- Email: [email protected]
Free choline formation by cerebral cortical slices from rat brain
Vol. 45, No. 6, 1971
FREE
BIOCHEMICAL
CHOLINE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FORMATION BY CEREBRAL FROM RAT BRAIN
Edward
CORTICAL
SLICES
T. Browning
Department of Pharmacology Rutgers Medical School New Brunswick, New Jersey 08903
Received
October
7, 1971 SUMMARY
Changes in free choline levels of cerebral cortical slices and their incubation Free choline was found fluid were examined under conditions of active ACh synthesis. to increase tenfold during two hours of incubation. Most of the free choline, which was presumably formed from esterified choline precursors, was found in the extracellular medium. INTRODUCTION The amount
of free choline
equal to the amount 1 hour depends
ultimately
experiment and their
appears
was designed incubation
during
not to synthesize stream
to examine
fluid
before
per gm (1,2) is approximately
by slices
choline
cortex
--de novo (4,.5); rather
to supply choline
the free choline
of cerebral
(6-9).
in
the brain
The present
level in cerebral
cortical
slices
and after ACh synthesis.
show that rather
2 hours
0.1 pmole
can be synthesized
upon the blood
The results tenfold
of ACh which
The brain
(3).
in rat brain,
than being consumed
the free choline
increased
of incubation. METHODS
Slices described After
of cerebral
elsewhere
incubation
at 0” C.
cortex (3).
and these samples
Incubation
the slices
Supernatants were
were
were
were
from
conditions
separated
decanted,
placed
prepared
are described
by centrifugation
their
in a boiling
male Wistar
pH adjusted water
1586
rats (200-300
gm) as
in the table legend. at 33,000
to 4.3 with
bath for 10 min,
x g for 10 min 1 M acetic
rechilled
and
acid,
Vol. 45, No. 6, 1971
centrifuged
BIOCHEMICAL
as above.
The slices
disrupted
by sonication
The slice
extracts
potassium
AND BIOPHYSICAL
were
and centrifuged;
were
perchlorate
neutralized
enzyme
assay which
pyruvate
kinase,
and lactate
cuvette
as choline
by adding
3 M K2C03
with 0.5 ml PCA.
and the resulting
AND ACh ASSAY extracts
is based on coupling
in an enzymic
was then washed
acid (PCA),
by centrifugation.
and media
oxidation
3 ml 6% perchloric
to pH 4.3 with
was removed
of the slices
with
the pellet
CHOLINE Choline
treated
RESEARCH COMMUNICATIONS
system which
were
choline
by a fluorometric
phosphorylation
comprises
dehydrogenase
assayed
choline
(2).
acetylcholinesterase
to NADH
phosphokinase
(CK),
ACh was assayed
in the same
after
of samples
the choline
had
reacted. That the material aliquots
of extracts
experiments whereas
assayed
were
showed
was choline
made alkaline
that choline
into the chloroform(2). of 14C-choline amount initial
Second,
amount
which
eluted
of “choline11
by two means.
in the aqueous
a sample
this treatment
for CK, migrated
was mixed
on a 1 x 100 cm Dowex-50
by the fluorometric
Control
phase after
substrate
of extract
with the 14C-choline
First,
with chloroform.
the next most active
and chromatographed
of choline
and extracted
remained
dimethylethanolamine,
was verified
with a minute column
peak was compared
amount
(2,lO).
The
with the
assay.
RESULTS The changes
in choline
with the ACh synthesis. obtained
earlier
The purpose the slices progressively
levels
The amounts
by measuring
of the present during
experiment
2 hours
system
of ACh formed
the transfer
accumulation
during
by the slice
is depicted
in 1 hour are similar
of isotopic
acetyl
was to examine
of ACh. of incubation.
The free choline In addition,
1587
in the table along to those
units to choline
the free choline content larger
reserves
of the slices amounts
(3). of rises
of choline
BIOCHEMICAL
Vol. 45, No. 6, 1971
are released
into the incubation
In order
to verify
choline,
three
extracted
with
The choline
extraction
medium
during
that the material
120 min samples
from
concentration
of extracts
extract
whereas
accumulating
substrates before
RESEARCH COMMUNICATIONS
incubation.
the incubation
All other
chloroform.
4.6 wmoles/ml
AND BIOPHYSICAL
in the system fluid
were
was,
made alkaline
for CK are uncharged
and
at this pH.
alkali:chloroform
treatment
concentration
in the aqueous
the choline
in fact,
was 63. 8 i phase after
was 63.3 + 5.0 m,umoles/ml.
One 129 min media Ninety-five
percent
the column
as defined
sample
of the choline
was chromatographed sample
by the authentic
Choline
on a column
was recovered
14C-choline
in the choline
and ACh Formation by Cortical of Rat Brain
Medium
Slices Choline
87 k
60 120
Medium cortex
Slices mpmoles/gm
11 -+ 4 (8)
0
peak from
elution.
ACh Incubation min
of Dowex-50.
9 A 2 (6)
47 *
Slices
6 (8)
86 zlz 7 (9)
9 (8)
11 i 4 (6)
395 i 30 (8)
124 i
7 (8)
82 * 18 (8)
9 * 1 (6)
810 f 54 (8)
147 f 11 (8)
200 mg of slices were incubated at 37” C in 3 ml of a solution which contained 94 mM NaCl, 29 mM KCl, 1 mM KH2P04, 25 mM NaHC03, 1 mM MgSo4, 2.5 mM CaC12, 2 mM glucose, 0.01 M eserine sulfate pH 7.4. The atmosphere was 95% 02-5% C02. Data represent the pooled results of 3 experiments, each of which showed the essential features of the pooled data. DISCUSSION Brain several
relies
on peripheral
investigators
the brain
(7-9).
and later
it is present
Greenberg
and/or
have demonstrated
Once in the brain
demonstrated
tissues
substantially
diet as a source
choline
choline
transfnr
is rapidly
in a chloroform
that the rate of methyl
1588
from
acetylated soluble
transfer
of choline. the blood
stream
into
or phosphorylated
form.
to form
Recently
Bremer choline
and phospholipids
Vol. 45, No. 6, 1971
from
BIOCHEMICAL
ethanolamine
phospholipids
Ansell
of liver.
AND BIOPHYSICAL
is very low in brain,
and Spanner
showed
labeled
was associated
with choline
phospholipids
(5).
the choline
phospholipids
(11).
labeled
interpreted derived
only the ethanolamine
as substantiating from
presented
in this
a net formation
that the free choline catabolism occur
in brain
little
or no 1%
These
14C-choline
observations
of brain
were
phospholipids
above,
is
are phosphorylcholine,
phosphatidyl
glycerophosphorylcholine, of these contribute
The esterified
forms
choline,
from
a net which
to the choline
Iysophosphatidyl
and choline
to free choline
is advanced
of choline
significantly
sphingomyelin,
significantly
results
certain
cortical
hypothesis
work
enough to contribute
that under
in cerebral
the provisional
in the present
of choline.
large
demonstrate
may be observed
observed
forms
in amounts
accumulation
Which
Alternately,
communication
of free choline
formation
of esterified
choline,
was injected
phospholipids;
the idea that choline
In view of the data discussed
slices.
which
the blood stream.
The results conditions
less than lo/O of the activity
that 14C-ethanolamine
intracerebrally
injection
RESEARCH COMMUNICATIONS
plasmalogen.
formation
is under
investigation. In view of the rapid phosphatidyl
choline,
that the choline phospholipid present
of labeled
choline
both --in vivo (11) and in -- vitro
formation
catabolism,
results
conversion
observed possibly
emphasize
in the present phosphatidyl
the catabolic
segment
(12),
to phospholipids, it is reasonable
experiments
choline
mainly
results
catabolism,
of a process
to suggest from
and that the
of phospholipid
turnover. That free choline the amounts choline
reported
transport
precautions
relating
can accumulate
herein
in a system
poses an important
and metabolism to changes
in brain
consideration
and possibly
in cholinespecific
1589
of cerebral
in other
radioactivity
cortex
slices
in
for studies
of isotopic
tissues.
Appropriate
need to be taken.
Vol. 45, No. 6, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENT This work was supported by U. S. Public Health Service grants: General Research Support Grant No. FR 05576 and Grant No. NB 08665. REFERENCES Pharmacologist, 12, 1. Haubrich, D. R., Krishna, G. , and Reid, W. D., The 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
313 (1970). Browning, E. T., Fed. Proc -. , 30, 271 (1971); complete manuscript submitted. Browning, E. T., andSchulman, M. P., 2. Neurochem., 3 1391 (1968). Bremer, J. , and Greenberg, D. M., Biochim. Biophys. Acta, 46, 205 (1961). Ansell, G. B., and Spanner, S., 2. Neurochem., _1_4,873 (1967). Groth, D. P., Bain, J. A. , and Pfeiffer, C. C., 2. Pharmacol. Exptl. Therap., 124, 290 (1958). Schuberth, J., Sparf, B., and Sundwall, A., 2. Neurochem., 1_6, 6% (1969). Diamond, I. , A Arch Neurol 22, 333 (1971). L’ & , 122, 741 (1971). Ansell, G. B., and Spanner, S., Biochem. Pilgaram, L. 0. , Gal, E. M, , Sassenrath, E. N., and Greenberg, D. M., 2. Biol. Chem. , 22, 367 (1953). Ansell, G. B., and Spanner, S., Biochem. 2.) 110, 201 (1968). Lum, G. G., and Lapetfna, E. G., Brain RJ?s., 2, 451 (1970).
1590
FREE
BIOCHEMICAL
CHOLINE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FORMATION BY CEREBRAL FROM RAT BRAIN
Edward
CORTICAL
SLICES
T. Browning
Department of Pharmacology Rutgers Medical School New Brunswick, New Jersey 08903
Received
October
7, 1971 SUMMARY
Changes in free choline levels of cerebral cortical slices and their incubation Free choline was found fluid were examined under conditions of active ACh synthesis. to increase tenfold during two hours of incubation. Most of the free choline, which was presumably formed from esterified choline precursors, was found in the extracellular medium. INTRODUCTION The amount
of free choline
equal to the amount 1 hour depends
ultimately
experiment and their
appears
was designed incubation
during
not to synthesize stream
to examine
fluid
before
per gm (1,2) is approximately
by slices
choline
cortex
--de novo (4,.5); rather
to supply choline
the free choline
of cerebral
(6-9).
in
the brain
The present
level in cerebral
cortical
slices
and after ACh synthesis.
show that rather
2 hours
0.1 pmole
can be synthesized
upon the blood
The results tenfold
of ACh which
The brain
(3).
in rat brain,
than being consumed
the free choline
increased
of incubation. METHODS
Slices described After
of cerebral
elsewhere
incubation
at 0” C.
cortex (3).
and these samples
Incubation
the slices
Supernatants were
were
were
were
from
conditions
separated
decanted,
placed
prepared
are described
by centrifugation
their
in a boiling
male Wistar
pH adjusted water
1586
rats (200-300
gm) as
in the table legend. at 33,000
to 4.3 with
bath for 10 min,
x g for 10 min 1 M acetic
rechilled
and
acid,
Vol. 45, No. 6, 1971
centrifuged
BIOCHEMICAL
as above.
The slices
disrupted
by sonication
The slice
extracts
potassium
AND BIOPHYSICAL
were
and centrifuged;
were
perchlorate
neutralized
enzyme
assay which
pyruvate
kinase,
and lactate
cuvette
as choline
by adding
3 M K2C03
with 0.5 ml PCA.
and the resulting
AND ACh ASSAY extracts
is based on coupling
in an enzymic
was then washed
acid (PCA),
by centrifugation.
and media
oxidation
3 ml 6% perchloric
to pH 4.3 with
was removed
of the slices
with
the pellet
CHOLINE Choline
treated
RESEARCH COMMUNICATIONS
system which
were
choline
by a fluorometric
phosphorylation
comprises
dehydrogenase
assayed
choline
(2).
acetylcholinesterase
to NADH
phosphokinase
(CK),
ACh was assayed
in the same
after
of samples
the choline
had
reacted. That the material aliquots
of extracts
experiments whereas
assayed
were
showed
was choline
made alkaline
that choline
into the chloroform(2). of 14C-choline amount initial
Second,
amount
which
eluted
of “choline11
by two means.
in the aqueous
a sample
this treatment
for CK, migrated
was mixed
on a 1 x 100 cm Dowex-50
by the fluorometric
Control
phase after
substrate
of extract
with the 14C-choline
First,
with chloroform.
the next most active
and chromatographed
of choline
and extracted
remained
dimethylethanolamine,
was verified
with a minute column
peak was compared
amount
(2,lO).
The
with the
assay.
RESULTS The changes
in choline
with the ACh synthesis. obtained
earlier
The purpose the slices progressively
levels
The amounts
by measuring
of the present during
experiment
2 hours
system
of ACh formed
the transfer
accumulation
during
by the slice
is depicted
in 1 hour are similar
of isotopic
acetyl
was to examine
of ACh. of incubation.
The free choline In addition,
1587
in the table along to those
units to choline
the free choline content larger
reserves
of the slices amounts
(3). of rises
of choline
BIOCHEMICAL
Vol. 45, No. 6, 1971
are released
into the incubation
In order
to verify
choline,
three
extracted
with
The choline
extraction
medium
during
that the material
120 min samples
from
concentration
of extracts
extract
whereas
accumulating
substrates before
RESEARCH COMMUNICATIONS
incubation.
the incubation
All other
chloroform.
4.6 wmoles/ml
AND BIOPHYSICAL
in the system fluid
were
was,
made alkaline
for CK are uncharged
and
at this pH.
alkali:chloroform
treatment
concentration
in the aqueous
the choline
in fact,
was 63. 8 i phase after
was 63.3 + 5.0 m,umoles/ml.
One 129 min media Ninety-five
percent
the column
as defined
sample
of the choline
was chromatographed sample
by the authentic
Choline
on a column
was recovered
14C-choline
in the choline
and ACh Formation by Cortical of Rat Brain
Medium
Slices Choline
87 k
60 120
Medium cortex
Slices mpmoles/gm
11 -+ 4 (8)
0
peak from
elution.
ACh Incubation min
of Dowex-50.
9 A 2 (6)
47 *
Slices
6 (8)
86 zlz 7 (9)
9 (8)
11 i 4 (6)
395 i 30 (8)
124 i
7 (8)
82 * 18 (8)
9 * 1 (6)
810 f 54 (8)
147 f 11 (8)
200 mg of slices were incubated at 37” C in 3 ml of a solution which contained 94 mM NaCl, 29 mM KCl, 1 mM KH2P04, 25 mM NaHC03, 1 mM MgSo4, 2.5 mM CaC12, 2 mM glucose, 0.01 M eserine sulfate pH 7.4. The atmosphere was 95% 02-5% C02. Data represent the pooled results of 3 experiments, each of which showed the essential features of the pooled data. DISCUSSION Brain several
relies
on peripheral
investigators
the brain
(7-9).
and later
it is present
Greenberg
and/or
have demonstrated
Once in the brain
demonstrated
tissues
substantially
diet as a source
choline
choline
transfnr
is rapidly
in a chloroform
that the rate of methyl
1588
from
acetylated soluble
transfer
of choline. the blood
stream
into
or phosphorylated
form.
to form
Recently
Bremer choline
and phospholipids
Vol. 45, No. 6, 1971
from
BIOCHEMICAL
ethanolamine
phospholipids
Ansell
of liver.
AND BIOPHYSICAL
is very low in brain,
and Spanner
showed
labeled
was associated
with choline
phospholipids
(5).
the choline
phospholipids
(11).
labeled
interpreted derived
only the ethanolamine
as substantiating from
presented
in this
a net formation
that the free choline catabolism occur
in brain
little
or no 1%
These
14C-choline
observations
of brain
were
phospholipids
above,
is
are phosphorylcholine,
phosphatidyl
glycerophosphorylcholine, of these contribute
The esterified
forms
choline,
from
a net which
to the choline
Iysophosphatidyl
and choline
to free choline
is advanced
of choline
significantly
sphingomyelin,
significantly
results
certain
cortical
hypothesis
work
enough to contribute
that under
in cerebral
the provisional
in the present
of choline.
large
demonstrate
may be observed
observed
forms
in amounts
accumulation
Which
Alternately,
communication
of free choline
formation
of esterified
choline,
was injected
phospholipids;
the idea that choline
In view of the data discussed
slices.
which
the blood stream.
The results conditions
less than lo/O of the activity
that 14C-ethanolamine
intracerebrally
injection
RESEARCH COMMUNICATIONS
plasmalogen.
formation
is under
investigation. In view of the rapid phosphatidyl
choline,
that the choline phospholipid present
of labeled
choline
both --in vivo (11) and in -- vitro
formation
catabolism,
results
conversion
observed possibly
emphasize
in the present phosphatidyl
the catabolic
segment
(12),
to phospholipids, it is reasonable
experiments
choline
mainly
results
catabolism,
of a process
to suggest from
and that the
of phospholipid
turnover. That free choline the amounts choline
reported
transport
precautions
relating
can accumulate
herein
in a system
poses an important
and metabolism to changes
in brain
consideration
and possibly
in cholinespecific
1589
of cerebral
in other
radioactivity
cortex
slices
in
for studies
of isotopic
tissues.
Appropriate
need to be taken.
Vol. 45, No. 6, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENT This work was supported by U. S. Public Health Service grants: General Research Support Grant No. FR 05576 and Grant No. NB 08665. REFERENCES Pharmacologist, 12, 1. Haubrich, D. R., Krishna, G. , and Reid, W. D., The 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
313 (1970). Browning, E. T., Fed. Proc -. , 30, 271 (1971); complete manuscript submitted. Browning, E. T., andSchulman, M. P., 2. Neurochem., 3 1391 (1968). Bremer, J. , and Greenberg, D. M., Biochim. Biophys. Acta, 46, 205 (1961). Ansell, G. B., and Spanner, S., 2. Neurochem., _1_4,873 (1967). Groth, D. P., Bain, J. A. , and Pfeiffer, C. C., 2. Pharmacol. Exptl. Therap., 124, 290 (1958). Schuberth, J., Sparf, B., and Sundwall, A., 2. Neurochem., 1_6, 6% (1969). Diamond, I. , A Arch Neurol 22, 333 (1971). L’ & , 122, 741 (1971). Ansell, G. B., and Spanner, S., Biochem. Pilgaram, L. 0. , Gal, E. M, , Sassenrath, E. N., and Greenberg, D. M., 2. Biol. Chem. , 22, 367 (1953). Ansell, G. B., and Spanner, S., Biochem. 2.) 110, 201 (1968). Lum, G. G., and Lapetfna, E. G., Brain RJ?s., 2, 451 (1970).
1590