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Inhibition of [3H] MPTP binding to rat brain by pargyline.
BiochemicalPharmacology. Vol.33,No.24,pp 41054107, 1984
000~29S@d$3.00 + 0.W 0 1984Pergamon Press Ltd.
Printed inGreatBritain.
PRELIMINARY COMMUNICATION
INHIBITION
OF
MPTP BINDING
M.Del Zompo, A.Bocchetta, Clinical
Pharmacology,
M.P.Piccardi,
University
S.Pintus,
of Cagliari,
accxpted:
(Received: 8 August 1984;
MPTP
TO RAT BRAIN BY PARGYLINE.
Cagliari,
of MPPP
kinsonism
in man (l), rhesus monkey
Recently,
some authors demonstrated
a
1984)
binding
in
by-product
(l-Methyl-4-phenyl-4-propinoxy-piperidine),
saturable
high-affinity,
ITALY
September
10
(1-Methyl-4-phenyl-1,2,5,6_tetrahydropyridine),
synthesis
G.U.Corsini
the
induces par-
(2) and squirrel monkey (3). by quantitative
site
exists
autoradiography 3 H [
for
1
-MPTP
that a
in rat brain
(4). We
to
evaluated the
and report
mechanism
binding
MPTP
sites are present
amine oxidase tissue
from male
Tissue
toxicity.
Sprague-Dawley in 50 volumes
pellets
50
volumes
were
of
were used
for the samples.
cubation
binding
studies
of
(w/v) of ice-cold
Tris
HCl
buffer
the
were
M). The
pellets
assay. Aliquots
determined
assay on brain homogenates
(85 Ci/mmole,
New England
concentration
of 5 nM. Each
assay contained
50-70,ug
(pH 7.0).
out at 4OC for 30 min. and then 7 ml of ice-cold to each tube and the samples filters
under
low vacuum.
with ice-cold
buffer.
radioactivity
was measured
assay
was
difference unlabeled
performed between MPTP
were immediately The filtration
The filters
in total
with
liquid
triplicate. binding
and
(Aldrich). 4105
at a final
proteins.
The
was carried
Tris HCl buffer was added through Whatman
binding in
the
GF/B
by two 7 ml rinses
in scintillation
scintillation
binding
using an in-
Incubation
was followed
Specific
to Lowry et
Nuclear) tissue
filtered
were suspended a
finally
assay, of the aliquots
[I
in 0.05 M Tris HCl
twice
of homogenate
was measured
and
were performed
were
according
in the routine
(pH
at 17,300 x
and resuspended
of homogenate
assays
brain
was homogenized
homogenates
centrifuged
(0.05
for
Proteins
11 -MPTP
of mono-
0.05 M Tris HCl buffer
by centrifuging
volume of 500 ~1 consisting, 3
L-1
that inhibitors
The tissue
rats was used.
were washed,
ice-cold
in 50 volumes
L1
that can be related 3 H -MPTP confirm that
with these sites. In our experiments
prepared
suspended
al (5). 3 The H -MPTP
Our
properties
in rat brain and suggest
g for 10 min. Tissue pellets with
some molecular
(MAO) may interact
using a polytron 7.0).
of
here
fluid and
spectrometer. is
defined
presence
of
as 10
The the uM
4106
When
the brain homogenates were incubated with increasing concentrations of 37 Hj-XPTP CO.5 to 280 n%f, a specific binding was obtained. Saturation I isotherm was reached by the specific binding but not by the non-specific
one which increased linearly (data not shown) (Fig.I).
SATURATION
AND
OF [“H] MPTP
ON
SCATCHARD RAT
PLOTS
BRAIN
FIG.~. Saturation isatherm and Scatchard analysis (insert) of binding ta rat brain homogenate. are shown. Bound = fmoles of protein. Free = concentration of Scatchard analysis resulted in one straight line, indicating an apparently homogenous population af binding sites. The apparent KD was 87.7 nM and the Bmax was 4315 fmol/mg protein. At a concentration of 1 PM, a variety of compounds such as dopamine, apomorphine, (+) butaclamol, (-1 sulpiride, amphetamine, norepinephrine and 3 MPTP binding to brain homogenates. verapamil failed to inhibit the K II On the contrary, pargyline, clorgyline and deprenyl displaced specific 3 H MPTP binding as shoxvnin Fig.;?. Ci It is noteworthy that pargyline and the selective inhibitor of type A 3 MPTP binding at concentrations MAD, clorgyline, inhibited specific ClH similar to those of unlabeled MPTP. Moreover, other data from our lab demonstrated that MPTP decreases DOPAC formation both
in
viva and in vitro, suggesting that tnis
compound
Preliminary communication
Fig.2.
Inhibition
producing
50%
of
3
[I H
.3 + .Ol ,uM; Pargyline =
different
PI
of specific
H
=
MPTP
drugs. The concentration
binding
(IC50) was: MPTP =
.52 -+ .04 ,uM; Clorgyline = .66 _+ .03&M; Deprenyl above data approximately 74% of total binding was are mean values (+ - SD) of at least three deter-
In the .7,uM. These values
9,
MPTP binding
inhibition
4107
specific.
minations.
inhibits
MAO
paration)./ sites
may
crucial
activity
In
(M.P.Piccardi
conclusion,
be
related
these
to MAO
and data
activity
G.U.Corsini, suggest
and MAO
manuscript
in pre-
the
binding
that activity
MPTP
may
therefore
be
for MPTP neurotoxicity.
REFERENCES
1) J.W.LANGSTON, 2) R.S.BURNS,
P.BALLARD,
C.C.CHIUEH,
Proc.Natl.Acad.Sci.USA 3) J.W.LANGSTON, 390,
J.W.TETRUD,
S.P.MARKEY. 80, 4546,
L.S.FORNO,
I.IRWIN.
M.H.EBERT,
Science,
219, 979 (1983).
D.J.JACOBOWITZ,
I.J.KOPIN.
(1983).
C.S.REBERT,
I.IRWIN.
Brain Research
292,
(1984).
4) C.M.WIECZOREK, 51 O.H.LOWRY,
B.PARSONS,
N.J.ROSENBROUGH,
265, (1951).
T.C.RAINBOW. A.L.FARR,
Eur.J.Pharmacol. R.J.RANDALL.
98, 453,
J.Biol.Chem.
(1984). 193,
000~29S@d$3.00 + 0.W 0 1984Pergamon Press Ltd.
Printed inGreatBritain.
PRELIMINARY COMMUNICATION
INHIBITION
OF
MPTP BINDING
M.Del Zompo, A.Bocchetta, Clinical
Pharmacology,
M.P.Piccardi,
University
S.Pintus,
of Cagliari,
accxpted:
(Received: 8 August 1984;
MPTP
TO RAT BRAIN BY PARGYLINE.
Cagliari,
of MPPP
kinsonism
in man (l), rhesus monkey
Recently,
some authors demonstrated
a
1984)
binding
in
by-product
(l-Methyl-4-phenyl-4-propinoxy-piperidine),
saturable
high-affinity,
ITALY
September
10
(1-Methyl-4-phenyl-1,2,5,6_tetrahydropyridine),
synthesis
G.U.Corsini
the
induces par-
(2) and squirrel monkey (3). by quantitative
site
exists
autoradiography 3 H [
for
1
-MPTP
that a
in rat brain
(4). We
to
evaluated the
and report
mechanism
binding
MPTP
sites are present
amine oxidase tissue
from male
Tissue
toxicity.
Sprague-Dawley in 50 volumes
pellets
50
volumes
were
of
were used
for the samples.
cubation
binding
studies
of
(w/v) of ice-cold
Tris
HCl
buffer
the
were
M). The
pellets
assay. Aliquots
determined
assay on brain homogenates
(85 Ci/mmole,
New England
concentration
of 5 nM. Each
assay contained
50-70,ug
(pH 7.0).
out at 4OC for 30 min. and then 7 ml of ice-cold to each tube and the samples filters
under
low vacuum.
with ice-cold
buffer.
radioactivity
was measured
assay
was
difference unlabeled
performed between MPTP
were immediately The filtration
The filters
in total
with
liquid
triplicate. binding
and
(Aldrich). 4105
at a final
proteins.
The
was carried
Tris HCl buffer was added through Whatman
binding in
the
GF/B
by two 7 ml rinses
in scintillation
scintillation
binding
using an in-
Incubation
was followed
Specific
to Lowry et
Nuclear) tissue
filtered
were suspended a
finally
assay, of the aliquots
[I
in 0.05 M Tris HCl
twice
of homogenate
was measured
and
were performed
were
according
in the routine
(pH
at 17,300 x
and resuspended
of homogenate
assays
brain
was homogenized
homogenates
centrifuged
(0.05
for
Proteins
11 -MPTP
of mono-
0.05 M Tris HCl buffer
by centrifuging
volume of 500 ~1 consisting, 3
L-1
that inhibitors
The tissue
rats was used.
were washed,
ice-cold
in 50 volumes
L1
that can be related 3 H -MPTP confirm that
with these sites. In our experiments
prepared
suspended
al (5). 3 The H -MPTP
Our
properties
in rat brain and suggest
g for 10 min. Tissue pellets with
some molecular
(MAO) may interact
using a polytron 7.0).
of
here
fluid and
spectrometer. is
defined
presence
of
as 10
The the uM
4106
When
the brain homogenates were incubated with increasing concentrations of 37 Hj-XPTP CO.5 to 280 n%f, a specific binding was obtained. Saturation I isotherm was reached by the specific binding but not by the non-specific
one which increased linearly (data not shown) (Fig.I).
SATURATION
AND
OF [“H] MPTP
ON
SCATCHARD RAT
PLOTS
BRAIN
FIG.~. Saturation isatherm and Scatchard analysis (insert) of binding ta rat brain homogenate. are shown. Bound = fmoles of protein. Free = concentration of Scatchard analysis resulted in one straight line, indicating an apparently homogenous population af binding sites. The apparent KD was 87.7 nM and the Bmax was 4315 fmol/mg protein. At a concentration of 1 PM, a variety of compounds such as dopamine, apomorphine, (+) butaclamol, (-1 sulpiride, amphetamine, norepinephrine and 3 MPTP binding to brain homogenates. verapamil failed to inhibit the K II On the contrary, pargyline, clorgyline and deprenyl displaced specific 3 H MPTP binding as shoxvnin Fig.;?. Ci It is noteworthy that pargyline and the selective inhibitor of type A 3 MPTP binding at concentrations MAD, clorgyline, inhibited specific ClH similar to those of unlabeled MPTP. Moreover, other data from our lab demonstrated that MPTP decreases DOPAC formation both
in
viva and in vitro, suggesting that tnis
compound
Preliminary communication
Fig.2.
Inhibition
producing
50%
of
3
[I H
.3 + .Ol ,uM; Pargyline =
different
PI
of specific
H
=
MPTP
drugs. The concentration
binding
(IC50) was: MPTP =
.52 -+ .04 ,uM; Clorgyline = .66 _+ .03&M; Deprenyl above data approximately 74% of total binding was are mean values (+ - SD) of at least three deter-
In the .7,uM. These values
9,
MPTP binding
inhibition
4107
specific.
minations.
inhibits
MAO
paration)./ sites
may
crucial
activity
In
(M.P.Piccardi
conclusion,
be
related
these
to MAO
and data
activity
G.U.Corsini, suggest
and MAO
manuscript
in pre-
the
binding
that activity
MPTP
may
therefore
be
for MPTP neurotoxicity.
REFERENCES
1) J.W.LANGSTON, 2) R.S.BURNS,
P.BALLARD,
C.C.CHIUEH,
Proc.Natl.Acad.Sci.USA 3) J.W.LANGSTON, 390,
J.W.TETRUD,
S.P.MARKEY. 80, 4546,
L.S.FORNO,
I.IRWIN.
M.H.EBERT,
Science,
219, 979 (1983).
D.J.JACOBOWITZ,
I.J.KOPIN.
(1983).
C.S.REBERT,
I.IRWIN.
Brain Research
292,
(1984).
4) C.M.WIECZOREK, 51 O.H.LOWRY,
B.PARSONS,
N.J.ROSENBROUGH,
265, (1951).
T.C.RAINBOW. A.L.FARR,
Eur.J.Pharmacol. R.J.RANDALL.
98, 453,
J.Biol.Chem.
(1984). 193,