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Direct Inhibitory Effect of Hemicholinium-No. 3 on Acetylcholine Release
DIRECT
INHIBITORY No.
Fumiaki
3 ON
HATA,
EFFECT
OF
HEMICHOLINIUM
ACETYLCHOLINE
Tomohiro
RELEASE
MATSUDA
and Hiroshi
YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, Kita-kit , Osaka, Japan Accepted February
It is well known choline
was demonstrated of choline hibits
that hemicholinium
(ACh) in both central
that the reduced
into neuronal
uptake
(1-7)
synthesis
of ACh in the presence
a direct inhibitory
3 (HC-3) reduces
and peripheral
cells (7, 11-13).
findings suggest that HC-3 reduces ducing the ACh content
No.
24, 1975
the synthesis
(1, 8-10) nervous
of ACh was due to inhibition It was also found
of an inhibitor
However,
It
of the transport
that in brain slices HC-3 in
of cholinesterase
the release of ACh from nervous
of nervous tissue.
of acetyl
tissues in vitro.
(14, 15).
These
tissue indirectly
by re
in this study HC-3 was found to have
effect on the release of ACh from brain slices under certain experimental
conditions. Sprague-Dawley
strain rats, weighing
and then chilled in cold Krebs Ringer. KCI,
1.34 mM
MgSO4,
10 mM glucose.
1.34 mM
In experiments
the lateral surface. suspended
CaCl2, 20 mM
using a Stadie-Rigg's The slices (about
in 4 ml of Krebs Ringer,
Ringer with or without ing 90 mM NaCl
were decapitated,
sodium
phosphate
on the effect of anticholinesterase,
was added to the Krebs Ringer solution. brain hemisphere
200-300g
The Krebs Ringer contained
Three cerebral slicer:
cortex
the brains
134 mM NaG, buffer,
removed 5.4 mM
pH 7.4 and
25 1 M eserine sulfate slices were cut from each
two from the apical surface and one from
160-200 mg wt.) were weighed on a torsion and then preincubated
balance,
at 37'C for 30 min in Krebs
eserine sulfate, or in a medium of high. K+ concentration
and 50 mM KCI and other ions at the same concentrations
contain
as in Krebs
SHORT Ringer.
After rinsing
COMMUNICATIONS
with cold Krebs
for 20 min with 4 ml of medium 20 1cM HC-3 was added
Ringer
Japan. J. Pharmacol. 25, 355 (1975) the slices were incubated
of high K+ concentration.
to the incubation
25 1 M eserine sulfate
supernatant
was neutralized
frog rectus muscle by the method cholinesterase Microsomal
(AChE) fraction
activity obtained
tion and it was preincubated
and boiled
with NaOH
at 37'C
medium.
For assay of total ACh of the slices, they were homogenized containing
further
To test the effect of HC-3,
with 5 ml of Frog Ringer
at pH 4.0 for 10 min and centrifuged.
and then the ACh concentration
of Chang
and Gaddum
was carried out according from rat brain homogenate with or without
inhibitors
(16).
was assayed
Measurement
to the procedure
on
of acetyl
of Hestrin
was used as the enzyme
The
(17).
prepara
at 37'C for 20 min.
FIG. 1. (A) Effects of high K ion concentration on ACh release from slices pre incubated with or without eserine. (B) Effects of high K ion concentration in presence of HC-3 on ACh release from slices preincubated with or without eserine. ------Krebs Ringer, -•-•-•-•-• Krebs Ringer containing eserine , High K+ medium Values are means -S.D. and the numbers of experiments are shown in brackets.
SHORT There were significant incubated normal
in Krebs Krebs
increases
Ringer
Ringer
Japan. J. Pharrnacol. 25, 356 (1975)
in the amount
containing
medium
Birks and MacIntosh and of Potter
COMMUNICATIONS
(Fig.
of ACh in the slices, which were pre
25 /-tM eserine 1, A).
sulfate
This finding
in comparison
is in agreement
(8) and of Collier and Katz (18) using cat's superior
(10) using the nerve diaphragm
preparation.
The extra
to value
on
with results
of
cervical ganglion ACh accumulated
by presence of AChE inhibitor such as eserine or tetraethylpyrophosphate was termed "Surplus ACh" b y Birks and MacIntosh (8). On the other hand, high K' concentration is widely known to cause a release of ACh from the brain slices resulting in a reduction of ACh content
in the slice.
tion of ACh content
Effect of HC-3 was examined
in the slices preincubated
As shown in Fig. 1, A and B, reduction slices preincubated But its reduction
in normal
ACh" by preincubation (P<0.01). ACh"
in medium
Our results,
were transferred
of HC-3
Krebs
shown
influence
containing
that
influenced
by
by HC-3
"Surplus in presence
--S.D.,
This effect of HC-3 cannot be explained
HC-3.
significantly
was higher
slices, mean
from
"Surplus
when slices containing
their ACh content ACh/g
of eserine.
had accumulated
eserine was depressed
1, indicate
(8.8-1-1.6 m,amoles
in presence by its known
on ACh synthesis.
As the higher ACh content an inhibitory
of slices which
to high K+ medium,
than in its absence
and absence
reduc
by high K' concentration
was not significantly
content
in Fig.
of HC-3 and 6.5 + 1.8 in its absence). inhibitory
in the presence
of ACh content
Ringer
by K+ in the ACh
here on the K+-induced
of slices in the presence
effect of HC-3 on AChE activity,
rat brain microsomes
was examined.
be due to
the influence of HC-3 on AChE activity
As shown
described
of HC-3 might possibly
in Table 1, HC-3 slightly inhibited
of
AChE.
However,
in the experiments
Therefore,
even if HC-3 readily enters the brain slices (19) it seems unlikely that the higher
level of ACh in the slices observed the weak inhibitory has no significant on uptake
effect on activity
of an increase
of choline
of HC-3 is due to
it is kno%vn that
HC-3
(2) and an inhibitory
effect
are
represented
inhibitors.
of the slices.
A more
release of ACh from
th
likely explanation so-called
"Surplus
In the small intestine also, HC-3 has been shown to inhibit
TABLE 1. Inhibition
Data
acetyltransferase
in ACh content it may inhibit
in the brain slices.
without
of this concentration
On the other hand
into brain slices (7, 11-13), but these effects of 1-IC-3 do not indicate
of the effect of HC-3 is that ACh Pool"
in the presence
effect of HC-3 on AChE.
of choline
the possibility
above on release of ACh, 20 ,uM HC-3 was used.
as per
of AChE by HC-3 or eserine.
cent
of
inhibition
to
enzyme
activity
SHORT spontaneous
COMMUNICATIONS
and stretch-induced
The nature the cytoplasm. of "Surplus
of "Surplus
ACh"
release
is not clear, but it may represent
ACh" may be a carrier mediated with uptake
HC-3 (14, 15).
Several
mulation
release
workers
of "Surplus
physiological
tion increased
ACh in a free form in
transmembrane
transport
postulate
ACh"
significances,
release of "Surplus
(21-23)
having by
that the ACh in the pool available
for
form in the synaptic
which was inhibited there
release
to be inhibited
not a bound
though
by HC-3,
phenomenon,
of ACh by the slices which is also known
release is in a free form in the cytoplasm,
important
of ACh (20).
If this is so and if its release from the slices is inhibited
a close relation
this meaning,
Japan. J. Pharmacol. 25, 357 (1975)
by HC-3
is a report
ACh" from the sympathetic
vesicles.
In
may have rather
that
high K+ concentra
ganglion
while electrical
sti
did not (18).
REFERENCES 1) MACINTOSH,F.C., BIRKS, R.I. AND SASTRY,P.B.: Nature 178, 1181 (1956); 2) GARDINER, J.E.: Blochem. J., 81, 297 (1961); 3) BHATNAGAR,S.P. AND MACINTOSH,F.C.: Canad. J. Phy siol. Pharmacol. 45, 249 (1967); 4) SLATER,P.: Int. J. Neuropharmacol., 7, 421 (1968); 5) BROWNING,E.T. AND SCHULMAN,M.P.: J. Neurochemn. 15, 1391 (1968); 6) MARCHBANKS, R.M.: Biochem. Pharmacol. 18, 1763 (1969); 7) GUYENET,P., LEFRESNE,P., ROSSIER,J., BEAU JOUAN,J.C. AND GLOWINSKI,J.: Mol. Pharmacol. 9, 630 (1973); 8) BIRKS, R.I. AND MAC INTOSH,F.C.: Canad..1. Biochem. PhysioL 39, 787 (1961) 9) COLLIER,B. AND MACINTOSH,F.C.: Canal. J. Physiol. Pharmacol. 47, 127 (1969); 10) POTTER,L.T.: J. Physiol. 206,145 (1970); 11) SCHUBERTH, J., SUNDWALL,A., SoRBO, B. ANDLINDELL,J-O.: J. Neurochem. 13, 347 (1966); 12) MARCHBANKs,R.M.,: Biochem. J. 110, 533 (1968); 13) YAMAMURA,H.I. AND SNYDER,S.H.: J. Neuro(-hem. 21, 1355 (1973); 14) SCHUBERTH,J. AND SUNDWALL,A.: J. Neurochem. 14, 807 (1967); 15) PoLAK, R.L.: Brit..I. Pharmacol. 36, 144 (1969); 16) CHANG, H.C. ANDGADDUM, J.H.: J. Physiol. 79, 255 (1933); 17) HESTRIN,S.: J. biol. Chem. 180, 249 (1949); 18) COL LIER,B. AND KATZ, H.S.: J. Physiol. 214, 537 (1971); 19) SLATER,P. AND STONIER,P.D.: J. Neurochem. 20, 637 (1973); 20) YAGASAKI,0., TAKFWAKi,T., UTSUMI, S. AND YANAGIYA, I.: Fo!ia Pharmacol. Japon. 65, 171§ (1969) (in Japanese); 21) WHITTAKER,V.P.: Prog. Brain Res. 31, 211 (1969); 22) RICHTER,J.A. AND MARCHBAKNS, R.M.: J. Neurochem. 18, 691 (1971); 23) RICHTER,J.A. AND MARCHBANKS,R.M.: J. Neurochem. 18, 705 (1971)
INHIBITORY No.
Fumiaki
3 ON
HATA,
EFFECT
OF
HEMICHOLINIUM
ACETYLCHOLINE
Tomohiro
RELEASE
MATSUDA
and Hiroshi
YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, Kita-kit , Osaka, Japan Accepted February
It is well known choline
was demonstrated of choline hibits
that hemicholinium
(ACh) in both central
that the reduced
into neuronal
uptake
(1-7)
synthesis
of ACh in the presence
a direct inhibitory
3 (HC-3) reduces
and peripheral
cells (7, 11-13).
findings suggest that HC-3 reduces ducing the ACh content
No.
24, 1975
the synthesis
(1, 8-10) nervous
of ACh was due to inhibition It was also found
of an inhibitor
However,
It
of the transport
that in brain slices HC-3 in
of cholinesterase
the release of ACh from nervous
of nervous tissue.
of acetyl
tissues in vitro.
(14, 15).
These
tissue indirectly
by re
in this study HC-3 was found to have
effect on the release of ACh from brain slices under certain experimental
conditions. Sprague-Dawley
strain rats, weighing
and then chilled in cold Krebs Ringer. KCI,
1.34 mM
MgSO4,
10 mM glucose.
1.34 mM
In experiments
the lateral surface. suspended
CaCl2, 20 mM
using a Stadie-Rigg's The slices (about
in 4 ml of Krebs Ringer,
Ringer with or without ing 90 mM NaCl
were decapitated,
sodium
phosphate
on the effect of anticholinesterase,
was added to the Krebs Ringer solution. brain hemisphere
200-300g
The Krebs Ringer contained
Three cerebral slicer:
cortex
the brains
134 mM NaG, buffer,
removed 5.4 mM
pH 7.4 and
25 1 M eserine sulfate slices were cut from each
two from the apical surface and one from
160-200 mg wt.) were weighed on a torsion and then preincubated
balance,
at 37'C for 30 min in Krebs
eserine sulfate, or in a medium of high. K+ concentration
and 50 mM KCI and other ions at the same concentrations
contain
as in Krebs
SHORT Ringer.
After rinsing
COMMUNICATIONS
with cold Krebs
for 20 min with 4 ml of medium 20 1cM HC-3 was added
Ringer
Japan. J. Pharmacol. 25, 355 (1975) the slices were incubated
of high K+ concentration.
to the incubation
25 1 M eserine sulfate
supernatant
was neutralized
frog rectus muscle by the method cholinesterase Microsomal
(AChE) fraction
activity obtained
tion and it was preincubated
and boiled
with NaOH
at 37'C
medium.
For assay of total ACh of the slices, they were homogenized containing
further
To test the effect of HC-3,
with 5 ml of Frog Ringer
at pH 4.0 for 10 min and centrifuged.
and then the ACh concentration
of Chang
and Gaddum
was carried out according from rat brain homogenate with or without
inhibitors
(16).
was assayed
Measurement
to the procedure
on
of acetyl
of Hestrin
was used as the enzyme
The
(17).
prepara
at 37'C for 20 min.
FIG. 1. (A) Effects of high K ion concentration on ACh release from slices pre incubated with or without eserine. (B) Effects of high K ion concentration in presence of HC-3 on ACh release from slices preincubated with or without eserine. ------Krebs Ringer, -•-•-•-•-• Krebs Ringer containing eserine , High K+ medium Values are means -S.D. and the numbers of experiments are shown in brackets.
SHORT There were significant incubated normal
in Krebs Krebs
increases
Ringer
Ringer
Japan. J. Pharrnacol. 25, 356 (1975)
in the amount
containing
medium
Birks and MacIntosh and of Potter
COMMUNICATIONS
(Fig.
of ACh in the slices, which were pre
25 /-tM eserine 1, A).
sulfate
This finding
in comparison
is in agreement
(8) and of Collier and Katz (18) using cat's superior
(10) using the nerve diaphragm
preparation.
The extra
to value
on
with results
of
cervical ganglion ACh accumulated
by presence of AChE inhibitor such as eserine or tetraethylpyrophosphate was termed "Surplus ACh" b y Birks and MacIntosh (8). On the other hand, high K' concentration is widely known to cause a release of ACh from the brain slices resulting in a reduction of ACh content
in the slice.
tion of ACh content
Effect of HC-3 was examined
in the slices preincubated
As shown in Fig. 1, A and B, reduction slices preincubated But its reduction
in normal
ACh" by preincubation (P<0.01). ACh"
in medium
Our results,
were transferred
of HC-3
Krebs
shown
influence
containing
that
influenced
by
by HC-3
"Surplus in presence
--S.D.,
This effect of HC-3 cannot be explained
HC-3.
significantly
was higher
slices, mean
from
"Surplus
when slices containing
their ACh content ACh/g
of eserine.
had accumulated
eserine was depressed
1, indicate
(8.8-1-1.6 m,amoles
in presence by its known
on ACh synthesis.
As the higher ACh content an inhibitory
of slices which
to high K+ medium,
than in its absence
and absence
reduc
by high K' concentration
was not significantly
content
in Fig.
of HC-3 and 6.5 + 1.8 in its absence). inhibitory
in the presence
of ACh content
Ringer
by K+ in the ACh
here on the K+-induced
of slices in the presence
effect of HC-3 on AChE activity,
rat brain microsomes
was examined.
be due to
the influence of HC-3 on AChE activity
As shown
described
of HC-3 might possibly
in Table 1, HC-3 slightly inhibited
of
AChE.
However,
in the experiments
Therefore,
even if HC-3 readily enters the brain slices (19) it seems unlikely that the higher
level of ACh in the slices observed the weak inhibitory has no significant on uptake
effect on activity
of an increase
of choline
of HC-3 is due to
it is kno%vn that
HC-3
(2) and an inhibitory
effect
are
represented
inhibitors.
of the slices.
A more
release of ACh from
th
likely explanation so-called
"Surplus
In the small intestine also, HC-3 has been shown to inhibit
TABLE 1. Inhibition
Data
acetyltransferase
in ACh content it may inhibit
in the brain slices.
without
of this concentration
On the other hand
into brain slices (7, 11-13), but these effects of 1-IC-3 do not indicate
of the effect of HC-3 is that ACh Pool"
in the presence
effect of HC-3 on AChE.
of choline
the possibility
above on release of ACh, 20 ,uM HC-3 was used.
as per
of AChE by HC-3 or eserine.
cent
of
inhibition
to
enzyme
activity
SHORT spontaneous
COMMUNICATIONS
and stretch-induced
The nature the cytoplasm. of "Surplus
of "Surplus
ACh"
release
is not clear, but it may represent
ACh" may be a carrier mediated with uptake
HC-3 (14, 15).
Several
mulation
release
workers
of "Surplus
physiological
tion increased
ACh in a free form in
transmembrane
transport
postulate
ACh"
significances,
release of "Surplus
(21-23)
having by
that the ACh in the pool available
for
form in the synaptic
which was inhibited there
release
to be inhibited
not a bound
though
by HC-3,
phenomenon,
of ACh by the slices which is also known
release is in a free form in the cytoplasm,
important
of ACh (20).
If this is so and if its release from the slices is inhibited
a close relation
this meaning,
Japan. J. Pharmacol. 25, 357 (1975)
by HC-3
is a report
ACh" from the sympathetic
vesicles.
In
may have rather
that
high K+ concentra
ganglion
while electrical
sti
did not (18).
REFERENCES 1) MACINTOSH,F.C., BIRKS, R.I. AND SASTRY,P.B.: Nature 178, 1181 (1956); 2) GARDINER, J.E.: Blochem. J., 81, 297 (1961); 3) BHATNAGAR,S.P. AND MACINTOSH,F.C.: Canad. J. Phy siol. Pharmacol. 45, 249 (1967); 4) SLATER,P.: Int. J. Neuropharmacol., 7, 421 (1968); 5) BROWNING,E.T. AND SCHULMAN,M.P.: J. Neurochemn. 15, 1391 (1968); 6) MARCHBANKS, R.M.: Biochem. Pharmacol. 18, 1763 (1969); 7) GUYENET,P., LEFRESNE,P., ROSSIER,J., BEAU JOUAN,J.C. AND GLOWINSKI,J.: Mol. Pharmacol. 9, 630 (1973); 8) BIRKS, R.I. AND MAC INTOSH,F.C.: Canad..1. Biochem. PhysioL 39, 787 (1961) 9) COLLIER,B. AND MACINTOSH,F.C.: Canal. J. Physiol. Pharmacol. 47, 127 (1969); 10) POTTER,L.T.: J. Physiol. 206,145 (1970); 11) SCHUBERTH, J., SUNDWALL,A., SoRBO, B. ANDLINDELL,J-O.: J. Neurochem. 13, 347 (1966); 12) MARCHBANKs,R.M.,: Biochem. J. 110, 533 (1968); 13) YAMAMURA,H.I. AND SNYDER,S.H.: J. Neuro(-hem. 21, 1355 (1973); 14) SCHUBERTH,J. AND SUNDWALL,A.: J. Neurochem. 14, 807 (1967); 15) PoLAK, R.L.: Brit..I. Pharmacol. 36, 144 (1969); 16) CHANG, H.C. ANDGADDUM, J.H.: J. Physiol. 79, 255 (1933); 17) HESTRIN,S.: J. biol. Chem. 180, 249 (1949); 18) COL LIER,B. AND KATZ, H.S.: J. Physiol. 214, 537 (1971); 19) SLATER,P. AND STONIER,P.D.: J. Neurochem. 20, 637 (1973); 20) YAGASAKI,0., TAKFWAKi,T., UTSUMI, S. AND YANAGIYA, I.: Fo!ia Pharmacol. Japon. 65, 171§ (1969) (in Japanese); 21) WHITTAKER,V.P.: Prog. Brain Res. 31, 211 (1969); 22) RICHTER,J.A. AND MARCHBAKNS, R.M.: J. Neurochem. 18, 691 (1971); 23) RICHTER,J.A. AND MARCHBANKS,R.M.: J. Neurochem. 18, 705 (1971)