Table Of ContentReviews of
201 Physiology,
Biochemistry and
y g o l o c a m r a h P
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Contents
Relation Between Mitochondrial Calcium Trans-
port and Control of Energy Metabolism.
ByR . G. ,DROFSNAH Baltimore, Maryland/USA.
With 91 Figures . . . . . . . . . . . . . .
Acetylcholine-Receptor-Mediated Ion Fluxes in
surohportcelE sucirtcele and odeproT acinrofilac
Membrane Vesicles.
By D. J. ,HSAC St. Louis, Missouri/USA,
H. ,AMIHSOA Yamaguchi/Japan, E. ,B. ELAUQSAP
San Diego, California/USAa nd Parma/Italy,
and G. P. ,SSEH Ithaca, New York/USA.
With 91 Figures ...... ........ 73
Organization of the Lumbar Sympathetic Outflow
to Skeletal Muscle and Skin of the Cat Hindlimb
and Tail.
By W. JXNIG, Kiel, Federal Republic of
Germany. With 20 Figures . . . . . . . . . 911
Author Index . . . . . . . . . . . . . . . . 215
Subject Index . . . . . . . . . . . . . . . . 228
dexednI Current in stnetnoC
Rev. Physiol. Biochem. Pharmacol., Vol. 102
© by Springer-Verlag 1985
Relation Between Mitoehondrial Calcium tropsnarT
and Control of Energy Metabolism
RICHARD G. HANSFORD
Contents
Introduction ............................................ 2
The Ca2÷-Sensitive Dehydrogenases ............................. 4
2.1 Pyruvate Dehydrogenase Phosphate Phosphatase ................ 4
2.2 NAD-Isocitrate Dehydrogenase ........................... 6
2.3 2-Oxoglutarate Dehydrogenase ........................... 8
2.4 Other Ca2+-Sensitive Intramitochondrial Enzymes ............... 10
2.5 Glycerol 3-Phosphate Dehydrogenase ....................... 12
Ca +R Sensitivity of Dehydrogenase Activity in Intact Mitochondria ......... 13
3.1 Dependence on [Ca 2+ ]o ................................ 13
3.2 Kinetics of Dehydrogenase Activation and Inactivation by Changes
in [Ca2÷]o ......................................... 19
3.3 Dependence of Dehydrogenase Activation on Mitochondrial Total Ca
and [Ca2÷]m ........................................ 21
The Calcium Content of Mitochondria in Situ ....................... 24
4.1 Ca Content of Muscle Mitochondria ........................ 24
4.2 Ca Content of Liver and Kidney Mitochondria ................. 26
4.3 Ca Content of Nervous Tissue Mitochondria ................... 29
4.4 Consequences for the Model of Dehydrogenase Regulation ......... 30
Values of [Ca2÷]c: Relevance to the Model of Dehydrogenase Regulation ..... 31
5.1 Heart Muscle ....................................... 32
5.2 Nervous Tissue ...................................... 33
5.3 Liver and Kidney ..................................... 34
Does Mitochondrial Ca ÷2 Transport Control [Ca2+]m or [Ca2+]c? .......... 36
6.1 The Kinetics of Ca +2 Efflux in Heart and Liver Mitochondria and the
Relationship Between [Ca2+]c and [Ca2+]m ................... 36
6.2 A Simple Model ..................................... 42
The Physiology of Dehydrogenase Regulation by Ca ÷2 ................. 45
7.1 The Role of Ca ÷2 in the Response of Cardiac PDH A Content to
Increased Work Load and ~-Adrenergic Stimulation .............. 45
7.1.1 Background ........................................ 45
7.1.2 Recent Definitive Experiments ............................ 46
7.2 The Role of Ca ÷2 in the Response of PDH A Content to the
Stimulation of Nervous Tissue ............................ 49
National Institute on Aging, National Institutes of Health, Gerontology Research
Center, Baltimore City Hospitals, Baltimore, Maryland 21224, USA
2 .G.R drofsnaH
7.3 The Response of Liver Ca2+-Sensitive Dehydrogenases to
al-Adrenergic Stimulation, Vasopressin, and Angiotensin II ........ 15
7.3,1 Evidence for Dehydrogenase Activation by Ca ÷2 ................ 51
7.3.2 Results of the Direct Measurement of Mitochondrial Ca Content in
Response to al -Agonists, Vasopressin ....................... 53
7.4 The Response of Ca2+-Sensitive Dehydrogenases to Stimulation by
Other Hormones ..................................... 57
8 Some Thoughts on the Biological Advantages of Dehydrogenase Level Control. 59
9 Summary ............................................... 61
References ................................................ 62
List of Abbreviations
c]÷2aC[ Free Ca +2 concentration in the cytosol
o]+~aC[ Free Ca ÷2 concentration in the extramitochondrial phase of
in vitro experiments
[Ca2+]m Free Ca ÷2 concentration of the mitochondrial matrix
Ca content The total Ca of a tissue, both bound and free
+ A~-/H The proton electrochemical gradient across the inner mito-
chondrial membrane
+4A The membrane potential across the inner mitochondrial
membrane
FCCP Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - an
uncoupling agent or proton ionophore
EGTA Ethyleneglycol-bis-(O-aminoethyl ether)-N,N 1 -tetraacetic acid
Hepes N-2-hydroxyethylpiperazin-N 1-2-ethanesulfonic acid
Pi Orthophosphate, inorganic phosphate
1 Introduction
Mitochondrial Ca, ÷2 uptake si a process of great biochemical and physiolo-
gical interest. It has been shown to be linked directly to the fundamental
energy transducing system in the inner mitochondrial membrane, to have
the capacity to transport rapidly large amounts of calcium from cytosol to
the inner mitochondrial matrix space, and to be ubiquitous in mammalian
mitochondria. It has been the subject of a vast amount of research work,
much of which is summarized in several recent cogent reviews (Fiskum
and Lehninger 1982; Nicholls and ~kerman 1982; namrek~. and sllohciN
1983). In addition to this uptake process, there is a parallel effiux path-
way by which Ca ÷2 leaves the mitochondrion, and it has been realized
rather recently that there is in fact a continuous cycling of the ion across
the mitochondrial membrane. The energetics of this calcium-cycling pro-
Between Relation lairdnohcotiM muiclaC Transport dna Energy msilobateM 3
cess and the potential that it provides for control of cellular events have
been the subject of a recent review in this series (~kerman and Nicholls
1983). The emphasis of their article was on the role that mitochondrial
Ca ÷2 transport may play in the regulation of the free Ca ÷: concentration
of the cytosol ([Ca:+]c). The present article has an entirely different per-
spective and puts forward and examines the idea that mitochondrial Ca ÷2
transport serves to regulate the free Ca +2 concentration of the mito-
chondrial matrix ([Ca2÷]m). This has the potential advantage to the animal
that the activity of major catabolic pathways, which are Ca +: sensitive and
intramitochondrial, can be adjusted to the energy demands placed upon
the tissue and signalled through changes in cytosolic Ca .÷2 Indeed, a case
will be made that in muscle and in nervous tissue the phenomenon of
respiratory control, long thought of in terms of the availability of ADP to
the mitochondria (Chance and Williams 1956), should instead be thought
of in terms of the availability of both ADP and Ca ÷: ions. Each of these
serves to activate enzymes of the Krebs tricarboxylate cycle, the final
common pathway of catabolism, and thus to help the working cell main-
tain the adenine nucleotide phosphate potential and allow efficient per-
formance of cellular work. Further, the possibility exists that control of
catabolism by Ca +2 may function independently of control by ADP,
allowing mitochondria in hormone-sensitive tissue to accelerate energy
transduction in response to a hormone which elevates [Ca2*]c, without the
need to disturb the cellular phosphate potential. This possibility will be
developed in some detail below. Similar views on mitochondrial Ca *2
transport have been expressed in excellent short reviews by Denton and
McCormack (1980, 1981).
The model to be presented requires that each of the following condi-
tions be satisfied:
.1 Intramitochondrial enzymes catalyzing rate-limiting reaction steps in
catabolic pathways are activated by Ca .÷2
2. Values of [Ca2+]c increase in response to conditions requiring higher
rates of energy transduction in the mitochondrion.
3. Transport processes exist in the mitochondrial membrane which are
capable of raising [Ca2*]m in response to the change in the cytosol.
This requires a net inward flux of Ca ÷: across the mitochondrial mem-
brane, which must reverse when the change in the [Ca:*]c reverses.
4. The values of [Ca2*]m occurring in vivo must be in the range where
such changes elicit changes in enzyme activity, i.e., the model would be
invalidated if these concentrations were so high as to continually
saturate the Ca2*-sensitive intramitochondrial enzymes.
Each of these assumptions will be scrutinized in turn. In addition, the
separate possibility will be considered that [Ca2+]m can be altered without
4 .G.R drofsnaH
the necessity of a prior change in [Ca2+]c, if the relative balance of the
activities of the mitochondrial uptake and release pathways is altered as
might occur in the response of a cell to a hormone.
Finally, this article will examine the idea, recently reviewed and sup-
ported by Fiskum and Lehninger (1982) and ~kerrnan and Nicholls (1983),
that mitochondria accurately buffer [Ca2+]c. Although not necessarily the
antithesis of the thesis developed in the current article, such buffering
may not be compatible with the concept that changes in [Ca2÷]m regulate
the activity of intramitochondrial enzymes. This arises because values of
[Ca2÷]m allowing enzyme regulation are not sufficiently high to allow
accurate buffering of [Ca2*]c by the mitochondria, i.e., mitochondria can
buffer [Ca2+]c or allow the control by Ca ÷2 of intramitochondrial enzymes,
but they cannot do both. The question of which paradigm better describes
the behavior of mitochondria in the animal can only be answered by a
knowledge of the Ca content of the mitochondria in vivo. The current
state of our knowledge of this important parameter will also be discussed.
2 The evitisneS-÷2aC sesanegordyheD
This section discusses evidence that Ca ÷2 ions activate key catabolic
enzymes. The first to be considered will be three intramitochondrial
dehydrogenases which catalyze nonequilibrium, and thus rate-limiting,
reactions in the terminal oxidations of carbohydrates and fatty acids.
Following this, other Ca2÷-sensitive enzymes which are intramitochon-
drial but which appear to respond to higher concentrations of Ca ÷2 will
be discussed. Finally, the enzyme glycerol 3-phosphate dehydrogenase,
which is Ca ÷2 sensitive and forms an integral part of the inner mitochon-
drial membrane, will be considered.
2.1 Pyruvate Dehydrogenase Phosphate Phosphatase
The pyruvate dehydrogenase complex catalyzes a nonequilibrium reaction
committing carbon from carbohydrate to the formation of fat or to com-
plete oxidation to CO2. Part of the metabolic control of this enzyme si
achieved by a phosphorylation/dephosphorylation cycle, catalyzed by a
kinase and a phosphatase respectively. The dephosphoenzyme (PDH n) is
the catalytically active form. These relations have been reviewed recently
by Hansford (1980), Reed (1981), and Wieland (1983). Denton et al.
(1972) discovered that the phosphatase activity was enhanced by micro-
molar concentrations of Ca ÷2 and this has been attributed to a decrease in
Transport Relation Calcium Metabolism Mitochondrial and Between Energy 5
~A
tu~.~ '~"o
(1. 0 // I I I
I°' < -9 - -7 -6 -5 ./-F4
~ o... o
<'6 O4
~ E O2
a / 0 t tI I L 1 I I
-9 -8 -7 -6 -5 -4
i.
- ~ t~~
. < 10
o
x
0 ~ 0 i I I L I I
-9 -8 -7 -6 -5 -4 -3
log I[M2"](M)I
Fig. la-c. Activation of a pyruvate dehydrogenase phosphate phosphatase, b NAD-
isocitrate dehydrogenase, and c 2-oxoglutarate dehydrogenase by Ca ÷2 (e) and Sr ÷2
(o) ions. Enzyme activity was measured in mitochondrial extracts (a,b) or with the
purified rrotein (c), as described in the original papers. The indicated values of [Ca 2÷]
and [St 2 ] were maintained with EGTA buffers. (a, McCormack and Denton 1980;
b, Denton et al. 1978 ; c, McCorraack and Denton 1979)
the Km of the enzyme for its substrate, pyruvate dehydrogenase phosphate
(Pettit et al. 1972; Randle et al. 1974). Although Ca ÷2 causes a change in
the affinity of the enzyme for its substrate, as in the other cases to be dis-
6 Hansford R.G.
cussed, the net effect is the generation of more PDH A and thus an in-
creased Vma x of the PDH complex.
It is seen in Fig. 1 that the Ko.s for Ca +2 activation of the isolated phos-
phatase is approximately 1 .41d~ These data are derived from studies
employing the phosphatase of white adipose tissue mitochondria, but the
sensitivity of the enzyme from heart is similar (Denton et al. 1975). How-
ever, there is also the possibility that the pyruvate dehydrogenase kinase
from heart is inhibited by Ca ÷2 with approximately the same sensitivity
(Cooper et al. 1974): this would be expected to result in a still steeper
dependence of PDH A content on [Ca2÷]m in the heart. Presented together
with the sensitivity of the phosphatase to Ca ÷2 in Fig. 1 is the sensitivity
to Sr ,÷2 which has been found by Denton and coworkers to activate the
trio of Ca2÷-sensitive intramitochondrial dehydrogenases. Activation by
Sr ÷~ requires a concentration approximately one order of magnitude
higher than that needed for activation by Ca +2 (Fig. 1).
2.2 NAD4socitrate Dehydrogenase
The enzyme NAD-isocitrate dehydrogenase catalyzes a nonequilibrium
reaction in the tricarboxylate cycle and is activated by a decline in the
ratios ATP/ADP and NADH/NAD ÷ (see Hansford 1980, for a review). The
discovery that NAD-isocitrate dehydrogenase is also activated by micro-
molar concentrations of Ca +2 (Denton et al. 1978) raised the intriguing
possibility that flux through this reaction could be adjusted to the
demands of the work load not only by the availability of ADP to the
mitochondria but also by the availability of Ca .÷2
Again, the Ko.s for stimulation by Ca ÷2 is approximately 1/aM in experi-
ments with mitochondrial extracts (Fig. 1), with Sr +2 being effective at
somewhat higher concentrations. Ca ÷~ acts as an allosteric activator,
decreasing the apparent m K of the enzyme for its substrate isocitrate
(Fig. 2). These observations have been extended recently to the purified
enzyme from bovine heart, with the finding that the Ko.s value for Ca ÷~
decreases with increasing concentrations of the substrate, magnesium
isocitrate, with the limiting value of the K0.s for Ca ÷2 being 0.29 4IM
(Aogaichi et al. 1980). The apparent Km for isocitrate is decreased sharply
with decreasing pH and with increasing ADP concentration, such that a
wide range of values is obtained under different experimental conditions.
These changes have been tied to changes in aggregation of the enzyme sub-
units, at least for the effect of adenine nucleotides (Giorgio et al. 1970).
It is noteworthy that Ca ÷~ was found to be an activator only in the pre-
sence of ADP (Denton et al. 1978; Aogaichi et al. 1980), lending credence
to the idea of respiratory control by ADP plus Ca .÷2 It would be interesting
Relation Between Mitochondrial Calcium Transport and Energy Metabolism 7
60 J, ,
¢ 2
40
E 20
.<
, II,
0 0.2 , o.4 , 0.6 o 8 2.
Ithreo-Ds-lsocitratel (mM)
100
m m
75
.:_.,ac
~' 25
o
©
I l l I 1 // I
0 1 2 3 4 5 10
b I Oxoglutaratel (raM)
Fig. 2a,b. Effect of Ca ÷2 on the apparent substrate affinity of a NAD-isocitrate dehy-
drogenase and b 2-oxoglutarate dehydrogenase. Enzyme activity was determined using
a mitochondrial extract (a) or the purified protein (b) as described in the original
papers. Symbols: a ,A 5 mM EGTA; ,A 5 mM EGTA plus 5 mM 21CaC ([Ca ]÷2 approx.
30/aM); % 5 mM EGTA plus 1 mM ADP; e, 5 mM EGTA plus 5 mM 21CaC plus 1 mM
ADP ([Ca ]÷2 approx. 30/aM); b e, 5 mM EGTA; u, 5 mM EGTA + 5 mM 21CaC ([Ca ]÷2
approx. 30 p.M). (a, Denton et al. 1978 ; b, McCormack and Denton 1979)
to know whether the enzyme shows Ca +2 sensitivity at ATP/ADP ratios
plausible for the mitochondrial matrix (around 1-4, see Davis and Lumeng
1975; Akerboom et al. 1978), in view of the proposal that hormonal
stimulation may be mediated via changes in [Ca2+]c in the absence of
changes in ATP/ADP ratio (Denton and MeCormaek 1980, 1981 ; and see
below).
