same as for (a), though the EGTA was addedbefore the suspending medium had become anaerobic, in order that the release of bound Ca2+ from the mito-chondria could be followed.

(a)

1965

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Vol. 95 CALCIIUM-INDUCED SWELLING 385et al. 1963, and the Discussion section of thepresent paper).

DISCUSSION

The respiration-dependent accumulation of Ca2+together with phosphate or arsenate led to anextensive mitochondrial swelling. If the accumula-tion was prevented by inhibition of respiration orby uncoupling agents, then swelling did not occur.Chelating agents also prevented accumulation andthe associated swelling. However, the addition ofEGTA to mitochondria that had accumulatedCa2+ did not prevent the swelling that occurred onthe addition of a respiratory inhibitor or uncouplingagent.Once accumulation had occurred, a variety of

factors either induced swelling or increased thetendency of mitochondria to swell. These factorsincluded excess of Ca2+, uncoupling agents,respiratory inhibitors and anaerobic conditions.Under each of these circumstances it would bereasonable to assume that the concentration of'high-energy' intermediates, which are perhapsnecessary for the maintenance of the structuralintegrity of the mitochondrial membrane, would berelatively low.

Evidence has been presented by Chappell(1963) that the alkylguanidines interact with anon-phosphorylated 'high-energy' intermediate inmitochondria, causing inhibition ofthose oxidationsinvolving the use ofNAD. The alkylguanidines failto act in the presence of uncoupling agents, underanaerobic conditions, or in the presence of Ca2+.The conditions for the inhibitory action of alkyl-guanidines and for the prevention of Ca2+-inducedswelling after accumulation are therefore similar.These conditions are also those under which Mn2+accumulated by liver mitchondria in the absence ofadded phosphate is released. Considerable quan-tities of Mn2+ can be accumulated by liver mito-chondria in the absence of added phosphate in arespiration-dependent process that has the sameinhibitor sensitivity as the uptake that occurs in thepresence of phosphate. However, when theseinhibitors are added to mitochondria that haveaccumulated Mn2+ in the absence of phosphate, theMn2+ is released into the solution again, but withoutswelling of the mitochondria (Chappell et al. 1963).The effects of dinitrophenol on Ca2+-induced

swelling of the type discussed in the present paperare similar, at least superficially, to the effects ofdinitrophenol on the phosphate-induced swelling ofmitochondria (Chappell & Greville, 1959). Theaddition of dinitrophenol at zero time to mito-chondrial suspensions in buffered 0-3M-sucroseprevented phosphate-induced swelling, but thedelayed addition of dinitrophenol accelerated theprocess. However, although antimycin or Amytal

inhibited phosphate-induced swelling, they did notcause swelling when added subsequently, in contrastwith the results obtained with Ca2+ reported in thepresent paper.From previous work it has been concluded that,

in order that mitochondria should swell, not onlythe presence of a swelling agent (e.g. Ca2+, phos-phate, thyroxine), but also some measure ofelectron transport through the respiratory chain, isnecessary (see Chappell & Greville, 1963a). How-ever, in many instances in the present investigationthe most rapid swelling was observed on inhibitionof respiration, e.g. on the suspension becominganaerobic or on the addition of respiratory inhibi-tors. It is emphasized, however, that coupledrespiration was necessary at a previous stage inorder that Ca2+ accumulation should occur.The physiological significance of Ca2+-induced

swelling is not immediately apparent. However,preliminary experiments in collaboration withD. Tillotson (Dunn Nutritional Laboratories,Cambridge) have indicated that mitochondriafrom the livers and kidneys of vitamin D-deficientrats are much less prone to Ca2+-induced swellingthan are the mitochondria from vitamin D-dosedrats. There was no difference in rates of Ca2+uptake (as judged by H+ production) or of respira-tion between mitochondria from vitamin D-deficient and vitamin D-dosed rats. These observa-tions may account for the lowered rates of Ca2+release from mitochondria from vitamin D-deficientrats observed by DeLuca, Engstrom & Rasmussen(1962).

We thank Miss Jenny Flemans for her expert assistanceand the Medical Research Council for financial support.

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