Bioscience Reports 4, 189-194 (1984)
Printed in Great Britain
Inhibition
Ig9
by d i c y c l o h e x y l c a r b o d i i m i d e of ATP
s y n t h e s i s in i s o l a t e d rat h e p a t o c y t e s
S. EMAMI~ A. LODOLA, M, PARTIS, and D. R. SANADI ~
Biological Laboratory, University of Kent,
Canterbury, Kent CT2 7NJ, U.K.
(Received 9 January 1984)
D i c y c l o h e x y l c a r b o d i i m i d e (DCCD) inhibits, by 50%,
ATP synthesis in isolated hepatocytes.
This inhibition
is a s s o c i a t e d with D C C D - b i n d i n g to a proteolipid
fraction present in submitochondrial particles.
The oligomycin- and DCCD-sensitive ATPase complex catalyses the
s y n t h e s i s of ATP during m i t o c h o n d r i a l respiration (1).
At low
c o n c e n t r a t i o n s , DCCD inhibits both the hydrolytic and synthetic
activities of the ATPase with associated covalent modification of a
low-molecular-weight
s u b u n i t of the A T P a s e c o m p l e x ( 2 - 4 ) .
DCCD-binding proteolipid has been isolated from both mitochondria
(5)~ and the purified ATPase complex (3,6).
Although the exact
function of this protein in ATP synthesis is unknown it has been
implicated in proton translocation in mitochondria. Support for this is
provided by the finding that it is capable of proton transfer across
lipid bilayers (7).
Modification of the 8=subunit of the soluble Ft-ATPase has also
been reported (8).
Under slightly a c i d i c condition% and using higher
levels of DCCD, both the DCCD-binding proteolip[d and the 13-subunit
of F I are labelled; at alkaline pH only the proteollpid is modified.
Iff this paper we report the use of DCCD to inhibit ATP synthesis
in isolated hepatocytes. At low concentrations of DCCD~ ATP levels
in rat hepatocytes fall by approximately 50% without apparent loss of
cell viability; at higher conentrations ATP synthesis can be inhibited
completely with concurrent increase in the r a t e of l a c t a t e dehydrogenase release in the cells. This inhibitory e f f e c t has been shown to be
a s s o c i a t e d with DCCD binding to a p r o t e o l i p i d whose e l u t i o n
characteristics on thin-layer chromatography are comparable to the
DCCD-binding protein present in submitochondrial particles.
M a t e r i a l s and M e t h o d s
D i c y c l o h e x y l [ t ~ C ] c a r b o d i i m i d e (t~-C_DCCD) was obtained from
CEA ( F r a n c e ) .
C o l l a g e n a s e was purchased from the Boehringer
Corporation (London) Ltd.
All other chemicals were obtained from
Sigma Chemical Co. and were of the purest grade available.
*Present address: INSERM U.55, H6pita! Saint-Antoine, 184 rue du
Faubourg Saint-Antoine, 75571 Paris Cedex 12, France.
**Boston Biomedical Research Institute, Department of Cell
Physiology, 20 Stanford Street, Boston, Massachusetts 02114, USA
01984
The Biochemical Society
190
EMAMI
ET
AL.
Hepatocyte preparation
Hepatocytes were prepared as described in (9). After isolation the
ceils were washed (xl) and resuspended in Krebs-Henseleit buffer (10)
supplemented with 2% (w/v) bovine albumin, 0.1% glucose, and 25
mM Hepes buffer, pH 7.3. Ceils were diluted to a density of 6 x 106
cells/ml with this buffer just prior to use.
Cell incubations
Incubations were in paired 250-ml (siliconized) Erhlenmeyer flasks
at 37~
in a shaking water bath at a frequency of ltt0 strokes/rain,
using either 50 or 20 ml of cell suspension.
Reaction was initiated by addition of DCCD, dissolved in methanol,
to the reaction flask.
The final concentration of methanol in the
reaction flask did not exceed 0.001% (v/v) in any experiment.
Sampling
At appropriate time intervals cells were recovered by centrifugation
(50 g, 3 min) from aliquots (2 ml) of the incubating suspension. The
supernatant was carefully removed and stored at -20~
the cell pellet
was quenched by addition of ice-cold 6% (w/v) perchloric acid (0.2
ml) or directly frozen unquenched, prior to storage at -20~
Assays
The activity of lactate dehydrogenase (EC 1.1.1.27; LDH) in the
cell supernatant was determined as described in reference l l.
ATP
concentrations in the cell pellets were determined using the hexokinase
(EC 2.7.1.i) method described in reference i2.
Proteolipid isolation and analysis
After incubation with I~C-DCCD (100 l~M; 10 I~mol/pCi; 60 min),
cells from 50 ml of incubating suspension were recovered by centrifugation (50 g, 3 rain) and the proteolipid extracted as described in
reference 5.
Analysis of the proteolipid mixture was by thin-layer
chromatography, as described in reference 13.
Scintillation counting
Cell samples for scintillation counting were dissolved directly in a
phase combining scintillation fluid (1 ml; PCS; Radiochemical Center,
Amersham), prior to counting.
Bands from the thin-layer-chromatography plates were scraped off
into a scintillation vial prior to addition of PCS and counting.
Counting was performed using a Packard Tri-Carb liquid-scintillation
spectrometer, model 3375.
DCCD labelling of submitochondrial particles
These were prepared as described in reference 14.
R e s u l t s and D i s c u s s i o n
Data for the uptake and DCCD-binding to a suspension of isolated
hepatocytes are shown in Fig. 1A. There was an immediate association of DCCD with the hepatocytes, followed by a slow (transient)
DCCD
I
AND
HEPATIC
ATPase
191
9
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r'
100,
sol- \
0.25ramt 0
9
~
0
'
~
68
-
J
u
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30
60
S
0
o.1 ~M ....i
30
~
TIME (Minul.es)
Fig. i.
E f f e c t s of dicyclohexylcarbodiimide on
isolated rat hepatocyte suspensions.
(A) Uptake of
1 4 C - D C C D by hepatocyte suspensions.
Methods are
described
in the text.
(B) Effect of various
concentrations of DCCD on hepatocyte ATP concentration.
ATP measurements are described in ref. 12.
(C) Rate of lactate dehydrogenase release in the
presence and absence of DCCD. Lactate dehydrogenase
activity was determined as in reference II.
i n c r e a s e and then a rapid loss of labelled D C C D f r o m the cell f r a c t i o n
a f t e r 20 min. O v e r the s a m e t i m e period, m e a s u r e m e n t s of cell ATP
c o n c e n t r a t i o n s w e r e m a d e (Fig. I B ) .
S t a r t i n g ATP (4.2 n m o l / m g )
c o n c e n t r a t i o n s w e r e c o n s i s t e n t with published values ( I 5 ) .
In c o n t r o l
incubations, a f t e r an initial fall in cell ATP levels, t h e r e was a rapid
r e c o v e r y to s t a r t i n g values.
In the p r e s e n c e of D C C D t h e r e was a
rapid fall in ATP levels.
At the two l o w e s t D C C D c o n c e n t r a t i o n s
t e s t e d (30 and 100 pM) t h e r e was no d i f f e r e n c e b e t w e e n e x p e r i m e n t a l
and c o n t r o l ATP v a l u e s for the f i r s t 20 rain of i n c u b a t i o n .
A f t e r this
t i m e t h e r e w a s a r a p i d f a l l in A T P c o n c e n t r a t i o n in the cells
incubated in the p r e s e n c e of D C C D .
At 50 tJM D C C D the ATP
c o n c e n t r a t i o n felt to approx. 40% of c o n t r o l values while with 100 pM
DCCD t h e r e was a c o m p l e t e loss of ATP.
At t h e highest D C C D
c o n c e n t r a t i o n (250 t~M) t h e r e was c o m p l e t e loss of ATP within 40 rain
of incubation.
S i n c e h e p a t i c ATP c o n c e n t r a t i o n s a r e a s e n s i t i v e index of cell
viability (13) the ATP losses o b s e r v e d could h a v e been the result of
D C C D - i n d u c e d v i a b i l i t y loss.
As an i n d e p e n d e n t a s s e s s m e n t of ceil
viability, measurement
o f LDH was m a d e c o i n c i d e n t with D C C D
t r e a t m e n t (Fig. 1C). At 50 M DCCD, inhibition of ATP s y n t h e s i s was
not a c c o m p a n i e d
by an i n c r e a s e in the r a t e of LDH r e l e a s e , as
c o m p a r e d to the c o n t r o l s .
No s i g n i f i c a n t d e p a r t u r e of LDH values
from t h e c o n t r o l was o b s e r v e d at the higher D C C D c o n c e n t r a t i o n s
until c o m p l e t e loss of ATP had o c c u r r e d .
This would be e x p e c t e d on
the grounds t h a t ceils devoid of ATP a r e by d e f i n i t i o n nonviable.
In order to d e t e r m i n e t h e n a t u r e of the D C C D inhibition e f f e c t ,
and m o r e s p e c i f i c a l l y to a s c e r t a i n w h e t h e r the D C C D - s e n s i t i v e p r o t e i n
192
EMAM[
ET
AL,
of the ATPase complex was involved~ the hepatocyte proteolipid
protein was extracted and analysed by thin-layer chromatography (Fig.
2 A-C). There was a rapid labelling of a fast-moving component (RI
= 0.86) which showed a Joss of label as the incubation proceeded,
This f r a c t i o n quantitatively contained the largest fraction of total
hepatocyte-associated label. There was also a time-dependent labelling
of a slower-moving component (Rf = 0.#) which appears to correspond
to the DCCD-binding protein of the ATPase associated with submitochondria] particles (Fig. 2D).
The slight differences in mobility can
be accounted for by the v i a b i l i t y inherent in this system.
No
fast-moving component was found in the submitochondria/ particles to
correspond with that in the hepatocytes.
"~
|
~= 100
IUU
U
a:
~
GO
E
t ' 0 rain
60
~
~
2C
t : 2 0 rain
2(3
o
Fraction Number
|
f,,.
Q
s|
r
60
E
cr
E
t : 4 0 min
20
DCCD-BIndm 9 Pr'ote,n
,-,
i
~
60
~
~
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O
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Fraction Number
Fig. 2.
Labelling of the hepatocyte proteolipid
protein by DCCD.
Hepatocyte suspensions were
incubated
w i t h 14C-DCCD for 60 min prior to
proteolipid extraction and thin-layer-chromatography
analysis as described in references 5 and 13. Bands
from the thin-layer plate were recovered and
counted,
as" described in the text, the 14Cdistribution data over the first 40 min of incubation are shown in A to C. Also shown in D is the
result of proteolipid analysis of DCCD-labelled
submitochondrial particles.
A major peak is found
in fraction 6 (Rf = 0~
associated with the
DCCD-binding protein.
DCCD
AND
HEPATIC ATPase
193
These findings, taken in conjunction with the DCCD uptake and
ATP m e a s u r e m e n t s , s u g g e s t a possible scheme for inhibition of
hepatocyte ATP production. On addition of DCCD to the hepatocyte
suspension there is a rapid and presumably nonspecific association of
DCCD with the hepatocyte. This may account for the rapidly labelled
fast-moving component. This would be followed by the slow uptake of
DCCD and its conveyance to the mitochondria, an event consistent
with the delay prior to the fall in the celt ATP concentrations after
DCCD addition.
At the mitochondrion the DCCD forms a complex
with a specific protein of the ATPase complex (the DCCD-binding
protein), resulting in a rapid loss of ATP, since the hepatocyte ATP
pool (and nucleotide pool in general) is in a constant and rapid state
of flux. At the same time there is presumably a rapid degradation or
dissociation of DCCD (perhaps by equilibration of DCCD with the
bovine albumin in the incubation buffer).
Although inhibitory e f f e c t s of DCCD on giycolytically mediated
membrane transport have been noted in bacteria (t6) and yeast (17),
no a t t e m p t s have been made previously to determine whether the
inhibition in intact cells resulted from nonspecific interaction of the
highly r e a c t i v e DCCD with several essential cell constitutents or
specifically with the mitochondrial proteolipid. The temporal correlation of decline of ATP levels with labelling of proteolipid, but not of
other bands, favors the latter interpretation, and also shows that at
iow concentrations of DCCD, the cellular ATP levels can be reduced
effectively without loss of cell viability. Such controlled perturbation
is useful for simulating the ATP changes in aging ( l g ) , hypoxia, and
ischemic cell death. In myocardial ischemia cell death, for example,
w h e t h e r irreversible cell injury results from ATP loss, lactic acid
a c c u m u l a t i o n (19), or p a l m i t y l - c a r n i t i n e accumulation (20) is an
unsettled issue.
Acknowledgements
A. Lodola is s u p p o r t e d by grants from the Medical Research
Council.
The research was supported by grants from the National
Institutes of Health (GM 2676# and RR 05711). The authors wish to
thank Mrs D. Lhenry for the preparation of the manuscript.
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EMAMI
ET
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