VOLUME CHANGES IN LIVER MITOCHONDRIA* BY GIOVANNI FELICE AZZONE AND ANGELO AzzI - PNAS
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VOLUME CHANGES IN LIVER MITOCHONDRIA*
BY GIOVANNI FELICE AZZONE AND ANGELO AzzI
UNIT " G. VERNONI" FOR THE STUDY OF PHYSIOPATHOLOGY, INSTITUTE OF GENERAL PATHOLOGY,
UNIVERSITY OF PADUA, ITALY
Communicated by Britton Chance, March 1, 1965
Animal mitochondria show modifications of shape and size in the intact cell.'
After isolation, animal mitochondria show variations of their optical density which
are defined, in general terms, as swelling-shrinking phenomena.2-7 Swelling-shrink-
ing phenomena involving large changes of the optical density are denoted as large-
amplitude or Phase II swelling.8-" Swelling-shrinking phenomena involving small
changes of the optical density are known as low-amplitude, or Phase I swelling.8-"
The large-amplitude swelling-shrinking process involves considerable increases or
decreases of the mitochondrial volume and is accompanied by movement of water
in or out of the mitochondrion." 6, 12-15 In the case of the low-amplitude swelling-
shrinking process it has not yet been established whether the changes in optical
density are an expression of variations of the mitochondrial volume and of the water
content of the mitochondrion, or are consequent of molecular rearrangements of the
mitochondrial membrane.8' 10 Large-amplitude swelling is caused by several
agents or conditions (for recent reviews, see refs. 8, 11). Only the large- and small-
amplitude swelling induced by inorganic phosphate will be considered in the
present study.
Inorganic phosphate was reported to induce large-amplitude swelling by Hunter
and Ford,7 and by Price, Fonnesu, and Davies.6 Swelling did not occur when the
mitochondria were kept under anaerobic conditions,'6' 1' or supplemented with cy-
anide.'8 In subsequent investigations it was established that electron transport
through any portion of the respiratory chain could support swelling.'9' 20 It has
then been concluded that electron transport is an absolute requirement for this
type of swelling.8 11
Swelling induced by inorganic phosphate has been reported to be either in-
hibited6' 21, 22 or stimulated6' 20, 22 by uncoupling agents depending on the time of
addition of the uncoupler, on the concentration of the uncoupler, and on the
type of uncoupler. More recently, Chappell and Greville20 have reported that the
swelling induced by inorganic phosphate was prevented by oligomycin and the in-
hibitory effect of oligomycin was removed by dinitrophenol. It has been concluded
that phosphorylated compounds are not involved in the swelling process, since it
was difficult to imagine that the synthesis of these compounds was stimulated in the
presence of dinitrophenol and oligomycin."
The low-amplitude swelling was first reported by Chance and Packer.23 Heart
mitochondria supplemented with substrate and inorganic phosphate underwent a
slight decrease of optical density. Addition of ADP induced, parallel to the acti-
vation of the respiration, an increase of optical density which was reversed when
ADP was exhausted. In subsequent studies Packer9 suggested that the mito-
chondria contained a nonphosphorylated high-energy compound which was de-
noted as "shrinkage intermediate." When this compound is destroyed by the ad-
dition of uncoupling agents or of arsenate, swelling of the mitochondria should Oc-
1084
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cur. In independent studies, Beechey and Holton24 reported the occurrence of a
small-amplitude reversible swelling-shrinking phenomenon, following aeration and
oxygen exhaustion of muscle mnitochondrial suspensions. The phenomenon was
defined as "autonomic reversible swelling."
In the present article we shall report experiments suggesting that the swelling
phenomena of large and small amplitude induced by inorganic phosphate are de-
pendent only on the supply of energy from the respiratory chain or from external
ATP and not on electron transport per se. It will be proposed that the swelling
processes induced by inorganic phosphate may be distinguished on the basis of the
source of energy required for the shrinkage phase rather than on the extent of the
optical density changes.
Experimental Methods.7-Rat liver mitochondria have been used in all the experi-
ments reported. The mitochondria were suspended in a saline medium of the
following composition: 0.125 M KC1, 0.02 M tris buffer pH 7.4, 0.1 per cent serum
albumin, and, when indicated, 0.005 M 13-hydroxybutyrate or 0.003 M succinate.
In the experiments on the low-amplitude changes, 0.003 M EDTA was also added.
The changes in optical density were measured with a Beckman spectrophotometer
or with an Eppendorf photometer equipped with a recording apparatus. Intra-
mitochondrial pyridine nucleotides were measured with an Eppendorf fluorimeter.
Experimental Results.-Formation of phosphorylated compounds in the large-ampli-
tude swelling induced by inorganic phosphate: That the phenomenon of large ampli-
tude swelling is not dependent on electron transport but only on the formation of
phosphorylated compounds is strongly suggested by the following data concerning
the effects of Pi, of inhibitors of energy transfer, of uncoupling agents, and of
adenine nucleotides:
(a) Effect of Pi: Half-maximal rate of swelling was obtained at 0.6 mM Pi.
This figure is close to that found for stimulation of respiration and pyridine nucleo-
tide oxidation by Pi.25
(b) Effect of inhibitors of energy transfer: The two best-known inhibitors of
energy transfer, oligomycin and atractyloside, are assumed to act at first, before,26
and second, after27 the formation of phosphorylated compounds. This difference
in site of action is correlated with a difference in effects on large-amplitude swelling:
oligomycin inhibits and atractyloside does not.
(c) Effect of uncoupling agents: As mentioned above, uncoupling agents have
been reported to inhibit, to activate, and to have no effect on swelling. The ques-
tion was carefully investigated in our studies, and evidence was obtained in favor of
the conclusion that the uncoupling agents have only an inhibitory effect on swelling
and that the lack of effect or stimulatory effect sometimes reported is to be ascribed
to synthesis of ATP through substrate level phosphorylation linked to the oxidation
of a-ketoglutarate. In fact, swelling in the presence of dinitrophenol could never
be observed when either internal ATP was removed by adding external ADP (Fig.1)
or when the substrate level phosphorylation was abolished by using succinate as
substrate in the presence of rotenone.
(d) Effect of adenine nucleotides: It has been reported that large-amplitude
swelling is prevented by addition of adenine nucleotides.6' I The protecting effect
of the adenine nucleotides has been ascribed to the shrinking effect of ATP either
added as such or formed from AMP and ADP.8 From our studies we conclude that
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OLIGOMYCIN 333,iM AOP
oM
10- X0ju ON
E - WITHOUT
AOP
0 2 4 6 S 10
MINUTE S
FIG. 1.-Prevention by ADP of activation effect on swelling by dinitrophenol. Liver mito-
chondria were suspended in a medium of the following composition: 0.125 M KCl, 0.02 M Tris
buffer pH 7.4, 0.1% serum albumin, and 0.005 mM ,3-hydroxybutyrate. Final volume 3 ml.
Temperature, 300C.
the protecting effect of the adenine nucleotide is to be ascribed only to ADP.
Furthermore, it was found that addition of ATP to liver mitochondria under anaero-
bic conditions resulted in large-amplitude swelling when either Pf and low Ca++
concentrations were present or the mitochondria were treated with the SH-group
inhibitor N-ethylmaleimide. The swelling effect of ATP was abolished by dinitro-
phenol, oligomycin, and ADP. The apparent Km for the swelling effect of ATP
was 100MlM.
Requirement for energy supply in the case of small-amplitude swelling is
suggested by results similar to those mentioned above for the large-amplitude
process. Low-amplitude swelling with liver mitochondria was obtained by adding
3-6 mM EDTA, which was probably required to bind the mitochondrial magnesium.
Addition of equivalent amounts of magnesium completely abolished the low-ampli-
tude swelling process. Dinitrophenol prevented swelling, the extent of pre-
vention being proportional to the extent of the uncoupling effect. ADP prevented
swelling but not ATP. The protection by ADP was removed by atractyloside. Pf
could not be replaced by arsenate. Under anaerobic conditions ATP induced swell-
ing, and an effect which was abolished by dinitrophenol, oligomycin, and ADP.
The apparent Km for the ATP-induced low-amplitude swelling was 65 MuM.
In one aspect, however, the low-amplitude swelling was different from the large-
amplitude swelling, namely, in the effect of oligomycin, which did not prevent
phosphate-induced low-amplitude swelling. The possibility that oligomycin was
not acting in the EDTA-supplemented mitochondria was excluded by ascertaining
that (a) the State 4-State 3 transition induced by the addition of ADP was abolished
in the EDTA-oligomycin-supplemented mitochondria, and (b) the synthesis of
ATP was completely abolished in the EDTA-oligomycin-supplemented mitochon-
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dria. Support for the hypothesis of an oligo- -
mycin-insensitive pathway in the low-ampli-
tude swelling was given by the observation z 0.096-
that addition of P1 to the EDTA-oligomycin- X
supplemented mitochondria resulted, parallel Z
to the swelling process, in a stimulation of the =
respiration and an oxidation of the pyridine 0.032-
nucleotide. Addition of magnesium, which
abolished the low-amplitude swelling, inhibited 0
also the stimulation of the respiration and the
oxidation of the pyridine nucleotide induced by (ONP)J, M
FIG. 2.-Concentrations of dinitro-
inorganic phosphate.
inorganic phosphate. phenol required to induce mitochon-
Abolition of both large- and low-amplitude drial shrinkage after low-amplitude
swelling by uncoupling concentrations of di 1-in swelling. Experimental
Fig. 1, except
conditions as
that 0.0033 M EDTA,
nitrophenol indicates that the nonphosphoryl- 1 MM rotenone, and 0.003 M succinate
ated high-energy intermediates are not re- were also added
quired for maintaining the shrunken state of the the mitochondrion.
The reversibility of the swelling process: As mentioned in the introduction, large-
and low-amplitude swelling have been distinguished by definition on the basis of the
extent of the optical density changes. The changes were about 2 per cent of the
optical density in the case of the low-amplitude swelling and 50 per cent of the opti-
cal density in the case of the large-amplitude swelling. However, this distinction is
rendered inadequate from the present findings that liver mitochondria supple-
mented with EDTA show a 10-15 per cent decrease of optical density after addition
of inorganic phosphate. Furthermore, as concluded above, both large- and low-
amplitude swelling appear to be dependent on the formation of phosphorylated
compounds. The requirements for reversing the swelling process and therefore
regaining the original shrunken state of the mitochondria appear to offer a better
criterion to distinguish between swelling processes occurring through different
reaction pathways and leading to a different final state of the mitochondrion.
Mitochondrial shrinkage after addition of ATP was first observed in pigeon breast
mitochondria by Chappell and Perry.28 The conditions for the ATP-induced
shrinkage were further investigated by Lehninger.29 It was suggested that external
ATP was required to supply energy for extruding water from the mitochondrion.
Thus, liver mitochondria after large-amplitude swelling can regain the original
shrunken state only on the expense of an external source of energy.
An opposite situation exists in the case of the low-amplitude swollen state. In
general, we have found that all conditions which lower the energy level of the
mitochondrion cause a shrinkage phase after the low-amplitude swelling. The
evidence in favor of this conclusion is as follows: (1) Mitochondria in the low-
amplitude swollen state under aerobic conditions underwent a shrinkage phase after
the addition of respiratory inhibitors such as cyanide or antimycin, or of uncouplers
such as dinitrophenol or dicoumarol, of ADP, or, after exhaustion, of oxygen. The
concentrations of dinitrophenol required for shrinkage were equal to those required
to uncouple oxidative phosphorylation (see Fig. 2). Swelling-shrinking cycles
were obtained by repeated aerations of an anaerobic mitochondrial suspension (see
Fig. 3). (2) Mitochondria in the low-amplitude swollen state due to addition of
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IOAg OLIGOMYCIN OXYGEN OXYGEN OXYGEN
| 1ml Pi111
t02=0 t
o0o t 02=
Lo I .IO25
Zg(20 mm)
t02-
60 sec
FIG. 3.-Reversible anaerobic shrinkage of liver mitochondria. Experimental conditions as in
Fig. 1, except that 0.0066 M EDTA, 1 JAM rotenone, and 0.003 M succinate were also added.
Amount of mitochondrial protein was 8 mg.
ATP under anaerobic conditions shrank upon the addition of ADP, oligomycin, or
dinitrophenol.
Tightly coupled mitochondria are required in order to obtain low-amplitude
swelling-shrinking cycles. Damage to the mitochondria leads either to swelling
which cannot be reversed by uncoupling agents and respiratory chain inhibitors, or
to loss of the capacity to perform low-amplitude swelling-shrinking cycles. Thus,
the capacity for low-amplitude swelling-shrinking cycles is lost due to the simulta-
neous occurrence of a large-amplitude swelling process. When the mitochondria are
completely swollen, only ATP plus Mg++ is able to induce mitochondrial shrinkage.
After the shrinkage phase due to ATP and Mg++ is terminated, the mitochondria
are again able to undergo low-amplitude swelling-shrinking cycles.
It appears, therefore, that inorganic phosphate can induce two kinds of swelling
processes, one which can be reversed by the addition of uncoupling agents, respira-
tory chain inhibitors, or ADP, and another which can be reversed only by external
ATP. If we assume that the extrusion of water from the mitochondrion always
requires an expenditure of energy, it follows that the shrinkage phase initiated by
uncouplers or respiratory chain inhibitors utilizes some form of energy which must
be available within the mitochondrion. From the above considerations we con-
clude that a swollen state requiring external ATP for its reversal indicates a "low-
energy" state of the mitochondrion. A swollen state requiring addition of ADP
or of uncouplers for its reversal indicates a "high-energy" state of the mitochon-
drion. The response of the swollen mitochondrion to uncoupling agents or to re-
spiratory chain inhibitors is therefore of utmost significance for the characteriza-
tion and the interpretation of the swelling process.
* This investigation was aided by a grant from the Muscular Dystrophy Associations of America,
Inc.
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NONEMPIRICAL MOLECULAR ORBITAL THEORY FROM
MOLECULAR HARTREE-FOCK THEORY
BY M. D. NEWTON, F. P. BOER, W. E. PALKE, AND W. N. LIPSCOMB
DEPARTMENT OF CHEMISTRY, HARVARD UNIVERSITY
Communicated March 29, 1965
A recent comparison' of molecular self-consistent field (SCF) theory2 and extended
Hiickel (EH) theory3 has now resulted in a nonempirical method for approximating
LCAO SCF wave functions of large molecules.
The separate and direct calculation of the kinetic energy and overlap integrals
for a complex molecule hasper mitted the replacement of the Wolfsberg-Helmholz4
approximation
fXaaH"PXbtdT = 1/2 KSaab I[fXaaH0PxaadT +fXb#H p XbdrI (1)
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