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INHIBITORY EFFECT OF ENDOGENOUS MACROMOLECULE ON THE Ca++-STIMULATED ACETYLCHOLINE RELEASE FROM THE CRUDE SYNAPTIC VESICLES
INHIBITORY ON
THE
EFFECT
OF
ENDOGENOUS
Ca++-STIMULATED FROM
THE
CRUDE
Fumiaki
HATA,
Che-Hui
MACROMOLECULE
ACETYLCHOLINE SYNAPTIC KUO, Tomohiro
and Hiroshi
RELEASE
VESICLES MATSUDA
YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, Kita-ku, Osaka 530, Japan Accepted July 19, 1976
There are many studies describing the release of acetylcholine (ACh) from nerve termi nals, but there is little evidence on the biochemical mechanism involved or the interaction of ACh in cytoplasm of nerve terminals with that in synaptic vesicles (1-3).
We carried out
studies on the release of ACh from isolated synaptic vesicles for the purpose of elucidating these problems.
We reported the stimulatory effect of ATP and MgC12 on ACh release
from the crude synaptic vesicles obtained from rat brain homogenate (4) and also that there
was a peptide-like factor in cell sap required for Call-stimulated
ACh release (5).
In the
present study, inhibitory effect of endogenous macromolecule on the Ca++-stimulated ACh release from isolated crude synaptic vesicles is described. Adult healthy Sprague-Dawley strain rats were used. and soluble supernatant,
Crude synaptic vesicle fraction
cell sap, were obtained by subjecting the crude mitochondrial
fraction to a hypotonic shock as described previously (5).
The crude synaptic vesicles
which were prepared from about l g of original brain tissue, were suspended in 6 ml of basic mixture containing 120 mM-KCI, 20 mM-NaCI, 10 mM-Tris HCI buffer (pH 7.4), 50p M-eserine sulphate and centrifuged at 100,000 x g for 40 min after standing for 20 min at 0°C. The resulting supernatant containing about 200 /tg protein per ml was used as "vesicular extract" . The pellet containing about 1.0 mpmole of ACh was used as crude synaptic vesicles suspended in 5 ml of basic mixture in a tube with other additions as indi cated.
Incubations were carried out at 20'C for 20 min.
After re-centrifugation, released
ACh was estimated by measuring the ACh remaining in the pellet as described previously (5) on a strip of rectus abdominis muscle of Rana Pipiens essentially according to the method of Chang and Gaddum (6). Cell sap stimulated ACh release in the presence of ATP, magnesium and calcium as reported previously.
On the other hand, we found here that "vesicular extract" inhibited
the stimulated ACh release as shown in Table 1.
Although the effect was rather weak,
heated "vesicular extract" which had been pretreated at 100'C for 20 min showed a greater inhibitory effect on the release indicating that the inhibitory factor was heat stable.
No
significant influence was observed on ACh release by ATP, magnesium and calcium in the absence of cell sap (Table 1). The "vesicular extract" was then filtrated through a col lodione membrane at 4'C and treated with heat.
TABLE 1.
Effects
magnesium,
of "vesicular calcium
extract"
This heated filtrate showed no inhibitory
on ACh
release
enhanced
by ATP,
and cell sap
Amount of ACh released was expressed as percentage of total ACh contained in the unincubated sample. The standard deviations and means are shown with the numbers of experiments in brackets. ATP : 2 mM, Mg++ : 2 MM-MgC12i Ca++ : 0.1 mM-CaC12, Sap : 2 ml * P 0.001 ; Significantly different from 1) ** P 0.001; Significantly different from 2) • P 0.05, •• P 0.001; Significantly different from 3) • vesicular extract was filtrated through a collodione membrane and treated with heat.
effect on the ACh release indicating
that the factor was impermeable
to a collodione
mem
brane perhaps as a result of its high molecular weight. These findings suggest that in "vesicular extract" there were at least two types of regulators on the Ca++ -stimulated ACh release,
a heat stable high molecular
stimulator
described
procedures
previously
with salt solution
ACh release (5). be extracted
inhibitor
and small amount
(5).
It was also reported
resulted
in an enhanced
Such is in parallel
of heat labile peptide-like
in our previous paper that washing
effect of cell sap on Ca'-stimulated
with the present results that an inhibitory
from the crude vesicle fraction
by a solution
having a certain
factor could
degree of ionic
strength. It is known that calcium binding protein is present in pig brain tissue (7, 8), porcine heart (9) and beef adrenal medulla (10) insoluble of this protein
and also in particle fractions
was studied and identified
terase (12) and of adenylate
as an activator
cyclase (11, 13).
(9, 11).
of cyclic nucleotide
One of its characteristics
The nature phosphodies
is that it is heat
stable (12, 14). The peptide-like tially calcium inhibitory through
ions.
stimulatory Accordingly
effect by decreasing its Ca++-binding
on the Ca++-stimulated
factor
on ACh release previously
"vesicular
concentration
ability,
though
extract"
treated
reported
with heat
requires
might
exhibit
of free calcium ion in the incubation
other possibilities
essen
such as direct inhibitory
an
mixture effect
ACh release should also be considered.
REFERENCES 1) WHITTAKER,V.P., MICHELSON,l.A. ANDKIRKLAND,J.A.: Biochem. J. 90,293 (1964); 2) DE ROBERTIS,E., RODRIGUEZDE LURESARNAIZ, G., SALGANICOFF, L., PELLEGRINO DE IRALDI,A. AND ZIEHER,L.M.: J. Neurochem. 10, 225 (1963); 3) MARCHBANKS, R.M. AND ISRAEL,M.: J. Neuro chem. 18, 439 (1971); 4) MATSUDA,T., HATA, F. ANDYOSHIDA,H.: Biochim. Biophys. Acta 150, 739(1968); 5) HATA, F., KUO, C.H., MATSUDA,T. AND YOSHIDA,H.: J. Neurochem. 27, 139 (1976); 6) CHANG, H.C. AND GADDUM,J.H.: J. Physiol. 79, 255 (1933); 7) WOLFF, D.J. AND SIEGEL, F.L.: Arch. Biochem. Biophys. 150, 578 (1972); 8) WOLFF, D.J. AND SIEGEL, F.L.: J. biol. Chem. 247, 4180 (1972); 9) WALLACE,G.A. AND HARARY,I.: Biochem. biophys. Res. Commun. 67, 810 (1975); 10) BROOKS,J.C. AND SIEGEL, F.L.: J. biol. Chem. 248, 4189 (1973); 11) CHEUNG, W.Y., BRADHAM,L.S., LYUCH, T.J., LIN, Y.M. AND TALLANT, E.A.: Biochem. biophys. Res. Commun. 66, 1055 (1975); 12) WOLFF,D.J. ANDBROSTROM, C.O.: Arch. Biochem. Biophys. 163, 349 (1974); 13) BROSTROM,C.O., HUANG, Y.C., BRECKENRIDGE, B. McL. ANDWOLFF,D.J.: Proc. natn. Acad. Sci. U.S.A. 72,64 (1975); 14) UZUNOV,P., REVUELTA, A. AND COSTA,E.: Mol. Pharmacol. 11, 506 (1975)
THE
EFFECT
OF
ENDOGENOUS
Ca++-STIMULATED FROM
THE
CRUDE
Fumiaki
HATA,
Che-Hui
MACROMOLECULE
ACETYLCHOLINE SYNAPTIC KUO, Tomohiro
and Hiroshi
RELEASE
VESICLES MATSUDA
YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, Kita-ku, Osaka 530, Japan Accepted July 19, 1976
There are many studies describing the release of acetylcholine (ACh) from nerve termi nals, but there is little evidence on the biochemical mechanism involved or the interaction of ACh in cytoplasm of nerve terminals with that in synaptic vesicles (1-3).
We carried out
studies on the release of ACh from isolated synaptic vesicles for the purpose of elucidating these problems.
We reported the stimulatory effect of ATP and MgC12 on ACh release
from the crude synaptic vesicles obtained from rat brain homogenate (4) and also that there
was a peptide-like factor in cell sap required for Call-stimulated
ACh release (5).
In the
present study, inhibitory effect of endogenous macromolecule on the Ca++-stimulated ACh release from isolated crude synaptic vesicles is described. Adult healthy Sprague-Dawley strain rats were used. and soluble supernatant,
Crude synaptic vesicle fraction
cell sap, were obtained by subjecting the crude mitochondrial
fraction to a hypotonic shock as described previously (5).
The crude synaptic vesicles
which were prepared from about l g of original brain tissue, were suspended in 6 ml of basic mixture containing 120 mM-KCI, 20 mM-NaCI, 10 mM-Tris HCI buffer (pH 7.4), 50p M-eserine sulphate and centrifuged at 100,000 x g for 40 min after standing for 20 min at 0°C. The resulting supernatant containing about 200 /tg protein per ml was used as "vesicular extract" . The pellet containing about 1.0 mpmole of ACh was used as crude synaptic vesicles suspended in 5 ml of basic mixture in a tube with other additions as indi cated.
Incubations were carried out at 20'C for 20 min.
After re-centrifugation, released
ACh was estimated by measuring the ACh remaining in the pellet as described previously (5) on a strip of rectus abdominis muscle of Rana Pipiens essentially according to the method of Chang and Gaddum (6). Cell sap stimulated ACh release in the presence of ATP, magnesium and calcium as reported previously.
On the other hand, we found here that "vesicular extract" inhibited
the stimulated ACh release as shown in Table 1.
Although the effect was rather weak,
heated "vesicular extract" which had been pretreated at 100'C for 20 min showed a greater inhibitory effect on the release indicating that the inhibitory factor was heat stable.
No
significant influence was observed on ACh release by ATP, magnesium and calcium in the absence of cell sap (Table 1). The "vesicular extract" was then filtrated through a col lodione membrane at 4'C and treated with heat.
TABLE 1.
Effects
magnesium,
of "vesicular calcium
extract"
This heated filtrate showed no inhibitory
on ACh
release
enhanced
by ATP,
and cell sap
Amount of ACh released was expressed as percentage of total ACh contained in the unincubated sample. The standard deviations and means are shown with the numbers of experiments in brackets. ATP : 2 mM, Mg++ : 2 MM-MgC12i Ca++ : 0.1 mM-CaC12, Sap : 2 ml * P 0.001 ; Significantly different from 1) ** P 0.001; Significantly different from 2) • P 0.05, •• P 0.001; Significantly different from 3) • vesicular extract was filtrated through a collodione membrane and treated with heat.
effect on the ACh release indicating
that the factor was impermeable
to a collodione
mem
brane perhaps as a result of its high molecular weight. These findings suggest that in "vesicular extract" there were at least two types of regulators on the Ca++ -stimulated ACh release,
a heat stable high molecular
stimulator
described
procedures
previously
with salt solution
ACh release (5). be extracted
inhibitor
and small amount
(5).
It was also reported
resulted
in an enhanced
Such is in parallel
of heat labile peptide-like
in our previous paper that washing
effect of cell sap on Ca'-stimulated
with the present results that an inhibitory
from the crude vesicle fraction
by a solution
having a certain
factor could
degree of ionic
strength. It is known that calcium binding protein is present in pig brain tissue (7, 8), porcine heart (9) and beef adrenal medulla (10) insoluble of this protein
and also in particle fractions
was studied and identified
terase (12) and of adenylate
as an activator
cyclase (11, 13).
(9, 11).
of cyclic nucleotide
One of its characteristics
The nature phosphodies
is that it is heat
stable (12, 14). The peptide-like tially calcium inhibitory through
ions.
stimulatory Accordingly
effect by decreasing its Ca++-binding
on the Ca++-stimulated
factor
on ACh release previously
"vesicular
concentration
ability,
though
extract"
treated
reported
with heat
requires
might
exhibit
of free calcium ion in the incubation
other possibilities
essen
such as direct inhibitory
an
mixture effect
ACh release should also be considered.
REFERENCES 1) WHITTAKER,V.P., MICHELSON,l.A. ANDKIRKLAND,J.A.: Biochem. J. 90,293 (1964); 2) DE ROBERTIS,E., RODRIGUEZDE LURESARNAIZ, G., SALGANICOFF, L., PELLEGRINO DE IRALDI,A. AND ZIEHER,L.M.: J. Neurochem. 10, 225 (1963); 3) MARCHBANKS, R.M. AND ISRAEL,M.: J. Neuro chem. 18, 439 (1971); 4) MATSUDA,T., HATA, F. ANDYOSHIDA,H.: Biochim. Biophys. Acta 150, 739(1968); 5) HATA, F., KUO, C.H., MATSUDA,T. AND YOSHIDA,H.: J. Neurochem. 27, 139 (1976); 6) CHANG, H.C. AND GADDUM,J.H.: J. Physiol. 79, 255 (1933); 7) WOLFF, D.J. AND SIEGEL, F.L.: Arch. Biochem. Biophys. 150, 578 (1972); 8) WOLFF, D.J. AND SIEGEL, F.L.: J. biol. Chem. 247, 4180 (1972); 9) WALLACE,G.A. AND HARARY,I.: Biochem. biophys. Res. Commun. 67, 810 (1975); 10) BROOKS,J.C. AND SIEGEL, F.L.: J. biol. Chem. 248, 4189 (1973); 11) CHEUNG, W.Y., BRADHAM,L.S., LYUCH, T.J., LIN, Y.M. AND TALLANT, E.A.: Biochem. biophys. Res. Commun. 66, 1055 (1975); 12) WOLFF,D.J. ANDBROSTROM, C.O.: Arch. Biochem. Biophys. 163, 349 (1974); 13) BROSTROM,C.O., HUANG, Y.C., BRECKENRIDGE, B. McL. ANDWOLFF,D.J.: Proc. natn. Acad. Sci. U.S.A. 72,64 (1975); 14) UZUNOV,P., REVUELTA, A. AND COSTA,E.: Mol. Pharmacol. 11, 506 (1975)