Regulation of Oxidative Phosphorylation
The role of intramitochondrial Ca2+in the regulation of oxidative
phosphorylation in mammalian tissues
James G. McCormack* and Richard M. Dentont
“Department of Pharmacology, Syntex Research Centre, Heriot-Watt Universi?y Research Park, Riccarton,
Edinburgh EH 14 4AP, Scotland, U K , and +Department of Biochemistry, University of Bristol School of Medical
Sciences, University Walk, Bristol BS8 ITD, U.K.
Introduction
r .
I he overall process of oxidative phosphorylation in
m;ininialian tissues allows the oxidation of respiratory friels to produce useful energy in the form of
ATP. It involves the formation of LAD1 1 and
reducrd f1;ivoproteins by mitochondrial dehydrogenases, ;ind their subsequent oxidation by molecular oxygen in the niitochondrial inner membrane
1 1 -41.T h e latter process is c.atalysed by the respiratory chain iind occurs in a stepwise manner, so that
the redox energy available in the electrons from
NAI )I I ; i d from flavoproteins can h e harnessed by
pumping protons from the matrix face of the inner
mrmbrane to the cytoplasmic side. l ’ h e resultant
protonmotive gr;idient is used t o drive the synthesis
o f ATI’. while the ATP-synthetase reaction is held
far from equilibrium so that subsequent A7’P
hydrolysis can provide usefill energy for the cell.
As witti any enzymic-based process, the net
rate of oxidative phosphorylation can be regulated
hy substrate supply, by end-product inhibition, and
by the activities of the enzymes involved. Thus,
c h m g e s in the mitochondrial redox status, the
ATI’/AI )P.PI ratio. the protonmotive gradient, the
supply of oxygen, and the activity of components of
the respiratory chain, may all play ;I role [ 1-41, T h e
sh;ire of control that is exerted by each mechanism
varies according t o the particular cellular circumstances. For instance. under ‘resting’ cellular conditions, it is probable that, as A‘I’P use will be at a
minimurn. the ATI’/AI )1’.1’,
ratio will achieve a
high steady-state level and so exert a feedback inhibitory influence on the protonmotive gradient. This
will then build up, arid so will have a similar effect
Abbreviations used: I’DH, pyruvate dehydrogenase complex; I’L)fia, active, non-phosphorylated form of I’DH;
I’I )F 1P-I’ase. I’DH phosphate phosphatase; NAD-ICDH,
N A I )+-linked isocitrate dehydrogenase; OGDH. 2-0x0glutarate dehydrogenase complex; CaBI, CaL+-binding
inhibitory subunit; fura-2, 1-[2-(5-carboxyoxazol-2-yl)-haiiiiiiobenzofiiran- S -oxyl-2-(2‘-aniino- S’-methylphenoxy)ethane-N, N, N, N-tetra-acetic acid; indo-1. 1-[2; i i n i n o - 5 - (6-carboxyindol-2 - yl)phcnoxy ] - 2-(2‘-aminoS’-methylphcnoxy)ethaiie-~,N, N, N-tetra-acetic acid.
*To whoni corrcspondence should be addressed.
on the oxidation state of the redox carriers. T h e
resultant high NAI>€I/NAI)’
and ATP/ADF’
ratios will then inhibit key dehydrogenases t o prevent wasteful fuel oxidation. Considering the stimulated cell, the ‘classical’ concept of respiratory
control [ S ] envisages that the increased use of A T P
in the cytosol would result in a fall in the AT€’/
AIlP.P, ratio, which would result in a relaxation of
the inhibitory forces that were acting on the process
of oxidative phosphorylation. This concept thus
involves an essentially passive or responsive form
of control to cell stirnulation, triggered by the activation of cytosolic ATP-requiring events, and
necessarily entails decreases in the two key metabolite ratios, ATP/AI)P and NAIII MVAI)+.
I Iowever, in many instances of cell stimulation, these key metabolite ratios d o not decrease,
and indeed may even increase (see If)]). l’here is
thus evidence that the response of oxidative phosphorylation to cell stimulation is, in fact, a n active
one, so that energy production is enhanced simultaneously with increased demand. In this way,
homeostasis of these key metabolite ratios can be
achieved. This homeostasis appears t o be acconiplished using the same intracellular signalling niolecule, namely C a L +16-12], within the mitochondrial
matrix, as is generally used in the cytosol t o bring
about the activation of the various energy-requiring
processes I 1 1 1.
Ca2 is the only second-messenger molecule
that is known to cross the permeability barrier of
the mitochondrial inner membrane. This membrane contains specific transporters for Ca’+ whose
primary role is to relay into the mitochondrial
matrix changes in the cytosolic concentration of
C a L + (see later). Oncts within the matrix, CaL+can
activate several key steps in the overall process of
oxidative phosphorylation, and thereby can bring
about a co-ordinated response to increased requirements for ATP in the cell (Figure 1).
+
Ca2+-sensitiveenzymes of the
mitochondrial matrix
Matrix Ca2 -sensitive dehydrogenases
+
C a l f activates three key dehydrogenases that are
exclusively intraniitochondrial in mammalian
I993
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Biochemical Society Transactions
794
tissues: pyruvate dehydrogenase (PD€I), NAI>+isocitrate dehydrogenase (NAD-ICDH) and
2-oxoglutarate (OGDI I) dehydrogenase. Cal+ activates PDH by increasing the amounts of the active
non-phosphorylated enzyme (PIIHa) through the
activation of PI)€I phosphate phosphatase (PDHPPase; Figure 1) [13]. NAD-ICDH and OG1)H are
activated more directly: Ca'+ causes marked
decreases in the K,,, values for their respective substrates, threo-i),-isocitrate and 2-oxoglutarate [ 14,
these lower organisms, however, the dehydrogenases concerned have Ca'+-sensitive sites on the
outside face of the inner membrane [ 171. Thus
some plant mitochondria have an external NAI 11 I
dehydrogenase, and some insect-muscle mitochondria have an a-glycerol phosphate dehydrogenase
that is activated by cytosolic Ca" [ 171; this enzyme
is also similarly located and similarly regulated in
mammalian mitochondria.
Initially it was thought that all three Ca" -sensitive intramitochondrial dehydrogenases were
151.
similarly sensitive to a range of [ C a L + ]around
These three dehydrogenases each catalyse
0.1-10 pM, with half-maximal effects (K,) around
irreversible oxidative decarboxylations and are key
sites of matrix NADH production. They are also
1 pM, when studied as extracted enzymes 11.3-151.
controlled through end-product inhibition, by
More recent data, however, indicates that the Ca'+sensitivity ranges for PDHP-Pase and for OCDI I
increased NAD€I/NAI>+ and ATPIADP ratios.
are similar and around 0.05-3 pM, with K , values
However, the regulation by C a L + as
, a mediator for
- 0.4-0.8 p M , whereas the corresponding values
extrinsic factors, can over-ride these local effects
for NAD-1CL)I I may approach an order of magniand thus can stimulate flux through these enzymes
tude higher [ 181. This may allow Ca" to influence
even when these ratios are high. In addition, PDH
NADH production over a wider concentration
and OGDH are subjected to end-product inhibition
range. However, this issue is complicated since,
by increased acetyl CoA/CoA and succinyl CoA/
it is clear that PDH and OC,I)H exhibit
although
CoA ratios, respectively, and NAD-ICDH and
O(;I)II can also be activated by increases in [H+] regulation by these ranges of [Ca'+] when located
within intact mitochondria (see later), there is, as
over the physiological range [ 161.
yet, no means of assaying NAD-ICDII activity
Ca'+ regulation of these dehydrogenases has
within intact mitochondria without ambiguity (see
been found in extracts of all mammalian and
[h]). It has now also been realized that increases or
vertebrate tissues studied so far, but not in extracts
decreases in the Al'P/AL>P ratio can correspondof invertebrate tissues, even though regulation by
ingly increase or decrease the (?a'+-sensitivity
nucleotide ratios has been found in some inverteranges and the K,, values both for NAI)-ICI)lI and
brates [ 161. This would appear to agree well with
for OGDH around 2-Hold [ 1x1. NAI)-ICI)I I
what is known in evolutionary terms about the
requires AI)P or ATI' to be present for Ca'+-sensiexistence of mitochondrial Ca' -transport systems
tivity to be shown [ 141.
[ 171. The concept of Ca'+-sensitive mitochondrial
These enzymes have all been shown to bind
dehydrogenases may, however, still apply to some of
+
Figure 1
Summary of how intramitochondrial Ca2+ may influence the overall process
of oxidative phosphorylation in a co-ordinated manner in mammalian
tissues
See text for the definitions of the abbreviations; mito, mitochondrial
PDHP-Pase
(0.03-3
PM)
-*
PDH
Pyrophosphatase
(matrix volume)
+
Respiratorychain rate
- \
\
\
\
\
\
CaBl -
Volume 21
-
- -
A
ATP-synthetase
I activity
Regulation of Oxidative Phosphorylation
C;$+ with the expected affinities [ 19, 201, however,
further details of their molecular interaction with
C;i2+ remain obscure [Is), 211. CaL+ may promote
the association of the subunits of NAII-ICDH [lc)],
and it may act as a bridging ligand between PIIHPI’ase and the transacetylase (EJ subunits that form
the core of the I’IIH complex [20\.With OGIIH, it
would appear that the CaL+-sensitivity resides
within the decarboxylase (E,) subunits of the complex (M. I m k e and R. M. Denton, unpublished
work) I22 1.
It is worth mentioning that there is evidence
that a further matrix-enzyme system that catalyses
an oxidative decarboxylation that produces NALIH
may be sensitive t o Ca’ : the glycine-cleavage
enzyme system 1231. IIowever, this is not a ma.jor
source of matrix NAIIl I.
+
Matrix pyrophosphatase and volume regulation
The work of I lalestrap and colleagues [ 10, 24 I has
shown that increases in matrix [Ch” 1. within the
same sub-micromol;i~to low micromolar range that
activates the dehydrogenases, also increases the
activity of the respiratory chain in liver mitochondri;i. This occurs at the level of electron flow into the
ubiquinone (try) pool and is brought about by
p 1 2 + causing an increase in matrix volume; fattyacid oxidation in p;irticular is dramatically stimulated [ 1O ] . The niechanisni involves the inhibition
b y Calf of a matrix pyrophosphatase, since CaL+is
a competitive inhibitor for its substrate, magnesium
pyrophosphate, at the concentrations of M g L + that
are found in the matrix (Figure 1) 1251. The
increased matrix concentration of pyrophosphate
then causes an increase in the inward permeability
of K + ions, perhaps by a mechanism that involves
the ;idenine-nucleotide translocase, and, by resultant anion and water flux, causes the swelling of the
m;itrix [ 10, 24I. Such increases in mitochondria1
volume not only result in a stimulation of the
respiratory chain and of fatty-acid oxidation, but
may also lead to the stimulation of pyruvate carboxylation. citrulline synthesis and glutaminase
activity I 101.
Matrix Ca’+ -binding inhibitory subunit of the
ATP synthetase
I Iarris (I). A. I larris, this colloquium) [ 121 and
J’amada and I Iuzel 1271 have described a Ca”binding inhibitory (CaHI) subunit of ATP synthetase, distinct from the inhibitor that was first
characterized by l’ullman and Monroy (see [f)]),
that may be dissociated from the enzyme by
increases in ICa‘+ I within the same sub-micromo-
lar to low micromolar range discussed for the other
enzymes above (Figure 1 ). However, evidence for
Ca‘ +-dependent changes in ATP synthetase activity has not yet been obtained with studies on isolated mitochondria.
Nevertheless, there is the clear potential for
increased matrix [Ca’+ ] within the sub-micromolar
to low micromolar range to effect the co-ordinated
activation of the overall process of oxidative phosphorylation in response to increased cytosolic
[Ca”] (Figure 1). T h e main advantage is that
ATP/AIIP does not need to be diminished when
the cell is activated.
Mitochondria1 Ca2+-transportand
matrix CaZ concentration
+
The relay of the Ca’+ signal to the matrix targets is
accomplished by a specific CaL+-transport system
in the mammalian mitochondria1 inner membrane
[h, 9? 211. Ca” enters mitochondria by a uniporter
that is driven electrophoretically by the mernbranepotential component of the respiratory protonmotive gradient. The major egress pathway is by
electroneutral exchange with 2Na , however, this
too is ultimately driven by the protonmotive
gradient through the subsequent involvement of the
much more active Na /I I exchanger. Although
both uptake and egress are active processes, presumably to allow the rapid relay of the Ca’+ signal,
it can be calculated that, under normal conditions,
no more than 1% of the respiratory capacity is used
to cycle CaL+across the membrane [9]. There has
been, as yet, little molecular characterization of
these transporters (see [21]). There may also be a
Na+-independent egress route, perhaps catalysing a
direct Ca’+/Z€I+ exchange, however, this is even
more poorly characterized and has only minor
activity at physiological CaL+1 [ 21 1.
The Ca’+-uniporter can be inhibited by
physiological concentrations of Mg” and can be
activated by sperniine; it can also be inhibited artificially by Ruthenium Red and by lanthanides
[h-101. The Na+/Ca’+ exchanger is also inhibited
by Mg” and by increases in [Ca” 1 in the physiological range; it can be inhibited by agents such as
diltiazem, but much more weakly than their effects
on the plasma membrane Ca’+-channel 16-101.
These effects are all on the extramitochondrial side
of the inner membrane, but it is not yet known if
changes in the cytosolic concentrations of the
physiological effectors ever affect the distribution of
Ca’+ across this membrane in intact cells. Nevertheless, it has been shown that changes in Mg”,
Na+ and spermine can alter this gradient in many
+
+
+
I993
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Biochemical Society Transactions
796
studies on isolated mitochondria from different
mammalian tissues 10-8, 28 1.
Many studies have confirmed that when isolated mitochondria are incubated in the presence of
physiological concentrations of Mg’+ and of Na+,
all of the above CaL+-sensitiveenzymes can be activated as extraniitochondrial [Ca” ] is increased
over the physiological range 16- 10, 12 I. The experiment in Iiigure 2 shows how increasing [Ca’+] in
Figure 2
Experiment showing that the stimulation of OGDH
by Ca2+ in isolated rat-heart mitochondria incubated
under probable physiological conditions can lead to
increases in NAD(P)H production, in 0, uptake and
in ATP synthesis, that is, in oxidative phosphorylation
Mitochondria were incubated at 30°C in KCI-based media,
initially containing 2 mM ADP, 0.5 mM malate, 10 rnM NaCI, 2
mM MgCI,, 0.5 rnM EGTA and 0.25 mM CaCI, (free extrarnitochondrial [Ca”] - 50 nM), that is, similar conditions to those
found in the cytosol of a resting cell. Where indicated, a subsaturating concentration of 2-oxoglutarate (OG)was added
(0.5 mM), followed either by no further additions ( - ; w ) , or by
5 mM tGTA plus 5 mM CaCI, ( + 0)to give a new [Ca’+] of
- 500 nM, that is, similar to that in a stimulated cell. Adapted
from data in (J. G.McCormack and R. M. Denton, unpublished
work) [ 17,28, 371 (see references for the details of units
involved).
~
f
ATP
content
this manner to activate 0CI)I I can lead t o increases
in the substrate supply t o the respiratory chain. in
the respiratory rate and in A T P production, that is,
the whole process of oxidative phosptioryIiition.
Some of these effects on mitochondrial C;I’ transport are worthy of further comment. ‘I’he sigmoidal positive co-operativity of Ca’+ uptake in the
presence of Mg”, and the inhibition of egress by
extramitochondriaI C:a’+, suggest that, 21s the latter
is increased, there will be an amplified increase in
matrix ICa” 1 [ I O ] . This has been confirmed
directly in heart mitochondria, which can be readily
loaded with fluorescent Ca’+-indicators, such as
fura-2 (for example [20j). This work also confirmed
the most recent estimates of the regulatory ranges
for I’1)I I and for O(;I)1 1 within their natural environment (see above). These studies further suggested that under conditions that are likely t o exist
i n unstirnu1;tted cells, that is, physiological IN;i+ I
and (Mg’+ I and
100 nM lCa’+ I, there would in
fact be a lower concentration of Ca’+ in the matrix
( - 30 nM), \vhich would be below the regulatory
ranges for the (’aL+-sensitive enzymes. In contrast,
at an extramitochondria1 ICla’+ ] of 1 p M , 21s in :I
maximally stimulated cell, the matrix [ Ca” I would
be
3 p M and would be near saturating for its
effects on the matrix enzymes 120 I.
A fascinating piece of work by Ilansford and
co-workers has recently established more directly
that this is the case in intact cells [30]. They f m n d
that in single cardiac myocytes loaded with indo- 1,
a significant amount of the indo-1 was located in the
mitochondrial matrix, and that the cytosolic signal
could be selectively quenched with low concentrations of MnL+. It was found that the gradient
(in:out) of CaL+ across the inner membrane was
less than one in unstimulated cells, approached one
at -600 nM Ca’+ and was greater than one at
cytosolic concentrations above 600 nM [ 301.
These and other [ 3 I , 321 studies suggest that,
over the regulatory ranges for the enzymes, about
0.lo/o of the matrix calcium is free (that is, similarly
to the situation in the cytosol), and that each 1 p M is
roughly equivalent to a total mitochondrial Ca’+
content of - 1 nmol/mg of protein (see Table I).
+
-
-
-
Evidence for the role of matrix [Ca”]
in regulating oxidative
phosphorylation
I
0
I
3
Time (min)
Volume 2 I
I
6
Evidence has been obtained mainly from studies in
rat and guinea-pig heart and rat liver. In such
tissues or in cells from these tissues, there are wellknown circumstances in which increases in cyto-
Regulation of Oxidative Phosphorylation
Table I
Effects of adrenalin pre-treatment of perfused rat
heart on amounts of PDHa, activity of O G D H and
total and free calcium contents in subsequently isolated mitochondria
All the values for pre-treatment with adrenalin were significantly different from the cori esponding control values Values
are tdken i t om * [36] j [35]and $ [38]
Ad renal in
Parameter (units)
Control
PDHa (fraction of total PDH;
%)*
OCDH (fraction of V,,,,, , %)*
Total Ca (nmolimg of protein) 1
Free matrix [Ca"] (nM)S
8
25
I .8
I72
pre-treated
20
47
4.2
916
solic [('a'+ I have been described and Lvhich are all
associ;ited with increased respiration (see [h-8,
10 I), in particular positive inotropic intervention in
the heart and exposure t o 'Ca-mobilizing' hormones
in liver.
'I'hc first indication that increases in matrix
[ Ca'+ I could form at least a partial link between
these tww phenoniena came with the observations
that increases in I'I)I la were also associated with
such events (see 'I'able 1 for an example). In heart
prep;ir;itions, it was ;ilso demonstrated that these
increases in 1'I)I la could be prevented by the
presence of liuthenium Kt:d, whereas cytosolic
Ca' -dependent responses were unaffected [ 33,
+
3-41.
r.
1 hen it was found that if tissues were rapidly
disrupted into appropriate media for rnitochondrial
isolation (ice-cold, Na -free and containing EGTA).
Ca' t-nloverneilts across the inner membrane could
be minimized and so the Ca content at the time of
disruption could be effectively maintained. In this
way, increases in the total Ca content of the mitochondria could be demonstrated as the result of the
pre-tre;itment of tissue ivith an agonist (for example,
( 3 2 , 351; see Table 1). Moreover, the Ca loss could
also be mininiized under incubation conditions
under which the Ca'+ -sensitive properties of PDH
and O(;I)I I could be exploited to use them as
matrix ICa'+ I probes. 1 Ience it was found, both in
1ie;irt and in liver, that pre-treatment of tissues with
agonists that increased cytosolic [ Ca'+ 1 caused the
persistent activations of these enzymes in isolated
mitochondria (Table 1) [ 32, 30, 371. Furthermore,
the persistent x-tivations of these enzymes could be
diminished by incubating the niitochondria with
+
Na' ions to activate <$+-egress, or with sufficient
extrainitochondrial Ca'+ to lead to saturation of the
effects of CaL+ on these enzymes. The conclusion
that the persistent effect on the enzymes was due to
persistently elevated matrix [ Ca'+] has recently
been confirmed directly by using a similar strategy
to isolate mitochondria from hearts that had been
perfused with fura-2 under conditions where some
of the ligand is located in the matrix (Table 1) [ 381.
The work on single cardiac myocytes where
the mitochondrial indo-1 signal can be specifically
measured has also demonstrated directly that
matrix (Ca'+ 1 is elevated under a variety of conditions where cytosolic [Ca'+] is elevated [ 3 0 ] .
Another recent piece of work (301 has used a completely different approach t o specifically and
directly measure niatrix ((?aL+1 within intact cells.
Rizzuto et al. [ 39) transfected bovine aortic-endothelial cells with a construct containing the Ca'+sensitive photoprotein aequorin and a pre-sequence
to target it to the mitochondrial matrix. Matrix
[Ca" ] was quickly elevated on the exposure of the
cells to ATP, which in these cells causes an increase
in cytosolic Ca'+ J 39J.
1 he consequences of such increases in matrix
[Ca" I have also 1)een explored. Studies on isolated
cells or with surface tluorescence of tissues indicate
that, both in heart and in liver. iXAl)(P)l I levels
increase under such stimulatory conditions 140, 41 I.
The use of ."I' ti.in.r., particularly in heart, has verified that, under stin)ulatory conditions where
increased respiration is clearly evident, there is no
change or there is even an increase in the A'I'I'/
ALIP ratio (42-441.
I h v e v e r , this wiis not the case if
Ruthenium Ked was present in the perfusions;
under these conditions, clear decreases in A T P /
A1 )P were observed, indicating that, if t h r ('aL+dependent mechanism is unavailable, then cells can
still increase respiration, but only at the cost of
decreases in the ATI'/AI)P ratio [43, 441.
The Ca' -dependent increases in matrix
Ipyrophosphate I, volume and respiratory-chain
activity that can be elicited in isolated liver niitochondria (see above) are similar t o those that are
evoked in intact liver cells by Ch'+ -mobilizing hormones. suggesting that such changes could be a key
part of the respiratory response in stimulated liver
cells [ 10, 2-11, 1Iarris (see I ). A. IIarris, this colloquium) has provided evidence, using cultured rat
cardiomyocytes, that the A'I'P synthetase may be
reversibly modulated in vivo by the association and
dissociation of CaHI [ 12, 4.51.Thus, for instance,
treatment with isoprenaline led to an increase in the
synthetic activity of the enzyme when subsequently
r
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+
I993
797
Biochemical Society I ransactions
798
extracted and assayed. 1)efects in this regulatory
mechanism may exist in some pathophysiological
conditions such as hypertension, and this may be
responsible for the associated known defects in
A‘I’P production [ 401.
I n summary, there is now a convincing body
of evidence that, when cytosolic [ C a l f ] is raised in
stimulated cells, there is an associated increase in
mitochondria1 matrix [Ca’+], which potentially
could play a key role in the stimulation of oxidative
phosphorylation under such conditions. Certainly,
there seems little doubt that PI111 and 0GI)II are
activated. One of the challenges for future research
is the development of approaches that will allow a
reliable assessment of the relative importance of the
various potential mechanisms for stimulating oxidative phosphorylation in stimulated cells. It is highly
likely that the relative importance will vary greatly
with different circumstances and Ivith different cell
types.
Studies from the authors‘ laboratories were supported by
grants from the British t k a r t Foundation, the Wellconic
Trust and the lister Institute of I’reventative Medicine;
J . (;. X l. was forrnerly a I,ister Institute Kesearch Fellcnv.
1. Erecinska, M. and b’ilson. I ). I;. (1082) J. Memhr.
Ijiol. 70, I - 14
-.7 Nicholls, I). (;, (1084) N. Coniprehcn. Hiochem. 9,
20-48
.>. f h n d . M. I ) . arid Murphy. Ill. 1’. (1087) Hiol. Rev.
Cambridge I’hilos. Soc. 62. 14 1 - 103
4.Slater. I<. C. (1OX7) Eur. J. Hiochern. 166, 480-504
.5. (’hance. H. J. and Williams, (;. K. ( 19.50) Adv.
Enzyniol. Kelat. Subj. I%iochem.17. 0.5-1 34
0 . McCormack. J. (;., l lalestrap, A. 1’. and L)enton. K.M.
(1000) I’hysiol. Rev. 70. 39-425
1. 1)enton. K. M. and Mc(’omiack, J. G. (108.5) Ani. J.
I’hysiol. 249, E.543-E.5.54
8. €lansford. K. (;. (1091) J. Hiocnerg. Hiomembr. 23.
823-8 5 4
0. (’rompton, M. ( I O O O ) in Calcium and the Fieart
(Imiger, (;. A,. ed.). pp. 107-108. liavcn I’ress. New
York
10. I lalestrap, A. 1’. ( I ( J X 0 ) Biochini. Hiophys. Acta 973,
-
35.5-382
11. Kasmussen. f 1. and Harrett, 1’. (1.(I(J84) I’hysiol. Kev.
64, 038-078
12. liarris, I). A. and I)as. A. M. (1001) Hiochem. J. 280,
501-573
13. I)enton, K. M., K;indle. 1’. J. and Martin. H. K. ( 1 072)
Hiochem. J. 128, 161-103
14. I lenton, K. M., Kichards. I). A. and Chin. J. (;. (1 978)
Hiochrm. J. 176. 801-000
Volume 21
1.5. McCorrnack. J. (;. and I)enton, K. M. (lO7(J)
Hiocheni. J. 180. 533-544
10. McCormack, J. G. and I)cnton, K. M. (1081)
I3iochcni. J. 196, hlO-h24
17. McCormack. J. G.and [lenton, K. M. (IWO) Trends
Hiocheni. Sci. 11,258-202
18. Kutter. (;. A. and Iknton, K. M. (1088) Hiochcm. J.
252, 181-180
10. Kutter, G. A. (1000) Int. J. Hiochein. 22. 1081-1088
20. Teague, W. M., I’ettit, F. 11.. Wu, T.-I,.?Silbernian,
S. K. and Keed, 1,. J. (10x2) l3iocheniistry 21,
5 j8 5-5SO2
21. McCormack, J. G.,L)aniel, K. I,., Osbaldeston, N. J.,
Iiutter, C. A. and I)enton. K. hl. (1002) fjiochcm. SOC.
Trans. 20. 153- 1 5 0
22. Idawlis, V. H. and Koche, T. E. (1081) I3ioclicniistry
20,25 12-25 18
23. Jois, M., I lall, H. and Hrosnan, J. T, (1900) J. Biol.
Cheni. 265. 1240- 1248
24. Davidson. A. M. and llalestrap, A. 1’. (1080)
Hiocheni. J. 258. X 17-82 1
25. Kutter, (;. A.. Osbaldeston, N. J., McCormack, J. (;.
and 1)enton. K. M. (1000) Hiochen1.J. 271. 027-034
20. Krfcrencr deleted.
27. Yamada. 13. W.and Huzel. N. J. (1088) J. l%iol.Chcm.
263, 1 1408- 1 1 503
28. [)enton, K. M., McCormack, J. (;. mid I<dgeII. N. J.
( 10x0) Hiocheni. J. 190. 107- 1 17
20. McCormack. J. (;., l3rownc. I I. M. and I )awes, N. J.
( 10x9) I3iochirii. Hiophys. Acta 973, 420-427
30. hliyata, ti.?Silverman, S., Sollott, S.J.. 1,akatt;i. E. (;.,
Stern, kl. I ). and I lansford. K. (;. (I00 I ) Ani. J.
I’hysiol. 261, 111 123-111 134
31. I lansford, K. (;. and Castro. F. (1082) J . I<iocncrg
Hiomcnib. 14. 371-370
32. Assiinacopoulos-Jeanii~~t,
F.%McCormack, J. (;. and
Jeanrcnaud. H. (1080) J. Hiol. Chem. 261, 8700-8804
.33. McCorrnnck. J. G.and I<ngland, 1’. J. (10x3) I h c h c m .
J. 214, 58 1-585
34. tlansford. K. (;. (19x7) I3iochcm. J. 241. 145-1 5 1
35. Cronipton, M., Kcssar. 1’. and Al-Nasser. I. (1083)
Hiochem. J. 216. 3.33-312
36. McCorniack. J. C. and I)cnton, K. M. (10x4)
Hiocheni. J. 218, 235-247
37. McCormack. J. G.(1085) Hiochem. J. 231. 5XI-.50.5
38. Allen. S. I’.. Stone. I). and IllcCormack, J. (;. (I(J(J2)J.
Mol. Cell. Cardiol. 24. 705-774
30. Kizzuto. K., Simpson, A. W. M., Hrini, M. and
I’ozzan. T. ( 1002) Nature (1,ondon) 358. 325-327
40. Halaban, K. S. and H l u n i , J. J. (1082) Ani. J. I’hysiol.
242. C 172-CI77
41. Katz, I,. A,,
Koretsky. A. 1’. and Halaban, K. S. (1087)
FEHS I,ett. 221, 270-2176
12. From. A. l l . . I’etein, M. A, Michurski, S. l’.. Zimmcr,
S. I). and Ilgurbil, K. (1086) FEHS I x t t . 206.
257-20 1
43. I’nitt. J. I;..McCormack. J. (;.. lieid, I)., M;tcI,achlan,
I,. K. and England, 1’. J. (1980) Hiochem. J. 262,
203-30 1
Regulation of Oxidative Phosphorylation
44. Lrtz, I,. A., Swain. J. A, l’oi-tman, M. A. and 1Mahan.
K.S. (1000) Am. J. I’hysiol. 256. ti265-kI274
4.5, I);IS* A. bl. ;ind I larris. I). A. (1000) Cardiovasc. Rrs.
4f). I hs , A. M. and I Iarris. I). A. (1000) Am. J. I’hysiol.
259, H 1264-1 I 1 200
Received h April 1093
24,411--117
799
long-term and short-term changes in mitochondrial parameters by thyroid
hormones
Sibylle Soboll
lnstitut fur Physiologische Chemie I, Universitat Dusseldorf, UniversitatstraOe I, 40225 Dusseldorf I, Germany
Introduction
Thyroid hormones (TI Is) affect cellular metabolism
via two different pathways. The first one is the socalled nuclear, long-term effect: there the TI I binds
to a nuclear receptor and regulates the transcription
of multiple cellular proteins f 11. T h e other pathway,
the short-term extranuclear pathway, is less w d l
defined and could in principle be similar to other
short-term signalling pathways, that is, the signal
originates in the binding of TI1 to a plasmamembrane receptor, which generates an intracellular messenger that induces changes in metabolism,
via phos~~horylatic,n-dephosphorylationof enzymes
and of proteins 121. I Iowever, with 7’Hs it is also
feasible that they interact directly with intracellular
proteins since they can enter the cell [ 3 ] .
Interestingly. several parameters. especially
those relating to bioenergetics, are affected in a
similar way in the short-term range as well as in the
long-term range. I hus, respiration is stimulated in
the long-term range in euthyroid or hyperthyroid
states, as well as within minutes in the presence of
1.-triiodothyronine (Ti) [ 41. This stirnulatory effect
of 7’1 1s is oligomycin-sensitive, indicating that it is
not due t o an uncoupling action of 7’Hs [ 41.Likewise glucose production is increased in liver in the
short-term range 1.51, as well as in euthyroid or
hyperthyroid states [ 4 1. W e also found that the distribution of adenine nucleotides is changed in a
similar way by TI Is. both in hyperthyroid livers
and in perfused liver after the direct infusion of T3
161. The niitochondrial A?’P/AI>P ratio is decreased both in vi710 and in vitro, whereas the cytor .
Abbreviations used: Ap, protonmotive force; Apll,],,
iiiitochondrial proton gradient; A Y , membrane potential;
c. cytosolic; in, niitochondrial; p, plasma membrane;
I I M O , 5.5-diniethyloxazolidinedione; 7 ’ ; ; 1.-triiodothyroninc; T H - thyroid hormone; ‘TI’MP, triphenylmethylphosphoniurn bromidc.
sotic ratio is increased in vivo, but is unchanged in
vitro.
In this study, the effect of TI Is on the mitochondrial proton gradient (ApH,,,), the mitochondrial-membrane potential (AY,,,) and on the
plasma-membrane potential (AY,,) in rat livers
were compared in the long-term and in the shortterm range. The influence on these parameters of
another two hormones, glucagon and vasopressin,
which are known to increase respiration and gluconeogenesis in liver via intracellular second messengers 121, was also related to the changes induced
by T,.
Materials and methods
For all experiments rnale Wistar rats (180-200 g)
were used, which were starved for 48 h before
experinients and which had free access to drinking
water. I Iypothyroidisni was induced by intraperitoneal injection of Na””1 (250 pCi/lOO g of body
weight) 2 1-28 days before the experiment. I Iyperthyroidism was produced by daily intraperitoneal
injections of thyroxine (SO pg/lOOg of body
weight) for 7 days. The hypo- and hyperthyroid
states were monitored by serum thyroxine levels
( < 10 and > 250 ng/ml of serum, respectively).
Rapid liver sampling (in vizm) of livers was performed on unrestrained unanaesthetized rats by the
double-hatchet method 171.
Liver perfusion was performed as described
in 1.51, and the calcium concentration was monitored in the effluent perfusate by using a calciumsensitive electrode. The perfusion was terminated
by freeze-clamping. The freeze-clamped livers (as
well ;is those obtained by the double-hatchet
method) were ground in liquid nitrogen. lyophilized
and fractionated in non-aqueous solvents I 81 for the
determination of mitochondrial and cytosolic
metabolite contents, as well as the specific radioactivities of marker compounds (see below).
I993