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Reprinted from Archives internationales de Pharmacodynamie et de Therapie
Vol. 209 - No. 2 - June 1974
Studies with Agents which
Influence Acetylcholine Metabolism
in Mouse Brain
J. K. SAELENS,
J. P. SIMM!,
J. SciIUMAN AND M. P. Al.LEN
BeteareH
Department, B—» Dmim, CrBAfVOY
Corporation,
Snnmti,
New Jersey , 07901
U.S.A.
Abstract—By using an intravenous single pulse injection of choline-
into
the
tail
vein
of mice and
measuring
the endogenous
levels
o
choline and acetylcholine, the effects of agents which influence ace y
-
choline metabolism
in mouse
brain were
studied.
<-
ies^ ts w{
j
rc
consistent with a feedback activation of cholinergic neurons with scopola-
mine
cholinc-O-acetyltransferase inhibition with naphthylvinylpyndine,
inhibition
of
acetylcholine
release
by
morphine
and
cholinesterase
inhibition with physostigmine. The interactions of these agents with the
cholinergic system further suggests certain characteristics of cholinergic
neurons,
e.g. that choline-O-acetyltransferase
activity
is accelerated by
a
decrease
in
intracellular
acetylcholine
levels
and
retarded
by
an
increase in intracellular acetylcholine
levels, that the choline transput
mechanism
is functioning at maximum capacity under normal physio -
ogical conditions and that the synaptic levels of endogenous choline play
a role in tracer choline capture.
Introduction
In 1969 Schuberth,
et at. (1) introduced a technique for measuring the rate
of synthesis of acetylcholine (ACh) in whole mouse brain. In their P^cdu e the
rate of radiactivc ACh formation in the brain was measured following an mtra
venous pulse injection of tritium-labelled choline (Ch). After determination o
the isotope dilution of the Ch in the brain, the ACh rate of synthesis, tnjvo
was
calculated. We have now
applied
this technique involving four
agen
purported to affect central cholinergic neurons in diftcrcn
ways. The agents are
the muscarinic cholinergic receptor blocking agent, scopolamine;
acetyltransferasc
inhibitor,
naphthylvinylpyndine
(2
3);
the
ch
inhibitor, physostigmine; and an agent reported to block the release of ACh
morphine (4, 5, 6) The results presented below are consistent with the proposed
250
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ACETYLCHOLINE METABOLISM
25 1
mechanisms of the various agents and help to further define certain character-
istics of central cholinergic neurons.
Methods
Administration of Choline-Methyl-C™(Ch-C14
). Male CF^ (Carworth Farms)
mice, 18-20g were used. Each mouse received 5 ^c (0.66
moles) of Ch-C14
chloride (New England Nuclear, specific activity = 7.6 pc/ju, mole) intravenously
in a tail vein. The tracer was dissolved in distilled water and administeied in a
volume of 0.1 ml with a gas-tight Hamilton syringe through a 30 gauge needle.
Preparation of Brain Samples. The mice were sacrificed at different times after
injection by rapid immersion of the whole animal into a Dewai flask containing
liquid N2 for 3.5 seconds. Individual mouse brains were removed as rapidly as
possible,
dissected on
a thermoelectric cold
plate maintained
at
1-2° C, and
frozen in liquid N2
. Where whole brain analysis was performed, the brain was
placed
directly
in
liquid Na
. Timing was
critical with
this method.
If the
immersion lasted for more than 4 seconds, the surface of the cortex began to
freeze and stick to the skull making removal of the tissue difficult. The frozen
brains or brain parts were weighed on a Mettler electronic balance and then
pulverized in a Thermovac (R) tissue crusher cooled with dry ice and liquid
nitrogen. The remainder of the procedure was as described for rats by Saelens
et al
(7).
Determination o/ACh and Ch. ACh and Ch were determined by the enzymatic
method of Feigcnson and Saelens (8) using the modifications recommended by
Saelens et ah
(9).
Statistical Analysis. The disposition of Ch-C-14
, and the levels of Ch and ACh
were determined in different groups of mice. In order to obtain the appropriate
parameters,
the product of two independent variables were analyzed by the
method of Goodman (10). At least 5-10 mice were used in
all measurements.
Agents.
Test
agents were
dissolved
in
distilled
water,
and
administered
subcutaneously, or intraperitoneally, in a volume of 0.1 ml/ 10 g of body weight
or intravenously in a volume of 0.05 ml/ 10 g of body weight. Mice received food
and water ad libitum up until the time of sacrifice.
The agents studied were scopolamine hydrobromide (Merck), naphthylvinyl-
pyridine (Aldrich), physostigmine sulfate (Merck) and morphine sulfate (Merck).
Results and Discussion
Control Studies: The Schuberth model for estimating the rate of synthesis of
ACh is essentially a single closed compartment model. To be valid, the level of
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254
J. K. SAELENS,
J. P. SIMKE,
J. SCHUMAN AND M. P. ALLEN
few minutes after administration of the tracer. ACh-C14/Ch-C14 ratios obtained
at one minute or
less were
clearly below the peak
ratios and therefore data
generated using this time interval satisfied the criteria of being below equilibrium
for product tracer formation. When the pharmacological agents were used, the
mice were always sacrificed 45 seconds after administration of the Ch-C14 and
30 minutes after administration of the agent.
As a working hypothesis,
it is suggested that the tracer was equilibrating with
a part of the free Ch pool which was readily available for ACh synthesis during
the rise of the ratio curve, and the peak and
fall were the result of the tracer
beginning to equilibrate with other parts of the Ch pool. This interpretation is
supported by the delayed appearance of organic soluble tracer (in the acetone-
chloroform extract usually discarded when measuring endogenous ACh and Ch).
If this hypothesis is correct, then the peaks in the ACh-C14/Ch-C14 ratio curves
should be reasonable,
albeit conservative,
estimates
of the
ratio between en-
dogenous ACh and the portion of the Ch pool suggested
to be available for
ACh synthesis. The peak ratios, therefore, allowed an indirect estimate of the
size of the available free Ch pool (AF Ch) using the following equation:
r Ch — C14 i
AF Ch =
[Xciw C14 ]
X ACh (end°genous)
at peak
Using this indirect technique, the sizes of the AF Ch pools and the synthesis
rates of ACh in the whole brain, cortex, midbrain, brainstem and cerebellum
were estimated and are shown in Table I. Under these conditions,
it was esti-
mated that the stores ofACh in mouse brain would be replaced every 2-3 minutes.
Using the direct technique,
it should be noted that the endogenous brain Ch
levels of control animals shown in Table 2 were approximately twice the AF Ch
estimated above. Recent investigators, particularly Ewetz el al (11), suggest that
post-morten increases in Ch levels due to catabolic destruction of Ch containing
compounds in the brain, are very difficult to avoid.
It
is possible that, despite
the precautions taken, the Ch levels in Table II include some Ch originating
from such sources. For this reason, when the single closed compartment calcula-
tions for rate of synthesis of ACh were applied to the data presented here the
results were expressed as “apparent rate of synthesis.”
Scopolamine: At 10 mg/kg i.p., scopolamine caused a 50 % decrease in endog-
enous ACh
levels. However,
the amount
of ACh-C14 found
in the brain
at
45 seconds was
actually
slightly increased. Thus, the
specific
activity of the
reduced endogenous ACh pool was 2.4 times the specific activity of the corre-
sponding control ACh pool. This dose of scopolamine also caused a statistically
significant decrease in the endogenous Ch levels and a
slight decrease in the
45 second Ch-C14
levels.
All the data with scopolamine
is consistent with
a
feedback activation of cholinergic neurons resulting from cholinergic receptor
blockade. The
conversion
of Ch-C14 to ACh-C14 was
clearly accelerated
as
illustrated by the large increase in ACh specific activity but the reduction in the
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ACETYLCHOLINE METABOLISM
255
endogenous Ch pool counterbalanced the significantly higher ACh-C14/Ch-C14
ratio resulting in no change in the apparent synthesis of ACh. The reduction in
ACh
levels caused by scopolamine
is most likely a matter of synthesis being
unable to keep up with the drug-induced facilitation of release. These results
suggest 2
characteristics
of
cholinergic
neurons.
First,
the
choline-0-acetyl-
transferase activity is accelerated by a decrease in ACh levels and/or Ch levels,
the former, alone, being the most logical. Second, Ch capture is not accelerated
by the decrease in ACh or Ch levels suggesting that the Ch transport mechanism
is functioning at maximum capacity under normal physiological conditions.
Table I
Estimates of the AFCholine Pools and Rates of Synthesis of Acetylcholine in Various Brain
Areas of Control Mice
Estimates
of Rate of
Mean
ACh - C14
Area
ACha mfi
Molesjg
Ch - C' 4
Whole Brain
Cortex
Midbrain
Brainstem
Cerebellum
13.6
12.5
18.5
19.7
6.9
0.550
0.635
0.720
0.661
0.201
b
Estimate
AF Ch c
mfi
ACh - C14
Ch - C14
Synthesis
of ACh
m/x Moles/
peak
Moles/g
24.7
19.6
25.6
29.8
33.6
0.263
0.339
0.244
0.189
0.040
1 minute
g/minute
6.50
6.64
6.25
5.63
1.34
a ACh — Acetylcholine endogenous.
ACh - C14
Ch - C14
= the peak ratios from Figure 2.
peak
c AF Ch—Available Free Choline—the
hypothetical
choline pool suggested
to be
available for acetylcholine synthesis.
NaphthylvinyIpyridine
(NVP).
The
chohne-0-acctyItransferase
inhibitor,
NVP, caused a dose related increase in endogenous ACh
levels. The a priori
expectation would be just the opposite, i.e. a decrease in endogenous ACh levels.
However, others have also failed to demonstrate ACh depletion with NVP (2, 3).
The increase in endogenous ACh was associated with a dose related decrease in
the 45 second ACh-C14 levels which was statistically significant at 40 mg/kg i.v.
The specific activity of ACh in NVP treated animals also showed a dose related
decrease which dropped to 40 % of control
at 40 mg/kg
i.v. Thus, NVP,
at
reasonable doses, was quite capable of depressing the conversion of Ch-C14 to
ACh-C14 but was incapable of reducing storage levels of ACh. The Ch transport
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Table
II
meet
of
Various
Agents
on
Acetylcholine
and
Choline
Levels
and
the
Disposition
of
Methyl-C
«
Choline
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J. K. SAELENS,
J.
P. SIMKE,
J. SCHUMAN AND M. P. ALLEN
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ACETYLCHOLINE METABOLISM
257
mechanism is also apparently independent of choline-O-acetyltransferase activity
as N VP clearly depressed the conversion of Ch-C14 to ACh-C14 but had no effect
on the capture of Ch-C14 from the bloodstream. Therefore, there is no evidence
here, that cholme-O-acetyltransferase
is a Ch acceptor inside the nerve ending
necessary for the Ch transport mechanism to function properly. NVP did cause
a significant reduction in the apparent rate of synthesis at 40 mg/kg i.v. Why the
ACh levels went up instead of down
is
still unclear.
Morphine: In a dose range of 1-10 mg/kg s.c. morphine was noteably ineffective
m altering a 1 parameters involving ACh but dramatically increased the 45 second
Ch-C
levels without altering endogenous Ch levels. The apparent rate of ACh
synthesis was significantly decreased by all doses of morphine tested but clearly
this was
a consequence
of increased Ch-C14
capture. There
is
considerable
evidence that morphine impairs the release of ACh in the brain (4
6)
There
is also some evidence that ACh released into the synaptic cleft is hydrolyzed by
cholinesterase and the resulting Ch transported back into the cholinergic nerve
ending (e.g. 7). Morphine is almost doubled in the 45 second Ch-C14 capture at
mg/ -g s.c. It is tempting to speculate that these 3 events are interrelated. The
tracer pulse most probably passes by the brain as a bolus where the peak specific
activity is exposed to the nervous tissue for a very short period of time. Once
into the extracellular space, including cholinergic neuron synapses,
it
is taken
bIJ
he Ch transP°rt mechanism into the neuron. At this juncture, the tracer
Ch-C
is suggested to undergo some amount of isotope dilution from the non-
radioactive Ch hydrolyzed from the non-radioactive ACh being continuously
leleased dunng transmission. If morphine impairs the release of ACh, the tracer
should undergo a lesser amount of isotope dilution as the source of dilution, the
released ACh,
is diminished. It is suggested that the cholinergic nerve endings
are not taking up a larger quantity of Ch but rather are taking up the same
quantity of Ch which has a higher proportion of Ch-C14
. This hypothesis
is
consistent with the fact that the endogenous Ch levels do not change under the
influence of morphine.
&
Physostigmine: The above hypothesis regarding morphine and Ch-C14 capture
was challenged by reducing the availability of synaptic Ch in a different way
Physostigmine does not impair the release of ACh
(2), but does prevent the
ydrolysis of ACh to Ch in the synapse. With 0.1 mg/kg s.c. of physostigmine
control It
SeT ?'C“ IeVdS WCre substantially higher than corresponding
the 45
se 7 a A nuT
€1
*°^ Aat Physostigmine significantly decrease
the 45
second ACh-C14
levels
and
specific
activity
of ACh
supporting
the
contention of Kaita and Goldberg (12) that feedback inhibition by intracellular
parametl?
I
T77^7^ efieCtS °f physostigmine on ACh related
paiameters also clearly distinguishes
it from morphine.
Min^ledgment—Thc authors would
like
to acknowledge the valuable
assistance
of
Vlr. C. Patel who performed the statistical analysis on the data presented here and the
invaluable skills and efficiency of Miss C. Laspina
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258
J. K. SAELENS,
J. P. SIMKE,
J. SCHUMAN AND M. P. ALLEN
References
1. Schuberth,
J., Sparf, B. and Sundwall, A. J. Neurochem.
16, 695 (1967).
2. Krell, R. D. and Goldberg, A. M. Trans. Amer. Soc. Neurochem. 4, 129 (1973).
3. Click, S. D., Mittag, T. W. and Green,
J. P. Neuropharmacol.
12, 291 (1973).
4.
Belesiiin, D. and Polaic, R. L. J. Physiol., Lond.> 177, 411 (1965).
5.
Belesiiin, D., Polar, R. L. and Sproull, D. H. J. Physiol , Lond.,
177, 420 (1965).
6. Sharkawi, M. and Sci-iulman, M. P. J. Pharm. Pharmacol 21, 546 (1969).
7. Saelens, J. K., Simke, J. P., Allen, M. P. and Conroy, C. A. Arch. int. Pharmacodyn.
203, 305 (1973).
8.
Feigenson, M. E. and Saelens,
J. K. Biochem, Pharmacol
18, 1479 (1969).
9. Saelens, J. K., Allen, M, P. and Simke, J. P. Arch. int. Pharmacodyn. 186, 279 (1970).
10. Goodman, L. A. J. Amer. Statist. Assoc. 55, 708 (1960).
11. Ewetz, L.; Sparf, B. and Sorbo, B. Symposium on in vitro Procedures With Radioiso-
topes in Medicine in Vienna , 1969. Ed. Int. Atomic Energy Agency SM- 124/39, 175
(1970).
12. Kaita, A. A. and Goldberg, A. M. J. Biochem.
16, 1185 (1969).
Received March 4 } 1974.
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