Vol. 73, No. 2, 1976
BIOCHEMICAL
DIVALENT
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CATION IONOPHORE A.23187:
A POTENT PROTEIN SYNTHESIS INHIBITOR
Jane
E. Bottenstein
and Jean de Vellis
Neuroscience Department Brain Research Institute and Department of Anatomy Mental Retardation Center and The Laboratory of Nuclear Medicine and Radiation Biology University of California Los Angeles, California 90024
Received
September
22,1976
SUMMARY: Divalent cation ionophore A23187 has a potent inhibitory effect on protein synthesis in the C6 rat glioma cell line. Treatment with 4 pM A23187 resulted in 93% inhibition of [l-14C]leucine label incorporation into proteins and a 61% increase in free pool labeling. Total RNA synthesis was not affected. Extracellular ionic calcium or magnesium are not required for these changes to occur. Therefore, these effects of A23187 may be a direct effect on protein synthesis or may result from release of internal stores By comparison, ionophore X537A (4 uM) has only a of divalent cations. slight inhibitory effect on protein synthesis. INTRODUCTION A23187 that
acts
is
as a freely
not monovalent bulk
phase
the alkali
Copyright All rights
is
in addition
processes
(1).
metal
utilized (4).
also
acid
antibiotic
carrier
Two molecules
plasma
and/or
cations to study
of the
mitochondrial weight
Both
of calcium
486
bind
for
a
a single
(2). acid
cation cation
X537A antibiotic,
has equal
affinity
compounds
have been
on various
a potent
but
into
can equilibrate
membranes
cations (3).
evidence
or magnesium,
monocarboxylic
divalent
the influence
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
that
weight
at pH 7.4
ionophore
complex
and H+ as well
We now present
calcium
medium buffered
soluble
a low molecular to transporting
of low molecular
to transport
an aqueous
lipid
across
(Lasalocid)
widely
from
an electroneutral
concentrations
which
mobile
cations,
organic
to form
a monocarboxylic
protein
cellular synthesis
for
Vol. 73, No. 2, 1976
inhibition A23187
BIOCHEMICAL
by A23187 as a tool
involving
to study
protein
levels
has retained
also
We found
of glycerol
or Mg*
in cellular
processes
e.g.,
enzyme
induction
and cell
differentiation.
the role induction
24 hr exposure
effect
for
of induced
nervous
(EC 1.1.1.8) of glycerol
in
glioma
cell Maximal
3 UM norepinephrine completely
blocked
To ascertain
dehydrogenase mediated
(EC 1.1.1.27)
tissue.
24 hr after
lactate
on the non-c-AMP
levels
a rat
dehydrogenase.
the
dehydrogenase
(5),
to 4 pM A23187
of lactate
of
in the adenosine
of normal attained
the usage
dehydrogenase
of C6 cells
are
was specific its
of calcium of lactate
many properties
that
phosphate
An 89% reduction
of Ca*
induction
effect
determined
the role
dehydrogenase
the norepinephrine the A23187
preclude
in the 2B subclone
of lactate
treatment.
findings
mediated
by norepinephrine
RESEARCH COMMUNICATIONS
These
investigating
3',5'-monophosphate
which
X537A.
synthesis,
We have been
line
and not
AND BIOPHYSICAL
whether
induction,
hydrocortisone the same cell
phosphate
we induction
line
dehydrogenase
(6,7). was
found. MATERIALS
AND METHODS
Cell Culture: In all the experiments described in this paper confluent monolayer cultures of C6 cells (2B subclone) were used of passage 1112-18. Cells grown in Ham's F-10 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Reheis, Chicago, IL) without antibiotics were used at 9-11 days of culture. Serum-free medium was employed during experiments. Chemicals: L-norepinephrine bitartrate was purchased from Sigma Chemical Company. Hydrocortisone, A grade, was obtained from Calbiochem. l[l-14Cl leucine and [Z-14Cluridine were purchased from Amersham/Searle. A23187 was the gift of Eli Lilly Company and X537A was the gift of Hoffman La Roche. Stock solutions of ionophores were made up in 5% acetone and 95% ethanol (100%) Assays: Cyclic AMP was assayed at 20 min with a competitive proteinbinding assay (8) with the charcoal modification of Brown --et al. (9) and lactate dehydrogenase or glycerol phosphate dehydrogenase at 24 hours in another set of cultures using a spectrophotometric method at 340 nm wavelength (10). [14C]leucine (0.5 Ci/3 ml) incorporation into protein: 5% trichloroarctic acid (0 "C) was used to precipitate the protein. Samples were then centrifuged (0 "C) and the pellet was washed 3 times with 5% trichloroacetic acid and once with 95% ethanol (0 "C) with recentrifugation each time. The final pellet was hydrolyzed in 0.5 N NaOH for one hour at 37 'C and then counted as was the initial supernatant on a liquid scintillation counter. Abbreviations:
c-AMP
adenosine
3'5'-monophosphate.
487
Vol. 73, No. 2,1976
BIOCHEMICAL
Table
1
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effects of A23187 or LaC13 on induced of LDH, c-AMP and GPDH in C6 cells
levels
Enzyme Activity Treatments Experiment
(units/mg
protein)
c-AMP % Control
1 (LDH) loo+1
Control
1661k24
4 uM A23187
1501+51
15 nM NE
2815+85a
169t5
A23187
1582+94b
95k6
Control
2333+18
100+1
10 @I LaC13
2303A33
9953
3 pM NE
3804c
164
LaC13 + NE
38702324
167+14
Control
75+15
100~20
4 uM A23187
98k7
131k9
346+12a
461?16
104+4d
139*5
Experiment
Experiment
% NE
+ NE
90+3 100+4 41_+2
2 (LDH)
100+7 59k6
3 (GPDH)
0.55 A23187 Abbreviations: dehydrogenase;
pM HC + HC
LDH, lactate dehydrogenase: GPDH, glycerol NE, norepinephrine; HC, hydrocortisone.
One unit of enzyme is conversion of 1 nanomole treatment are the means relative to control, bp< relative to control, and
phosphate
defined as that amount which catalyzes the of substrate/minute at 30 'C. Data for each i: s.d. from 3 samples. Significance: ap< 0.001 0.001 relative to norepinephrine alone, cp< 0.01 dp < 0.001 relative to hydrocortisone alone.
[14C]uridine (0.5 nCi/3 ml) incorporation into RNA: Harvest procedure was as in the leucine incorporation except that the hydrolyzed pellet was treated at 0 'C with 60% perchloric acid to precipitate DNA and protein; and the supernatant, containing the RNA, was read on a Gilford spectrophotometer (UV absorbance at 260 nm wavelength) and was corrected for protein contamination. An aliquot of the supernatant was counted on a liquid scintillation counter. Protein serum albumin
was determined as a standard.
by the method
488
of Lowry --et al.
(11)
using
bovine
Vol. 73, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
Izo Cl
Cl 0
0
CCNTROL
FREE POOL TCA PRECIPITABLE
FREE RNA
A.23187
NE
RESEARCH COMMUNICATIONS
POOL
A23187 +NE
ETHANOL
Figure 1. Effect of 4 pM A23187 and/or 3 UM norepinephrine (NE) on the incorporation of [14C]leucine or [ l4Cluridine into protein or RNA respectively. Samples were pretreated 1.5 hours (-NE) as indicated: NE was then added where indicated for 21 hours with a radioactive pulse for the last hour. a) Data for each treatment are the means ? S.D. of 3 samples. Significance: All treatments except NE or ethanol, p< 0.001 relative to control, and NE + A23187 relative to A23187 alone, p< 0.001. b) Data for each treatment are the means + s.d. of 4 samples. Significance: All treatments, p< 0.001 relative to control except for (1) the RNA fraction of the NE + A23187 treatment, p< 0.005 relative to control or NE and (2) the free pool fraction of the A23187 treatment, p< 0.005 relative to NE.
RESULTS AND DISCUSSION Table
1, experiment
no effect
on basal
norepinephrine significant
levels
induction suppression
1 shows
that
24 hr treatment
of lactate of this
dehydrogenase
enzyme.
of norepinephrine
ethylene-glycol-bis-(aminoethyl)tetraacetate inhibition
(52%)
dehydrogenase chloride
inhibited
(data
norepinephrine
effect
induced
without not
was not
c-AMP levels,
reversing
reverse the
shown).
Additionally,
induced
c-AMP levels
489
4 pM A23187
but completely
could
of c-AMP levels
induction
This
with
the
inhibition
has
blocks
the
due to a since
0.5 mM
ionophore of lactate
10 ~.IM lanthanum with
no change
in the
Vol. 73, No. 2,1976
induced with
BIOCHEMICAL
levels
A23187
of this
of lactate
Since
(Table
both
suppression on the
dehydrogenase
and hydrocortisone
enzyme
1, experiment
of protein of
Initial
inhibited,
labeling
(Figure
la).
of TCA insoluble drop
in uridine
free
pool
incorporation
labeling
treatment
also
labeling.
with
Thus,
RNA formation,
the but
pulse
pool
simply into
rather
the influence into
levels
the
hour,
last
reducing
was inhibiting
indicated
in
pool
the depression
lb).
A 40%
levels.
by a 40% drop
in the
Norepinephrine
(60%)
of free
leucine
protein
an 82%
the free
precursor
(Figure decrease
and RNA to A23187
was increased,
RNA was paralleled
was not
protein
of exposure
count
treatment
of A23187
20 hours
due to reduced
in a significant ionophore
of induced
and a 70% increase
the free
ionophore
resulted
examined
done with
counts
was not
Treatment
2).
a non-specific
and [ l4C]uridine
a radioactive
Since
counts
suggesting
we then
of TCA precipitable
1, experiment
in an 89% reduction
[14C]leucine
with
RESEARCH COMMUNICATIONS
3).
experiments
and norepinephrine, depression
were synthesis,
incorporation
(Table
results
inductions
respectively.
AND BIOPHYSICAL
pool
transport
synthesis
and RNA
nor
total
at some subsequent
point. These and thus mine
initial a time
effects
earliest for
increase
course
at earlier
time
pulse
experiments
point:
one hour. in free
pool
again
seen at one hour
small
decrease
pool
labeling.
changes
(12%) Longer
in labeling.
crenated,
Abbreviations:
very
granular
involved
experiment
long
was done
times.
Maximal
exposure (Table
times
2, experiment
inhibition
ionophore
treatment
In a separate
experiment
(Table
and decrease
exposure in protein exposure
to A23187, counts
When exposed and there
TCA, trichloroacetic
while
490
[14C]leucine
for
2), counts
4 uM X537A
increase
loss;
the
fraction
to 4 uM X537A
was some cell
1) to deter-
2, experiment
with
to 20 uM X537A
acid.
with
in protein
and a small
(21 hours)
ionophore
(93%) was seen at the
simultaneous
labeling
to the
there
(11%) reveals
one hour so that
the is is a
in a free no significant
the these
cells
were
decreases
2:
Experiment
4
21
X537A
X537A
X537A
A23187
A23187
A23187
A23187
A23187
-----~-----
0
21
-..-.-
20
4
21
1
0
21
4
4
14
1
0
14
4
4
4
1
0
4
0
4
1
1
0
1
Protein
times
-~.---.------.__--___
141.95k8.82
147.98?6.77
3.36+0.28s
37.60+4.@
3.99-co.53a
48.23k5.31
30.96f2.05a
111.09+8.62
25.80+1.19s
89.alk7.00
6.83+0.95s
44.9021.67
2.59+0.3aa
35.65k2.16
to ionophore
last
96
100
7
78
a
100
28
100
29
100
15
100
7
100
(mean)
% Control
pool (x10-3)
into
98
100
22
111
152
100
174
100
153
100
140
100
143
100
(mean)
% Control
protein
Data for each treatment to bP < 0.025 relative
26.35k5.83
26.91k12.13
1.61rf0.45s
8.o4+o.43b
10.97+0.41a
7.2420.33
27.12+1.26s
15.5akl.18
25.48+1.14s
16.691t0.75
16.65+0.07a
11.87ko.51
16.02?0.96=
11.2410.65
cpm/mg protein
Free
incorporation
(37 "C). to control;
on [ 14Clleucine
hour of incubation ep< 0.001 relative
(x10-3)
fraction
cpm/mg protein
exposure
[14C]leucine (0.5 pCi/3 ml) was added for the are the means ? s.d. of 4 samples. Significance: control.
-____-
1:
Experiment
(hrs)
(PM)
of various
time
Effect
Ionophore
2
Incubation
Table
Vol. 73, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0 FREE PCKIL 0 TCA PRECIPITABLE
I
01
2345
IO A23187 OJM)
15
20
Figure 2. Dose-dependence of A23187 effect on [14C]leucine incorporation into protein. A23187 at the indicated concentrations was added simultaneously with [14C]leucine (0.5 uCi/3 ml) for a one hour incubation (37 "C). Data for each treatment are the means t s.d. of 4 samples. Where the standard deviation is not shown, it is smaller than the size of the symbol. Significance: All treatments p< 0.001 relative to control except 20 uM A23187 (freepool) with p< 0.005 relative to control.
in counts
are
20 uM X537A
probably
toxic
resulted
in complete
A dose response (92%)
maximal
(88%)
(61%)
of free
2) indicated
into
at only pool
since
20 hour
exposure
to
death.
(Figure
incorporation
inhibition increase
particularly
cell
determination
of [14C]leucine
substantial
effects,
protein
maximal
with
4 PM A23187,
0.8 uM A23187. labeling
Figure
at 0.8
inhibition
2 also
uM A23187,
with shows
the
plateauing
thereafter. Using calcium that
4 JJM A23187
or magnesium
of complete
simultaneously free
medium
cellular
ionic
divalent
cations
cannot
which
can also
of X537A,
calcium
In conclusion, protein
synthesis
effect
on protein
divalent
cations.
medium (data
with results
not
A23187
out,
equilibrate is
in label
shown),
or magnesium. be ruled
but
now shown
at very
synthesis
or may result the
ions
for
across
the first
low concentrations. from release
ionophore
492
pulse
apparently
in
identical
no requirement
of mitochondrial
is weakened
these
[14C]leucine
distribution
suggesting
Release
inhibitor
In addition,
a one hour
for
stores
by the minimal
to extraof effect
membranes. time
to be a potent
This
may be a direct
of internal increases
stores leucine
of
BIOCHEMICAL
Vol. 73, No. 2, 1976
and decreases mental
uridine
conditions
attributable cations
transport
into
intact
cells,
using
to increased as previously
permeability
AND BIOPHYSICAL
the cells. the
effects
of plasma
RESEARCH COMMUNICATIONS
Therefore, of A23187 membranes
in some experimay not be solely to divalent
claimed. ACKNOWLEDGEMENTS
This We thank Diane Inglish for expert technical assistance. supported by ERDA Contract E(O4-l)GEN-12 and USPHS Grant HD-05615. 3. E. Bottenstein is an NIMH-MHTP predoctoral trainee (5TOlMH06415).
work
was
REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11.
H.A. (1970) J. Biol. Chem., 247, 6970-6977. Reed, P.W., and Lardy, Case, G.D., Vanderkooi, J.M., and Scarpa, A. (1974) Arch. Biochem. Biophysics, 162, 174-185. Pressman, B. (1973) Fedn. Proc., 32 (6), 1698-1703. McLaughlin, S., and Eisenberg, M. (1975) Ann. Rev. Biophysics and Engineering, 4, 350-353. G. (1973) in Tissue Culture of the Nervous de Vellis, J., and Brooker, System (Sato, G. ed.), pp. 231-245, Plenum, New York. de Vellis, J., and Inglish, D. (1973) Prog. Brain Res., 40, 321-330. McGinnis, J.F., and de Vellis, J. (1974) Nature, 250, 422-424. Gilman, A. (1970) Proc. Natn. Acad. Sci. U.S.A., 67, 303-312. Brown, B.L., Albano, J.D., Ekins, R.P., and Sgherzi, A.M. (1971) Biochem. J ., 121, 561-562. de Vellis, J., Schjeide, O.A., and Clemente, C.D. (1967) .I. Neurochem., 14, 499-511. Lowry, O., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275.
493
BIOCHEMICAL
DIVALENT
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CATION IONOPHORE A.23187:
A POTENT PROTEIN SYNTHESIS INHIBITOR
Jane
E. Bottenstein
and Jean de Vellis
Neuroscience Department Brain Research Institute and Department of Anatomy Mental Retardation Center and The Laboratory of Nuclear Medicine and Radiation Biology University of California Los Angeles, California 90024
Received
September
22,1976
SUMMARY: Divalent cation ionophore A23187 has a potent inhibitory effect on protein synthesis in the C6 rat glioma cell line. Treatment with 4 pM A23187 resulted in 93% inhibition of [l-14C]leucine label incorporation into proteins and a 61% increase in free pool labeling. Total RNA synthesis was not affected. Extracellular ionic calcium or magnesium are not required for these changes to occur. Therefore, these effects of A23187 may be a direct effect on protein synthesis or may result from release of internal stores By comparison, ionophore X537A (4 uM) has only a of divalent cations. slight inhibitory effect on protein synthesis. INTRODUCTION A23187 that
acts
is
as a freely
not monovalent bulk
phase
the alkali
Copyright All rights
is
in addition
processes
(1).
metal
utilized (4).
also
acid
antibiotic
carrier
Two molecules
plasma
and/or
cations to study
of the
mitochondrial weight
Both
of calcium
486
bind
for
a
a single
(2). acid
cation cation
X537A antibiotic,
has equal
affinity
compounds
have been
on various
a potent
but
into
can equilibrate
membranes
cations (3).
evidence
or magnesium,
monocarboxylic
divalent
the influence
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
that
weight
at pH 7.4
ionophore
complex
and H+ as well
We now present
calcium
medium buffered
soluble
a low molecular to transporting
of low molecular
to transport
an aqueous
lipid
across
(Lasalocid)
widely
from
an electroneutral
concentrations
which
mobile
cations,
organic
to form
a monocarboxylic
protein
cellular synthesis
for
Vol. 73, No. 2, 1976
inhibition A23187
BIOCHEMICAL
by A23187 as a tool
involving
to study
protein
levels
has retained
also
We found
of glycerol
or Mg*
in cellular
processes
e.g.,
enzyme
induction
and cell
differentiation.
the role induction
24 hr exposure
effect
for
of induced
nervous
(EC 1.1.1.8) of glycerol
in
glioma
cell Maximal
3 UM norepinephrine completely
blocked
To ascertain
dehydrogenase mediated
(EC 1.1.1.27)
tissue.
24 hr after
lactate
on the non-c-AMP
levels
a rat
dehydrogenase.
the
dehydrogenase
(5),
to 4 pM A23187
of lactate
of
in the adenosine
of normal attained
the usage
dehydrogenase
of C6 cells
are
was specific its
of calcium of lactate
many properties
that
phosphate
An 89% reduction
of Ca*
induction
effect
determined
the role
dehydrogenase
the norepinephrine the A23187
preclude
in the 2B subclone
of lactate
treatment.
findings
mediated
by norepinephrine
RESEARCH COMMUNICATIONS
These
investigating
3',5'-monophosphate
which
X537A.
synthesis,
We have been
line
and not
AND BIOPHYSICAL
whether
induction,
hydrocortisone the same cell
phosphate
we induction
line
dehydrogenase
(6,7). was
found. MATERIALS
AND METHODS
Cell Culture: In all the experiments described in this paper confluent monolayer cultures of C6 cells (2B subclone) were used of passage 1112-18. Cells grown in Ham's F-10 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Reheis, Chicago, IL) without antibiotics were used at 9-11 days of culture. Serum-free medium was employed during experiments. Chemicals: L-norepinephrine bitartrate was purchased from Sigma Chemical Company. Hydrocortisone, A grade, was obtained from Calbiochem. l[l-14Cl leucine and [Z-14Cluridine were purchased from Amersham/Searle. A23187 was the gift of Eli Lilly Company and X537A was the gift of Hoffman La Roche. Stock solutions of ionophores were made up in 5% acetone and 95% ethanol (100%) Assays: Cyclic AMP was assayed at 20 min with a competitive proteinbinding assay (8) with the charcoal modification of Brown --et al. (9) and lactate dehydrogenase or glycerol phosphate dehydrogenase at 24 hours in another set of cultures using a spectrophotometric method at 340 nm wavelength (10). [14C]leucine (0.5 Ci/3 ml) incorporation into protein: 5% trichloroarctic acid (0 "C) was used to precipitate the protein. Samples were then centrifuged (0 "C) and the pellet was washed 3 times with 5% trichloroacetic acid and once with 95% ethanol (0 "C) with recentrifugation each time. The final pellet was hydrolyzed in 0.5 N NaOH for one hour at 37 'C and then counted as was the initial supernatant on a liquid scintillation counter. Abbreviations:
c-AMP
adenosine
3'5'-monophosphate.
487
Vol. 73, No. 2,1976
BIOCHEMICAL
Table
1
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effects of A23187 or LaC13 on induced of LDH, c-AMP and GPDH in C6 cells
levels
Enzyme Activity Treatments Experiment
(units/mg
protein)
c-AMP % Control
1 (LDH) loo+1
Control
1661k24
4 uM A23187
1501+51
15 nM NE
2815+85a
169t5
A23187
1582+94b
95k6
Control
2333+18
100+1
10 @I LaC13
2303A33
9953
3 pM NE
3804c
164
LaC13 + NE
38702324
167+14
Control
75+15
100~20
4 uM A23187
98k7
131k9
346+12a
461?16
104+4d
139*5
Experiment
Experiment
% NE
+ NE
90+3 100+4 41_+2
2 (LDH)
100+7 59k6
3 (GPDH)
0.55 A23187 Abbreviations: dehydrogenase;
pM HC + HC
LDH, lactate dehydrogenase: GPDH, glycerol NE, norepinephrine; HC, hydrocortisone.
One unit of enzyme is conversion of 1 nanomole treatment are the means relative to control, bp< relative to control, and
phosphate
defined as that amount which catalyzes the of substrate/minute at 30 'C. Data for each i: s.d. from 3 samples. Significance: ap< 0.001 0.001 relative to norepinephrine alone, cp< 0.01 dp < 0.001 relative to hydrocortisone alone.
[14C]uridine (0.5 nCi/3 ml) incorporation into RNA: Harvest procedure was as in the leucine incorporation except that the hydrolyzed pellet was treated at 0 'C with 60% perchloric acid to precipitate DNA and protein; and the supernatant, containing the RNA, was read on a Gilford spectrophotometer (UV absorbance at 260 nm wavelength) and was corrected for protein contamination. An aliquot of the supernatant was counted on a liquid scintillation counter. Protein serum albumin
was determined as a standard.
by the method
488
of Lowry --et al.
(11)
using
bovine
Vol. 73, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
Izo Cl
Cl 0
0
CCNTROL
FREE POOL TCA PRECIPITABLE
FREE RNA
A.23187
NE
RESEARCH COMMUNICATIONS
POOL
A23187 +NE
ETHANOL
Figure 1. Effect of 4 pM A23187 and/or 3 UM norepinephrine (NE) on the incorporation of [14C]leucine or [ l4Cluridine into protein or RNA respectively. Samples were pretreated 1.5 hours (-NE) as indicated: NE was then added where indicated for 21 hours with a radioactive pulse for the last hour. a) Data for each treatment are the means ? S.D. of 3 samples. Significance: All treatments except NE or ethanol, p< 0.001 relative to control, and NE + A23187 relative to A23187 alone, p< 0.001. b) Data for each treatment are the means + s.d. of 4 samples. Significance: All treatments, p< 0.001 relative to control except for (1) the RNA fraction of the NE + A23187 treatment, p< 0.005 relative to control or NE and (2) the free pool fraction of the A23187 treatment, p< 0.005 relative to NE.
RESULTS AND DISCUSSION Table
1, experiment
no effect
on basal
norepinephrine significant
levels
induction suppression
1 shows
that
24 hr treatment
of lactate of this
dehydrogenase
enzyme.
of norepinephrine
ethylene-glycol-bis-(aminoethyl)tetraacetate inhibition
(52%)
dehydrogenase chloride
inhibited
(data
norepinephrine
effect
induced
without not
was not
c-AMP levels,
reversing
reverse the
shown).
Additionally,
induced
c-AMP levels
489
4 pM A23187
but completely
could
of c-AMP levels
induction
This
with
the
inhibition
has
blocks
the
due to a since
0.5 mM
ionophore of lactate
10 ~.IM lanthanum with
no change
in the
Vol. 73, No. 2,1976
induced with
BIOCHEMICAL
levels
A23187
of this
of lactate
Since
(Table
both
suppression on the
dehydrogenase
and hydrocortisone
enzyme
1, experiment
of protein of
Initial
inhibited,
labeling
(Figure
la).
of TCA insoluble drop
in uridine
free
pool
incorporation
labeling
treatment
also
labeling.
with
Thus,
RNA formation,
the but
pulse
pool
simply into
rather
the influence into
levels
the
hour,
last
reducing
was inhibiting
indicated
in
pool
the depression
lb).
A 40%
levels.
by a 40% drop
in the
Norepinephrine
(60%)
of free
leucine
protein
an 82%
the free
precursor
(Figure decrease
and RNA to A23187
was increased,
RNA was paralleled
was not
protein
of exposure
count
treatment
of A23187
20 hours
due to reduced
in a significant ionophore
of induced
and a 70% increase
the free
ionophore
resulted
examined
done with
counts
was not
Treatment
2).
a non-specific
and [ l4C]uridine
a radioactive
Since
counts
suggesting
we then
of TCA precipitable
1, experiment
in an 89% reduction
[14C]leucine
with
RESEARCH COMMUNICATIONS
3).
experiments
and norepinephrine, depression
were synthesis,
incorporation
(Table
results
inductions
respectively.
AND BIOPHYSICAL
pool
transport
synthesis
and RNA
nor
total
at some subsequent
point. These and thus mine
initial a time
effects
earliest for
increase
course
at earlier
time
pulse
experiments
point:
one hour. in free
pool
again
seen at one hour
small
decrease
pool
labeling.
changes
(12%) Longer
in labeling.
crenated,
Abbreviations:
very
granular
involved
experiment
long
was done
times.
Maximal
exposure (Table
times
2, experiment
inhibition
ionophore
treatment
In a separate
experiment
(Table
and decrease
exposure in protein exposure
to A23187, counts
When exposed and there
TCA, trichloroacetic
while
490
[14C]leucine
for
2), counts
4 uM X537A
increase
loss;
the
fraction
to 4 uM X537A
was some cell
1) to deter-
2, experiment
with
to 20 uM X537A
acid.
with
in protein
and a small
(21 hours)
ionophore
(93%) was seen at the
simultaneous
labeling
to the
there
(11%) reveals
one hour so that
the is is a
in a free no significant
the these
cells
were
decreases
2:
Experiment
4
21
X537A
X537A
X537A
A23187
A23187
A23187
A23187
A23187
-----~-----
0
21
-..-.-
20
4
21
1
0
21
4
4
14
1
0
14
4
4
4
1
0
4
0
4
1
1
0
1
Protein
times
-~.---.------.__--___
141.95k8.82
147.98?6.77
3.36+0.28s
37.60+4.@
3.99-co.53a
48.23k5.31
30.96f2.05a
111.09+8.62
25.80+1.19s
89.alk7.00
6.83+0.95s
44.9021.67
2.59+0.3aa
35.65k2.16
to ionophore
last
96
100
7
78
a
100
28
100
29
100
15
100
7
100
(mean)
% Control
pool (x10-3)
into
98
100
22
111
152
100
174
100
153
100
140
100
143
100
(mean)
% Control
protein
Data for each treatment to bP < 0.025 relative
26.35k5.83
26.91k12.13
1.61rf0.45s
8.o4+o.43b
10.97+0.41a
7.2420.33
27.12+1.26s
15.5akl.18
25.48+1.14s
16.691t0.75
16.65+0.07a
11.87ko.51
16.02?0.96=
11.2410.65
cpm/mg protein
Free
incorporation
(37 "C). to control;
on [ 14Clleucine
hour of incubation ep< 0.001 relative
(x10-3)
fraction
cpm/mg protein
exposure
[14C]leucine (0.5 pCi/3 ml) was added for the are the means ? s.d. of 4 samples. Significance: control.
-____-
1:
Experiment
(hrs)
(PM)
of various
time
Effect
Ionophore
2
Incubation
Table
Vol. 73, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0 FREE PCKIL 0 TCA PRECIPITABLE
I
01
2345
IO A23187 OJM)
15
20
Figure 2. Dose-dependence of A23187 effect on [14C]leucine incorporation into protein. A23187 at the indicated concentrations was added simultaneously with [14C]leucine (0.5 uCi/3 ml) for a one hour incubation (37 "C). Data for each treatment are the means t s.d. of 4 samples. Where the standard deviation is not shown, it is smaller than the size of the symbol. Significance: All treatments p< 0.001 relative to control except 20 uM A23187 (freepool) with p< 0.005 relative to control.
in counts
are
20 uM X537A
probably
toxic
resulted
in complete
A dose response (92%)
maximal
(88%)
(61%)
of free
2) indicated
into
at only pool
since
20 hour
exposure
to
death.
(Figure
incorporation
inhibition increase
particularly
cell
determination
of [14C]leucine
substantial
effects,
protein
maximal
with
4 PM A23187,
0.8 uM A23187. labeling
Figure
at 0.8
inhibition
2 also
uM A23187,
with shows
the
plateauing
thereafter. Using calcium that
4 JJM A23187
or magnesium
of complete
simultaneously free
medium
cellular
ionic
divalent
cations
cannot
which
can also
of X537A,
calcium
In conclusion, protein
synthesis
effect
on protein
divalent
cations.
medium (data
with results
not
A23187
out,
equilibrate is
in label
shown),
or magnesium. be ruled
but
now shown
at very
synthesis
or may result the
ions
for
across
the first
low concentrations. from release
ionophore
492
pulse
apparently
in
identical
no requirement
of mitochondrial
is weakened
these
[14C]leucine
distribution
suggesting
Release
inhibitor
In addition,
a one hour
for
stores
by the minimal
to extraof effect
membranes. time
to be a potent
This
may be a direct
of internal increases
stores leucine
of
BIOCHEMICAL
Vol. 73, No. 2, 1976
and decreases mental
uridine
conditions
attributable cations
transport
into
intact
cells,
using
to increased as previously
permeability
AND BIOPHYSICAL
the cells. the
effects
of plasma
RESEARCH COMMUNICATIONS
Therefore, of A23187 membranes
in some experimay not be solely to divalent
claimed. ACKNOWLEDGEMENTS
This We thank Diane Inglish for expert technical assistance. supported by ERDA Contract E(O4-l)GEN-12 and USPHS Grant HD-05615. 3. E. Bottenstein is an NIMH-MHTP predoctoral trainee (5TOlMH06415).
work
was
REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11.
H.A. (1970) J. Biol. Chem., 247, 6970-6977. Reed, P.W., and Lardy, Case, G.D., Vanderkooi, J.M., and Scarpa, A. (1974) Arch. Biochem. Biophysics, 162, 174-185. Pressman, B. (1973) Fedn. Proc., 32 (6), 1698-1703. McLaughlin, S., and Eisenberg, M. (1975) Ann. Rev. Biophysics and Engineering, 4, 350-353. G. (1973) in Tissue Culture of the Nervous de Vellis, J., and Brooker, System (Sato, G. ed.), pp. 231-245, Plenum, New York. de Vellis, J., and Inglish, D. (1973) Prog. Brain Res., 40, 321-330. McGinnis, J.F., and de Vellis, J. (1974) Nature, 250, 422-424. Gilman, A. (1970) Proc. Natn. Acad. Sci. U.S.A., 67, 303-312. Brown, B.L., Albano, J.D., Ekins, R.P., and Sgherzi, A.M. (1971) Biochem. J ., 121, 561-562. de Vellis, J., Schjeide, O.A., and Clemente, C.D. (1967) .I. Neurochem., 14, 499-511. Lowry, O., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275.
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