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Effects of ethanol on protein metabolism in hepatocyte primary cultures from adult rat
Ceil Biology
EFFECTS
International
OF
Reports,
ETHANOL
ON
Vol. 12, No. 8, August
PROTEIN
CULTURES
A.
Voci,
Istituto
FROM
Gallo,
G.
F.
Fisiologia
di
METABOLISM
16132
IN
ADULT
Genova
647
HEPPlTOCYTE
PRIMARY
RAT
Balestrero
and
Generale,
1988
E.
Fugassa
Universita
di
Genova,
(Italy)
ABSTRACT The
effect
ethanol
of
cultured
hepatocytes
The
presence
of
significantly
degradation
protein
synthesis in
However,
adult
100
mM
of
synthesis rat
has
in
rate. did
The not
the
culture
synthesis
protein
appear
medium
without
depressing
in
studied.
been
ethanol
degradation
and
effect
directly
affecting
of
ethanol
correlated
on
with
the
level.
ATP
an
systems was
from
decreased
protein
changes
protein
on
inhibition the
of
plasma
sodium-dependent
following
membrane
energy-requiring
and exposure
ethanol
to
observed.
INTRODUCTION Metabolic
responses
widely is fk
far
understood as
ethanol
(1,4-6).
large
variety
which
include
the
liver
to
mechanism
of
ethano
action
1
have
been
of
this
drug
(l-4).
effect
inhibition,
concerned, reported
although
documented
poorly
mammalian
of
stimulation
intact
liver and
conflicting
These of
on the
techniques
animals,
0309-1651/66/060647-14l$O3.0010
protein no
effect
reports and
at
may
be
organs
all
have
related
models
experimental
isolated
is
metabolism
or
cells,
@ 1999 Academic
been to
the
utilized tissues
Press Ltd.
648
Cell Biology
slices, In
homogenates, the
International
Reports,
Vol. 12, No. 8, August
1988
polyribosomes.
present
study
cultured
on collagen
evaluate
the
coated
effect
isolated
hepatocytes
of
the
dishes
for
ethanol
on
from
adult
several
days
liver
rats were
protein
and
used
to
synthesis
and
hepatocytes
in
degradation. Hepatocyte
monolayers
suspension
since
the
activities
enzyme
isolation
were
preferred
cells
may survive functions
and
involving
procedure
to
isolated for
many
which
days
are
recover
affected
perfusion
collagenase
and
by
of
the
the
liver
(7). results
The
protein
indicate
synthesis
MATERIALS
that
without
the affecting
2nd
male
(Correzzana,
Wistar-Nos
rats
previously
that
more
suspended
modified
Eagle's
Virginia,
USA),
cell
co1 lagen
dishes (acid
Hepatocytes
Mi,
and
(Flow
decreases rate.
soluble
cells/ml)
type
non
viable.
Cells
III,
Sigma
Industries,
and
Co.,
into
After
with
St
gentamycin
USA).
coated
of
McLean,
Chemicals
Lab.1
placed
Sigma).
acids
Inc., (Nova
previously
were (DMEM)
amino
Laboratories
were
indicated
medium
essential
Flow
from overnight
blue
Kenilworth,NJ,
Corporation,
3060)
fasted
Eagle's
@g/ml,
(0.25
prepared
trypan
were
(lpmol/l,
dexamethasone
(Costar
180-200)
insulin/ml
units
fungizone
were
of
modified
0.1
(5x10=
g
cells
the
medium
Schering
suspension
culture
of
essential
DK),
MO, USA), pg/ml,
1
ethano
degradation
Exclusion
Dulbecco’s
with
Copenhagen,
(8).
85%
than
supplemented
(SO
protein
Eulture:
described
in
Louis,
to
AND METHODS
l-l-l~a_~o_~y~~ jsolatjo_n
as
exposure
3 ml of
60 mm calf seeding,
tissue skin the
Cell Biology
International
culture
d i shes
were
incubator
(Forma
containing
5% COVain
fresh
medium
changed Protein
transferred
or
24
h
New
and
incorporation
precipitable
and ---
for
of
reverse-phase
HPLC
Cl8
column
Secretion
(5
culture
medium
already
reported
PEo__teln
370
as
analysis
cm,
Merck
were
ine
low
(294
Berlin,
D)
into
acid
incubation,
soluble
was
material
uptake
and
pool
was
for
using
the
isocratic with
chromatography) FRG)
proteins
at
protein
West
acid
Darmstadt,
to
(10).
evaluated
was
speed
Degradation
gf mono
which
remove
in
cell
the
debris
as
had
been
labelled 0.15
dose,
proteins
for
pCi/ml,
was
20
hours
294
mCi/mmol)
at
(9). nucleotide --_------.were
I ayers frozen were
long-lived
of
(tracer
adenLne
immediately
(11).
Media
[“Cl-valine
of
liquid
synthesized
described
concentrations
and
precursor
C”‘vCl-valine val
centrifuged
Determination ---_.._-- -----
and
25
C3”Clvaline
te
removed
evaluate mM
led
The
(high-performance
pm,
10
hours
(9). of
monolayers
previously
hepatocy
37oc
(9).
in
C with
added.
Products,
two
elsewere
d!Xl~S!S~i~E:
measured
To
label
the
after
newly
of
was
was
at
USA)
medium
ethanol
with
intracellular
determination
a
mM
the
Research
of
evaluation
the
min
labelled
Nuclear
England
described
as
90
humidified
a
OH,
5Cr_UgLOJ:
material,
measured
Marietta,
100
were
mCi/mmo.l,
to
thereafter.
synthesis,hepatocytes
used
After
without
1988
carefully
3163,
air.
~Y!n!i!?!S?i~
the
Vol. 12, No. 8, August
Scientific
with
every
Reports,
quantified
lEw%w~~:
trinsed in
three 1 iquid in
the
times
3 with
nitrogen. acid
day-cultured cold
saline
Nucleotide extract
by
HPLC
Cell Biology
650
International
Reports,
Vol. 12, No. 8, August
1988
.................... ............ ............. ................... ..........................................
..................
,.................,
.
.. . ..
In ..
..,................
............ .......
.
......
days In culture
Figure
1.
secret
Effects
ion
culture
rate
(ml
ethanol in
on
protein
hepatocyte
synthesis
CD)
and
as
a function
of
monolayers
time.
Cells
were
extracel
labelled
lular
Materials
and from
absence
of
Protein
secretion
expressed
with
10
mM C*‘+Cl-valine.
were
radioactivity
values
plus
of
Methods. contra
ethanol)
as
extracellular).
Protein 1
assumed refers
% of
cells
total
evaluated
synthesis (cultured as
to
Intracellular as is for
same
as
under % of
the
in
the
time
100.
extracellular
insoluble
described
expressed the
and
insoluble radioactivity
radioactivity (intracellular
Ceil Biology
Enzrme
International
Na-K
gsHYs_:
& Barting
(12).
Cathepsin
B
previously
described
and
5
for
at
37O C for
as
was
AND DISCUSSION
decreased
hepatocytes,
as
in
cells, was
not
employed corresponds
resulted
protein proteins the
drug.
of
with
reduced
figure
1,
control
the
the
and
containing
did
Hartree
uptake
was
in
in
also
shows
valine of
precursor
incorporation synthesis
rate.
monolayers
to
ethanol
of
the
of
protein
affect
the
to
22% of in
the the
a
by ethanol-
of
not
as
Such
intracellular
protein
the
ethanol-treated
C'-Cl-valine
the
in
table
concentration in
as well
The
uptake
high
medium
culture. uptake
1.
the
culture
C1‘+Cl-valine in
table
(14).
the
valine
exposure
amounted cells
saline
AIB
to
time
inhibition
ethanol which
the
of
decrease
a progressive
the
as
"oEel2Yers:
solution
of
valine
reduction
an actual
to
addition
in
the
secretion
extracts
with
AIB.
a reduced
to
Hence,
salt
incorporation
due
However,
in
ethanol
modified
in
Bakkeren
(13).
function
the
Earle's
mM unlabelled
radioactivity
in
secretion.
twice
specific
to
depicted
rinsed
as reported
of
(15).
cell
were
according
the
associated
spite the
pool
a
hepatocytes
that
in
kP~tC?LYt~~
in
1.5
that
progressively
cultured
assayed
to
BY
determined
1 demonstrates
was
according
uetake
elsewhere
RESULTS
AS
30 min
indicated
content
decrease
were
days
and
Protein
Figure
evaluated
LEG!1
three
fll'"l C1‘+Cl-labelled
evaluated
was
1988
(9). SEid
cultured
incubated
ATPase
Vol. 12, No. 8, August
D activities
waziQgisgQgtyrr& Cells
Reports,
rate percent newly ones
rate
of
synthesized treated
with
1.
Effect
of
ethanol
on
C *‘Cl-valine uptake
and
cpm
x
IO-“/mg
C”+Cl-valine
protein
uptake
cpm
x
C*%Zl-valine 10-9/mg
were
are
pool
Data
were
the
means
evaluated
radioactivity
The
Hepatocytes
as
soluble
with
of
10
triplicate
described
acid
2 SE
of
labelled
0.18
f
4.575
Ethano
1
+- 0.23
6.235
Control
under
and
plates
Material
insoluble
mM C”‘+Cl-valine
obtained
and
two
from
Methods.
material
for
14.17
24.04
--~----~----------~-------------~----~~~------~~---~~~~~~-~~~~~~~~~~~~~~-~~~-~~~~-~~~~~~~~~~~--~~~~~~~---
condition
Culture
0.91
two
and
hours.
by
specific
separate
the
+ 0.59
f
protein
incorporation
incorporation
----____-~---_-__-_-~-~-------~~~~~~~~-------~~~~~~~~---~~~--~~--~-----~---~--------~---~~---~~~~~~~~~~~~
Table
cpm
experiments.
radioactivity
hepatocytes
x
(S.A.)
11.58
for
mole
pool
12.05
10-=/F
Valine
cultured
of
valine
valine
S.6.
3 days.
*
P < 0.05
(StuaeritiB
The
represent
results
the
as
of
of
was
t
tssff.
for
were
mean
of
of
of
as
triplicate
triplicate
units/mg
assayed
experiment
enzyme
the
incubation
measured
days,
3.2
3.4
sampl
sampl
cells
DMEM
es
es
by
37O
for
obtained
100.
varied
cultured
as
at
val
less
-.
or
at than
of
8%.
least
as
value
of
1.15
lntracel
lular
separate
elsewere
labeiled
1 1 ‘, e r
mM
ir ij.19
D
- -.-.---
protein)
---
-c 0.24
100
ts/m.;
Cathebsiq
0.98
with
(AnI
.-. .-~
nmo 1 / ~mq
three
desrrlbed
753
_ .-.
supplemented
ir n.020
-fr 0.015
aroteinj
intracellular
days
The
from
content
0.333
G.043
ts/mg
5
days
~. .._.~_
three
::Jthepsiv
_.. .--.
for
from
three
C.
ine
unsuopiempnted
t uni
..____ -_.
~1.~1 tu:-ed
C"+C3-valine
cells
of
fractional
assumed
protein.
on
was
labelled
a
in
? 0.29
t
1 on
0.34
-..-.---.__---_---
hepatocytes
degradat
release
assuming
three
of
in ..______
metaboiism
-_---
turnover
% rate
calculated
was
cultured
radioactivity
expressed
The
+ SD.
start
hours
D activities
is
the
two
Activity
experiments
rate
degradation
(9).
and
Cathepsin
El
at
during
protein
(15).
synthesis
radioactivity
proteins
% rate
protein
protein
ethanol.
were
+ 0.020*
1.9
Protein
_______
protein
Ethano
of
-_---
on
synthesis
____
ethanol
k 0.029
monolayers
-_------
of
3.1
1
-___
Effect
Control
Hepatocyte
%
2.
Condition
------_---
Table
Cell Biology
To
evaluate
turnover, which in
the we
effects
no
Moreover,
lysosomal
enzymes
detected.
It
hepatocytes
were
control
cells
two
are
the
culture
the
since
protein Taken
act5
the
.
It
cultured
od.
In
the
protein
in
of of
postulated a
these
the
that
the
of
of
main
of
of
the
at
at
inhibits
ethanol present
ic
hepat
supply ATP
the
balance
effect
remains
affect
the
proteins.
which
energy
loss
rate
by
ethanol
should
in
be predominantly
machinery
the
ethanol
not
loss
mechanism
limitation
ethanol
that
to
were
the
in
protein
exposed
progressive
the
Earle's
synthesis
exceeded
to
be
with
modified
detectable
that
of
could
indicating
cells
a
level
experiments
protein
any
was
supplemented
rate
seems
has
case,
that
indicate
synthesis
the
D
turnover
However,
through
cathepsin
medium
the
in
protein been
in
degradation
results
level.
synthesis were
without
metabolism
protein
on
ante,
protein
our
B and
rate
reported
degradation total
degradation
resulting
synthesis
1 ear
this
(161.
If
level
in
hepatocytes.
verify
this
concentrations that
of
rate
on
protein
To
govern
together,
protein
ba per
synthesis,
ethanol
uric
in
which
processes
the
equalled
proteins,
the
DMEM
the
lived As
protein
aminoacids
cond i tions
these
protein
hepatocytes.
of
1988
overall
long
changes
in
essential
non
rate
stressed
cultured
processes
during
the
as cathepsin be
the of
in
appreciable
such must
and
In
no
on
proteins
on
Vol. 12, No. 8, August
degradation
of
effect
observed.
medium.
bulk
Reports,
ethanol
the
the 2
essential
of
investigated
represent
table
international
in
hypothesis in
ethanol
cultured treated
we measured cells hepatocytes
the
(table the
31. levels
adenine Our
of
nucleotide show
results ATP
and
the
Cell Biology
international
Reports,
Vol. 72, No. 8, August
I I I
I I I I I I I I I I I I I I I I I I I I I I ! I I I
I I I I I I i
I I I I I I I I I I I I I I
1988
655
Cell Biology
International
I I I I I I I
I I I
I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I I
Reports,
Vol. 12, No. 8, August
1988
Cell Biology
ATP/@DP agree
International
were
ratio
with
those
chronic
and
decrease At.
ATP
is
to
not
greatly
to
in
acute
also
to
4
table
Since of
treated
in
of (-42%)
agreement
energy
associated
supply
is
the
processes,
hepatocyte
reduction
hepatocytes
and
hepatocytes,
of
The
uptake
uptake
cell
cultured
of
addition
are
the
alteration(s) drug.
AIB
AIB
results
membrane
the
as the
both
These
effect
the
in
(17).
structural
ethanol
investigate such
treatment
to
data
whereas
reported
processes
(-40%).
exposure
(171,
been
impaired
energy-dependent
be due upon
to
shown
ethanol
These
(18-21).
reported
by
by Rubin have
worthwhile
activity
of
uptake
liver
AS
previously
impairment
membrane
was
monolayers
modified
could
in
657
1988
values.
control
administration
energy-requiring
Na-K-ATPase those
the
reported
activity.
ethanol
with
to
level it
on two
Na-K-ATPase
and
similar
previously
point
ethanol
Vol. 12, No. 8, August
ethanol
the
this
Repotis,
of
plasma the
line
in
valine
with
this
hypothesis. In
conclusion,
the
our
results
medium
culture
impairs
both
the
rate
of
The
depressing
could
in
of
protein protein
that
and
ethanol
monolayers
hepatocyte
synthesis
secretion
addition
to
significantly without
affecting
breakdown.
effect part
demonstrate
account
of
ethanol for
the
on aminoacid decreased
uptake
efficiency
by
the
of
cells
protein
synthesis. The
inhibitory
processes Na-K
&TPase
FITP levels.
effect associated does
of to
not
plasma
appear
on
other
membrane
such
ethanol
directly
correlated
energy-dependent as AIB with
uptake changes
and in
Cell Biology
658
International
Reports,
Vol. 72, No. 8, August
1988
ACKNOWLEDGEMENTS
Marchis
Dr
M.
de
of
adenine
We
also
the
preparation
is
gratefully
nucleotide thank
valine
and
Professor of
acknowledged
M.
for
his
determination
levels.
Orunesu
for
helpful
during
comments
manuscript.
the
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Cell Biology
15.
Seglen
P.O.
leupeptin rat 16.
on
(1978)
Effects
protein
synthesis
hepatocytes.
Williamson
rat
Reports,
of
M.H.
Scholz
(1969)
liver.
The
R.,
AT%,
Browning
E.T.,
effects
of
Biological
1988
ammonia
degradation
Journal
Metabolic
Journal
Vol. 12, No. 8, August
aminoacids and
Biochemical J.R.,
Fukami
International
in
and isolated
469-474. Thurman
of
R.G.
ethanol
in
Chemistry
and
perfused
244,
5044-
5054. 17.
Rosa
J.
and
acid
uptake
Rubin
E.
(1980)
rat
liver
cells.
by
Effects
of
ethanol
Laboratory
on
amino
Investigation
43,
366-372. 16.
Griffaton
G.,
Baron
administration en
aigue
nucleotides
internationales 19.
Hyenes
by
liver
20.
1196-1202.
Lieber
C-S.,
between fatty
De
et
Carli
dietary
fat
Journal
le
rat.
Biochimie.
teneurs Archives
29,
Prevention
75-79. of
fatty
triphosphate.
Duantitative
Clinical
Nature
relationship
severity
and of
d’une
sur
de
adenosine
(19701
L.M.
foie de
Effets
fructose
(1966)
K.J. of
American
liver.
de
Phisiologie
of
amount
et du
administration
(1971)
R.
adenyliques
Isselbacher
204 I
Lowry
d’ethanol
de D.E.,
P. ,
of Nutrition
alcoholic 23,
474-
478. 21.
Walker associated Journal
Received:
Gordon
J.E.C., with 112,
3.5.88
an
E.R.
ethanol-induced
(1970)
Biochemical fatty
511-518.
Accepted:
8.7.88
liver.
aspects Biochemical
EFFECTS
International
OF
Reports,
ETHANOL
ON
Vol. 12, No. 8, August
PROTEIN
CULTURES
A.
Voci,
Istituto
FROM
Gallo,
G.
F.
Fisiologia
di
METABOLISM
16132
IN
ADULT
Genova
647
HEPPlTOCYTE
PRIMARY
RAT
Balestrero
and
Generale,
1988
E.
Fugassa
Universita
di
Genova,
(Italy)
ABSTRACT The
effect
ethanol
of
cultured
hepatocytes
The
presence
of
significantly
degradation
protein
synthesis in
However,
adult
100
mM
of
synthesis rat
has
in
rate. did
The not
the
culture
synthesis
protein
appear
medium
without
depressing
in
studied.
been
ethanol
degradation
and
effect
directly
affecting
of
ethanol
correlated
on
with
the
level.
ATP
an
systems was
from
decreased
protein
changes
protein
on
inhibition the
of
plasma
sodium-dependent
following
membrane
energy-requiring
and exposure
ethanol
to
observed.
INTRODUCTION Metabolic
responses
widely is fk
far
understood as
ethanol
(1,4-6).
large
variety
which
include
the
liver
to
mechanism
of
ethano
action
1
have
been
of
this
drug
(l-4).
effect
inhibition,
concerned, reported
although
documented
poorly
mammalian
of
stimulation
intact
liver and
conflicting
These of
on the
techniques
animals,
0309-1651/66/060647-14l$O3.0010
protein no
effect
reports and
at
may
be
organs
all
have
related
models
experimental
isolated
is
metabolism
or
cells,
@ 1999 Academic
been to
the
utilized tissues
Press Ltd.
648
Cell Biology
slices, In
homogenates, the
International
Reports,
Vol. 12, No. 8, August
1988
polyribosomes.
present
study
cultured
on collagen
evaluate
the
coated
effect
isolated
hepatocytes
of
the
dishes
for
ethanol
on
from
adult
several
days
liver
rats were
protein
and
used
to
synthesis
and
hepatocytes
in
degradation. Hepatocyte
monolayers
suspension
since
the
activities
enzyme
isolation
were
preferred
cells
may survive functions
and
involving
procedure
to
isolated for
many
which
days
are
recover
affected
perfusion
collagenase
and
by
of
the
the
liver
(7). results
The
protein
indicate
synthesis
MATERIALS
that
without
the affecting
2nd
male
(Correzzana,
Wistar-Nos
rats
previously
that
more
suspended
modified
Eagle's
Virginia,
USA),
cell
co1 lagen
dishes (acid
Hepatocytes
Mi,
and
(Flow
decreases rate.
soluble
cells/ml)
type
non
viable.
Cells
III,
Sigma
Industries,
and
Co.,
into
After
with
St
gentamycin
USA).
coated
of
McLean,
Chemicals
Lab.1
placed
Sigma).
acids
Inc., (Nova
previously
were (DMEM)
amino
Laboratories
were
indicated
medium
essential
Flow
from overnight
blue
Kenilworth,NJ,
Corporation,
3060)
fasted
Eagle's
@g/ml,
(0.25
prepared
trypan
were
(lpmol/l,
dexamethasone
(Costar
180-200)
insulin/ml
units
fungizone
were
of
modified
0.1
(5x10=
g
cells
the
medium
Schering
suspension
culture
of
essential
DK),
MO, USA), pg/ml,
1
ethano
degradation
Exclusion
Dulbecco’s
with
Copenhagen,
(8).
85%
than
supplemented
(SO
protein
Eulture:
described
in
Louis,
to
AND METHODS
l-l-l~a_~o_~y~~ jsolatjo_n
as
exposure
3 ml of
60 mm calf seeding,
tissue skin the
Cell Biology
International
culture
d i shes
were
incubator
(Forma
containing
5% COVain
fresh
medium
changed Protein
transferred
or
24
h
New
and
incorporation
precipitable
and ---
for
of
reverse-phase
HPLC
Cl8
column
Secretion
(5
culture
medium
already
reported
PEo__teln
370
as
analysis
cm,
Merck
were
ine
low
(294
Berlin,
D)
into
acid
incubation,
soluble
was
material
uptake
and
pool
was
for
using
the
isocratic with
chromatography) FRG)
proteins
at
protein
West
acid
Darmstadt,
to
(10).
evaluated
was
speed
Degradation
gf mono
which
remove
in
cell
the
debris
as
had
been
labelled 0.15
dose,
proteins
for
pCi/ml,
was
20
hours
294
mCi/mmol)
at
(9). nucleotide --_------.were
I ayers frozen were
long-lived
of
(tracer
adenLne
immediately
(11).
Media
[“Cl-valine
of
liquid
synthesized
described
concentrations
and
precursor
C”‘vCl-valine val
centrifuged
Determination ---_.._-- -----
and
25
C3”Clvaline
te
removed
evaluate mM
led
The
(high-performance
pm,
10
hours
(9). of
monolayers
previously
hepatocy
37oc
(9).
in
C with
added.
Products,
two
elsewere
d!Xl~S!S~i~E:
measured
To
label
the
after
newly
of
was
was
at
USA)
medium
ethanol
with
intracellular
determination
a
mM
the
Research
of
evaluation
the
min
labelled
Nuclear
England
described
as
90
humidified
a
OH,
5Cr_UgLOJ:
material,
measured
Marietta,
100
were
mCi/mmo.l,
to
thereafter.
synthesis,hepatocytes
used
After
without
1988
carefully
3163,
air.
~Y!n!i!?!S?i~
the
Vol. 12, No. 8, August
Scientific
with
every
Reports,
quantified
lEw%w~~:
trinsed in
three 1 iquid in
the
times
3 with
nitrogen. acid
day-cultured cold
saline
Nucleotide extract
by
HPLC
Cell Biology
650
International
Reports,
Vol. 12, No. 8, August
1988
.................... ............ ............. ................... ..........................................
..................
,.................,
.
.. . ..
In ..
..,................
............ .......
.
......
days In culture
Figure
1.
secret
Effects
ion
culture
rate
(ml
ethanol in
on
protein
hepatocyte
synthesis
CD)
and
as
a function
of
monolayers
time.
Cells
were
extracel
labelled
lular
Materials
and from
absence
of
Protein
secretion
expressed
with
10
mM C*‘+Cl-valine.
were
radioactivity
values
plus
of
Methods. contra
ethanol)
as
extracellular).
Protein 1
assumed refers
% of
cells
total
evaluated
synthesis (cultured as
to
Intracellular as is for
same
as
under % of
the
in
the
time
100.
extracellular
insoluble
described
expressed the
and
insoluble radioactivity
radioactivity (intracellular
Ceil Biology
Enzrme
International
Na-K
gsHYs_:
& Barting
(12).
Cathepsin
B
previously
described
and
5
for
at
37O C for
as
was
AND DISCUSSION
decreased
hepatocytes,
as
in
cells, was
not
employed corresponds
resulted
protein proteins the
drug.
of
with
reduced
figure
1,
control
the
the
and
containing
did
Hartree
uptake
was
in
in
also
shows
valine of
precursor
incorporation synthesis
rate.
monolayers
to
ethanol
of
the
of
protein
affect
the
to
22% of in
the the
a
by ethanol-
of
not
as
Such
intracellular
protein
the
ethanol-treated
C'-Cl-valine
the
in
table
concentration in
as well
The
uptake
high
medium
culture. uptake
1.
the
culture
C1‘+Cl-valine in
table
(14).
the
valine
exposure
amounted cells
saline
AIB
to
time
inhibition
ethanol which
the
of
decrease
a progressive
the
as
"oEel2Yers:
solution
of
valine
reduction
an actual
to
addition
in
the
secretion
extracts
with
AIB.
a reduced
to
Hence,
salt
incorporation
due
However,
in
ethanol
modified
in
Bakkeren
(13).
function
the
Earle's
mM unlabelled
radioactivity
in
secretion.
twice
specific
to
depicted
rinsed
as reported
of
(15).
cell
were
according
the
associated
spite the
pool
a
hepatocytes
that
in
kP~tC?LYt~~
in
1.5
that
progressively
cultured
assayed
to
BY
determined
1 demonstrates
was
according
uetake
elsewhere
RESULTS
AS
30 min
indicated
content
decrease
were
days
and
Protein
Figure
evaluated
LEG!1
three
fll'"l C1‘+Cl-labelled
evaluated
was
1988
(9). SEid
cultured
incubated
ATPase
Vol. 12, No. 8, August
D activities
waziQgisgQgtyrr& Cells
Reports,
rate percent newly ones
rate
of
synthesized treated
with
1.
Effect
of
ethanol
on
C *‘Cl-valine uptake
and
cpm
x
IO-“/mg
C”+Cl-valine
protein
uptake
cpm
x
C*%Zl-valine 10-9/mg
were
are
pool
Data
were
the
means
evaluated
radioactivity
The
Hepatocytes
as
soluble
with
of
10
triplicate
described
acid
2 SE
of
labelled
0.18
f
4.575
Ethano
1
+- 0.23
6.235
Control
under
and
plates
Material
insoluble
mM C”‘+Cl-valine
obtained
and
two
from
Methods.
material
for
14.17
24.04
--~----~----------~-------------~----~~~------~~---~~~~~~-~~~~~~~~~~~~~~-~~~-~~~~-~~~~~~~~~~~--~~~~~~~---
condition
Culture
0.91
two
and
hours.
by
specific
separate
the
+ 0.59
f
protein
incorporation
incorporation
----____-~---_-__-_-~-~-------~~~~~~~~-------~~~~~~~~---~~~--~~--~-----~---~--------~---~~---~~~~~~~~~~~~
Table
cpm
experiments.
radioactivity
hepatocytes
x
(S.A.)
11.58
for
mole
pool
12.05
10-=/F
Valine
cultured
of
valine
valine
S.6.
3 days.
*
P < 0.05
(StuaeritiB
The
represent
results
the
as
of
of
was
t
tssff.
for
were
mean
of
of
of
as
triplicate
triplicate
units/mg
assayed
experiment
enzyme
the
incubation
measured
days,
3.2
3.4
sampl
sampl
cells
DMEM
es
es
by
37O
for
obtained
100.
varied
cultured
as
at
val
less
-.
or
at than
of
8%.
least
as
value
of
1.15
lntracel
lular
separate
elsewere
labeiled
1 1 ‘, e r
mM
ir ij.19
D
- -.-.---
protein)
---
-c 0.24
100
ts/m.;
Cathebsiq
0.98
with
(AnI
.-. .-~
nmo 1 / ~mq
three
desrrlbed
753
_ .-.
supplemented
ir n.020
-fr 0.015
aroteinj
intracellular
days
The
from
content
0.333
G.043
ts/mg
5
days
~. .._.~_
three
::Jthepsiv
_.. .--.
for
from
three
C.
ine
unsuopiempnted
t uni
..____ -_.
~1.~1 tu:-ed
C"+C3-valine
cells
of
fractional
assumed
protein.
on
was
labelled
a
in
? 0.29
t
1 on
0.34
-..-.---.__---_---
hepatocytes
degradat
release
assuming
three
of
in ..______
metaboiism
-_---
turnover
% rate
calculated
was
cultured
radioactivity
expressed
The
+ SD.
start
hours
D activities
is
the
two
Activity
experiments
rate
degradation
(9).
and
Cathepsin
El
at
during
protein
(15).
synthesis
radioactivity
proteins
% rate
protein
protein
ethanol.
were
+ 0.020*
1.9
Protein
_______
protein
Ethano
of
-_---
on
synthesis
____
ethanol
k 0.029
monolayers
-_------
of
3.1
1
-___
Effect
Control
Hepatocyte
%
2.
Condition
------_---
Table
Cell Biology
To
evaluate
turnover, which in
the we
effects
no
Moreover,
lysosomal
enzymes
detected.
It
hepatocytes
were
control
cells
two
are
the
culture
the
since
protein Taken
act5
the
.
It
cultured
od.
In
the
protein
in
of of
postulated a
these
the
that
the
of
of
main
of
of
the
at
at
inhibits
ethanol present
ic
hepat
supply ATP
the
balance
effect
remains
affect
the
proteins.
which
energy
loss
rate
by
ethanol
should
in
be predominantly
machinery
the
ethanol
not
loss
mechanism
limitation
ethanol
that
to
were
the
in
protein
exposed
progressive
the
Earle's
synthesis
exceeded
to
be
with
modified
detectable
that
of
could
indicating
cells
a
level
experiments
protein
any
was
supplemented
rate
seems
has
case,
that
indicate
synthesis
the
D
turnover
However,
through
cathepsin
medium
the
in
protein been
in
degradation
results
level.
synthesis were
without
metabolism
protein
on
ante,
protein
our
B and
rate
reported
degradation total
degradation
resulting
synthesis
1 ear
this
(161.
If
level
in
hepatocytes.
verify
this
concentrations that
of
rate
on
protein
To
govern
together,
protein
ba per
synthesis,
ethanol
uric
in
which
processes
the
equalled
proteins,
the
DMEM
the
lived As
protein
aminoacids
cond i tions
these
protein
hepatocytes.
of
1988
overall
long
changes
in
essential
non
rate
stressed
cultured
processes
during
the
as cathepsin be
the of
in
appreciable
such must
and
In
no
on
proteins
on
Vol. 12, No. 8, August
degradation
of
effect
observed.
medium.
bulk
Reports,
ethanol
the
the 2
essential
of
investigated
represent
table
international
in
hypothesis in
ethanol
cultured treated
we measured cells hepatocytes
the
(table the
31. levels
adenine Our
of
nucleotide show
results ATP
and
the
Cell Biology
international
Reports,
Vol. 72, No. 8, August
I I I
I I I I I I I I I I I I I I I I I I I I I I ! I I I
I I I I I I i
I I I I I I I I I I I I I I
1988
655
Cell Biology
International
I I I I I I I
I I I
I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I I
Reports,
Vol. 12, No. 8, August
1988
Cell Biology
ATP/@DP agree
International
were
ratio
with
those
chronic
and
decrease At.
ATP
is
to
not
greatly
to
in
acute
also
to
4
table
Since of
treated
in
of (-42%)
agreement
energy
associated
supply
is
the
processes,
hepatocyte
reduction
hepatocytes
and
hepatocytes,
of
The
uptake
uptake
cell
cultured
of
addition
are
the
alteration(s) drug.
AIB
AIB
results
membrane
the
as the
both
These
effect
the
in
(17).
structural
ethanol
investigate such
treatment
to
data
whereas
reported
processes
(-40%).
exposure
(171,
been
impaired
energy-dependent
be due upon
to
shown
ethanol
These
(18-21).
reported
by
by Rubin have
worthwhile
activity
of
uptake
liver
AS
previously
impairment
membrane
was
monolayers
modified
could
in
657
1988
values.
control
administration
energy-requiring
Na-K-ATPase those
the
reported
activity.
ethanol
with
to
level it
on two
Na-K-ATPase
and
similar
previously
point
ethanol
Vol. 12, No. 8, August
ethanol
the
this
Repotis,
of
plasma the
line
in
valine
with
this
hypothesis. In
conclusion,
the
our
results
medium
culture
impairs
both
the
rate
of
The
depressing
could
in
of
protein protein
that
and
ethanol
monolayers
hepatocyte
synthesis
secretion
addition
to
significantly without
affecting
breakdown.
effect part
demonstrate
account
of
ethanol for
the
on aminoacid decreased
uptake
efficiency
by
the
of
cells
protein
synthesis. The
inhibitory
processes Na-K
&TPase
FITP levels.
effect associated does
of to
not
plasma
appear
on
other
membrane
such
ethanol
directly
correlated
energy-dependent as AIB with
uptake changes
and in
Cell Biology
658
International
Reports,
Vol. 72, No. 8, August
1988
ACKNOWLEDGEMENTS
Marchis
Dr
M.
de
of
adenine
We
also
the
preparation
is
gratefully
nucleotide thank
valine
and
Professor of
acknowledged
M.
for
his
determination
levels.
Orunesu
for
helpful
during
comments
manuscript.
the
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of
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Voci
rat
Voci
A.
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primary
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of
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