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Reduced α2-Adrenoceptor Binding in Lower Brainstem of Stroke-Prone Spontaneously Hypertensive Rats
Short
Communications
Reduced ƒ¿2-Adrenoceptor
Binding
Stroke-Prone
YAMADA,
Department
of
is
accumulated
evidence
involvement
mechanisms
in
mental
of
the
adrenoceptors, and
especially
the
physiological functions
are
demonstrated
a
taneously little
hypertensive
ations
is
rats.
in To
study
central ƒ¿-adrenoceptors have
specific
Wistar SHR
rats
(SHRSP),
a specific
sites
loss
in the WKY
and killed
was
rapidly
20 twice
volumes
in
the
(20.4 (80.2 (8, 9). incubated
mg The
were (17.4
Ci/mmol, Briefly, with
performed NEN),
NEN) NEN), the
original
and as
brain varying
of
buffer.
assays
et al.
was for
using
blood
pressure
week
old
The
a3H-
3H-yohimbine
in
WKY
and
1.00•}0.06
of
3H-prazosin,
yohimbine of high
effects
specific
the
brain
higher
were of
prazosin
in
region.
higher
The
of
binding
times)
of the
adrenergic each
than
3H-
WKY was expected and ƒ¿2inhibitory drugs
on
radioligand a
to
selective ƒ¿1
showed affinity
sites
binding and
of ƒ¿1shows
Prazosin,
whole
0.77•}0.04
specific
brainstem exhibited
of
in
mmHg, of
was
antagonist, (1300
data (11).
age-matched
(g/rat)
SHRSP
values)
adrenoceptor
described
binding
SHRSP=231•}6
binding
of
albumin
markedly
specificity Table 1
(1050
the
of
serum
than
weight
lower and
a
method
3H-clonidine
pharmacological adrenoceptors.
in were
described
(n=15).
in the affinity
of
PPO),
assays
of
was
and
a
liter
g of
the
bovine
SHRSP
wet
of
Determination
previously
(WKY=132•}4,
ex-
ml 1
0.3
All
analysis
as
6
determined
by
using
The
was in
and
was
(10),
GF/B
toluene,
duplicate.
performed The
DPO
performed
standard.
heart
3H-clonidine
homogenates concentrations
Lowry
n=15).
The
tissue
previously
was
of
counter.
in
protein
WKY
washed
g of
scintillation
16-20
the for
15
filtra-
immediately of ice-cold
overnight
radioactivity
conducted
age
homogeCI buffer
was of
liquid
as
brain
sec), and 38,000•~g
at
binding
Ci/mmol,
Ci/mmol,
(2•~30
volume
5-10
assay.
mM
X-100,
rapid
were 4 ml
liters
the
phentolamine.
radioactivity
filters (2
as in
Whatman
Filters with
bound the
defined
by
through
fluid
the
was
brainstem
was Tris-H
Triton
10 ƒÊM
terminated
a vacuum
for
Specific
determined
of
was
40
buffer
was
binding
fiber filters. three times
and
there
the
lower
pellet
original
adrenoceptors prazosin
The
oblongata) of 50
under
for
3H-clonidine)
phosphate
presence
scintillation
and of
weeks and
tion
Tris-HCI
and
radioligand in
and reaction
mM
(3H-yohimbine).
each
of
SHRSP.
16-20
resulting
of per
at
of
The
50
Na/K
4•Ž
difference
Tissue
we
that
mM
from
binding
of
a Polytron was centrifuged
The
report
the
in
at
buffer.
stroke-prone
decapitation,
removed.
equivalent used
SHRSP
with
min.
now
brainstem
by
(pons+medulla nized in 40 (pH 7.7) homogenate
we
and
50
min
tracted
selective
of ƒ¿2-adrenoceptor
lower
were
of
3H-clonidine lower brainstem
(WKY)
and
hypertension,
binding
3H-prazosin, in the
Kyoto
significance
Sciences,
(3H-prazosin
of
alter-
brainstem
the in
examined
radioligands, 3H-yohimbine
the
of
HAYASHI,
25•Ž
in
glass washed
However,
regarding
in ƒ¿-adrenoceptors
hypertensive
is
(SHR).
available
spon-
(1984)
Rats
radioligand
absence have of
in
Brainstem
Pharmaceutical
binding
cardiovascular others (7)
468
Japan
at
120
the
35,
1984
and
experi-
enhancement
rats
information
25,
min
in
-adrenoceptors
of
422,
each
hypothalamus
of and
significant
hypothalamic ƒ¿1
indicate
implicated
regulation 5). We (6)
(4,
May
Central ƒ¿-
in the
brainstem,
Accepted
of
(1-3).
Pharmacol.
HAYASHI
College
Shizuoka
to
Lower
Machiko
Eiichi
Oshika,
catecholaminergic
pathogenesis
hypertension
and
Shizuoka
2-2-1
There
ISHIMA,
TOMITA
Pharmacology,
J.
Hypertensive
Tsuyoshi
Takako
possible
in
Spontaneously
Shizuo
a
Japan.
for
much
brain
yohimbine,
3Han
Short Communications Table 1. Inhibition of specific binding of {3H]prazosin, drugs in the lower brainstem of WKY
The
degree
of
inhibition
[3H]yohimbine concentrations for
of
displacing
value
of
(0.48 these
50%
represents
specific
nM)
the
binding
of
adrenergic
drugs.
of
the
by
The
specific
1050
from
was
values
binding
mean•}S.E.
of
3 to
which
each
of
value
represents
WKY
were
expressed
±3.2
fmol/mg
prazosin, in
the
on
ligand
of
in
prazosin while
of
of WKY
antagonist.
and
competing
Thus labels ƒ¿1 3H-clonidine
protein
with and these
On
clonidine the
specific
6
data
suggest
to
10
molar
were
by
nM)
ligand of
log
by adrenergic
(1.38
each
concentrations
calculated
animals. was
concentration
and
with
4 to
unlabled
probit
6
drugs
analysis.
fmol/mg and
SHRSP
the
other
were
more
in
WKY
Each
SHRSP
There
(WKY=62•}5,
than 3H-
selectively, label
α2-adrenoceptors.
The specific binding of each radioligand in the lower brainstem of WKY and SHRSP was measured using five or six concentrations of 3H-clonidine (0.34-4.13 nM), 3H-yohimbine (0 .19-1.89 nM) and 3Hprazosin (0.08-1.68 nM). The maximal binding sites (Bmax) and apparent dissociation constant (Kd) were determined by Scatchard analysis of the saturation isotherms. As shown in Table 2, the analysis demonstrated a significant reduction (23-29%) in the Bmax values for both 3H-clonidine and 3H-
from when
no
protein;
difference
SHRSP=61•}5
value values
SHRSP=60.3
fmol/mg
was
the Bmax
WKY=79.9•}3.5,
SHRSP=30.6•}1.6
protein).
and [3H]prazosin
different and
([3H]clonidine,
binding
that
[3H]yohimbine
*,**Significantly seen
WKY=39.9•}2.9,
3H-yohimbine
-adrenoceptors and 3H-yohimbine
the
[3H]clonidine
incubating
of [3H]clonidine,
significance
[3H]yohimbine,
brainstem
3H-clonidine
prazosin.
basis
nM), by
are
and [3H]yohimbine
5 animals.
from Similar
SHRSP=126•}7
lower
yohimbine
potent
the
protein;
α2-adrenoceptor
mean•}S.E.
**P<0.01).
WKY=133•}8,
protein
hand,
the
(*P<0.05,
(0.32
determined
Table 2. The B. and Ka for specific binding in the lower brainstem of WKY and SHRSP
Each
[3Hjclonidine
[3H]prazosin
drugs
Japan. J. Pharmacol. 35, 469 (1984)
in
mg/g
the
[3H]amount
tissue,
of
n=10).
yohimbine in SHRSP as compared to WKY, on the bases of both tissue weight and protein concentration. Since the Kd values for 3H-clonidine and 3H-yohimbine were not different from each control value, the decrease in specific binding of these radioligands in SHRSP brainstem is due mainly to a significant alteration in the density of α2-adrenoceptors
the
affinity.
Kd values the
On for
brain
with
those
of
the
and
rat
brain
were
and in
identical
of
induced an
by
it
of
binding
has
been
central ƒ¿1-
regulated
catechol6-hydroxy-
enhancement
3H-clonidine
of is
in
Bmax binding
SHRSP
destruction
(9),
number
noceptors
change
the
WKY.
causes
WB4101
a
3H-prazosin
neurons
dopamine
the
hand,
of
the
aminergic
than
other
specific
region
Because
rather
the
by
suggested and ƒ¿2-adre-
catecholaminergic
3H-
sites
in that
Short Communications neuronal
activity.
was
a
In
decrease
in
α1-adrenoceptors
The
its
are
a
in
reflects
in
adrenergic
two
(4)
an
that
on
and
in
have
the
being
baroreceptor of
clonidine
terms
of
its
noceptors provides that
the a
lower
and
reduction brainstem
is of
10 in
these ƒ¿-adrepresent
evidence
study to
possibly
involved
spontaneous
11
suggest
in ƒ¿2-adrenoceptors
pathogenesis
in of
explained
of
9
Stimu-
hypotensive
be
the
biochemical
the
activation
the
might
Thus,
two
located
an
stimulation (4).
that
pressors.
mimics
8
and
exist in depressors
probably
reflex,
effect
of
Bousquet
being
7
are
inhibition
suggested
brainstem,
an and
regions
and
of ƒ¿-adrenoceptors, lower
and
these
of ƒ¿-adrenoceptors the ƒ¿2-receptors
the
having functions,
Recently,
the ƒ¿1-receptors
lation
exist
pathway,
activation
(5)
populations rat brain:
there
hypothalamic
outflow.
Schwartz
nor-
6 that
adrenergic
both
sympathetic
5
(down
systems
a-adrenoceptors in
is
SHRSP
increased
cardiovascular
bulbar
involved
it
of
assumed
excitatory
inhibitory
SHR
of ƒ¿2-adre-
an
adrenergic
effects
namely,
to
4
activity. has
central
opposing
the
changes
response
neuronal
Hauesler
in
brainstem
compensatory
regulation)
of
and
therefore, loss
lower
not
brainstem
and
specific
the
there but
noradrenaline
reduced
13),
that
study, of ƒ¿2-
lower
of
reportedly (12,
noceptors
the
synthesis
brainstem unlikely
present
number
in
SHRSP. level
the
the
in
the
in
the
hypertension
in
SHRSP.
12 References 1
2
Axelrod,
J.:
Clin.
Mol.
Sci.
Chalmers, experimental 480
3
Catecholamines Med.
J.P.:
51,
Brain
hypertension.
and
hypertension.
415s-421s amines
(1976) and
Circ.
models
Res.
36,
of 469-
(1975)
Saavedra,
J.M.,
Grobecker,
H.
and
Axelrod,
J.:
13
Japan. J. Pharmacol. 35, 470 (1984)
Changes in central catecholaminergic neurons in the spontaneously(genetic) hypertensive rats. Circ. Res. 42, 529-534 (1978) Haeusler, G.: Cardiovascular regulation by central adrenergic mechanisms and its alteration by hypotensive drugs. Circ. Res. 36/37, 1-2231-232 (1975) Bousquet, P. and Schwartz, J.: Alpha-adrenergic drugs. Pharmacological tools for the study of the central vasomotor control. Biochem. Pharmacol. 32, 1459-1465 (1983) Hayashi, E., Yamada, S., Ishima, T. and Tomita, T.: Reciprocal alteration in cardiac and hypothalamic adrenergic receptors in spontaneously hypertensive rats. Japan. Heart J. 24, 804 (1983) Cantor, E.H., Abraham, S. and Spector, S.: Central neurotransmitter receptors in hypertensive rats. Life Sci. 28, 519-526 (1981) Yamada, S., Yamamura, HI and Roeske, W.R.: Alterations in central and peripheral adrenergic receptors in deoxycorticosteroneisalt hypertensive rats. Life Sci. 27, 2405-2416 (1980) U'Prichard, D.C., Bechtel, W.D., Rouot, B.M. and Snyder, S.H.: Multiple apparent alphanoradrenergic receptor binding sites in rat brain: Effect of 6-hydroxydopamine. Mol. Pharmacol. 16, 47-60 (1979) Lowry, O. H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275 (1951) Yamada, S., Yamamura, H.I. and Roeske, W.R.: Characterization of alpha-1-adrenoceptors in the heart using [3H]WB4101: Effect of 6-hydroxydopamine treatment. J. Pharmacol. Exp. Ther. 215, 176-185 (1980) Yamori, Y., Lovenberg, W. and Sjoerdsma, A.: Norepinephrine metabolism in brainstem of spontaneously hypertensive rats. Science 170, 544-546 (1970) Louis, W.J., Krauss, K.R., Kopin, I.J. and Sjoerdsma, A.: Catecholamine metabolism in hypertensive rats. Circ. Res, 27, 589-594 (1970)
Communications
Reduced ƒ¿2-Adrenoceptor
Binding
Stroke-Prone
YAMADA,
Department
of
is
accumulated
evidence
involvement
mechanisms
in
mental
of
the
adrenoceptors, and
especially
the
physiological functions
are
demonstrated
a
taneously little
hypertensive
ations
is
rats.
in To
study
central ƒ¿-adrenoceptors have
specific
Wistar SHR
rats
(SHRSP),
a specific
sites
loss
in the WKY
and killed
was
rapidly
20 twice
volumes
in
the
(20.4 (80.2 (8, 9). incubated
mg The
were (17.4
Ci/mmol, Briefly, with
performed NEN),
NEN) NEN), the
original
and as
brain varying
of
buffer.
assays
et al.
was for
using
blood
pressure
week
old
The
a3H-
3H-yohimbine
in
WKY
and
1.00•}0.06
of
3H-prazosin,
yohimbine of high
effects
specific
the
brain
higher
were of
prazosin
in
region.
higher
The
of
binding
times)
of the
adrenergic each
than
3H-
WKY was expected and ƒ¿2inhibitory drugs
on
radioligand a
to
selective ƒ¿1
showed affinity
sites
binding and
of ƒ¿1shows
Prazosin,
whole
0.77•}0.04
specific
brainstem exhibited
of
in
mmHg, of
was
antagonist, (1300
data (11).
age-matched
(g/rat)
SHRSP
values)
adrenoceptor
described
binding
SHRSP=231•}6
binding
of
albumin
markedly
specificity Table 1
(1050
the
of
serum
than
weight
lower and
a
method
3H-clonidine
pharmacological adrenoceptors.
in were
described
(n=15).
in the affinity
of
PPO),
assays
of
was
and
a
liter
g of
the
bovine
SHRSP
wet
of
Determination
previously
(WKY=132•}4,
ex-
ml 1
0.3
All
analysis
as
6
determined
by
using
The
was in
and
was
(10),
GF/B
toluene,
duplicate.
performed The
DPO
performed
standard.
heart
3H-clonidine
homogenates concentrations
Lowry
n=15).
The
tissue
previously
was
of
counter.
in
protein
WKY
washed
g of
scintillation
16-20
the for
15
filtra-
immediately of ice-cold
overnight
radioactivity
conducted
age
homogeCI buffer
was of
liquid
as
brain
sec), and 38,000•~g
at
binding
Ci/mmol,
Ci/mmol,
(2•~30
volume
5-10
assay.
mM
X-100,
rapid
were 4 ml
liters
the
phentolamine.
radioactivity
filters (2
as in
Whatman
Filters with
bound the
defined
by
through
fluid
the
was
brainstem
was Tris-H
Triton
10 ƒÊM
terminated
a vacuum
for
Specific
determined
of
was
40
buffer
was
binding
fiber filters. three times
and
there
the
lower
pellet
original
adrenoceptors prazosin
The
oblongata) of 50
under
for
3H-clonidine)
phosphate
presence
scintillation
and of
weeks and
tion
Tris-HCI
and
radioligand in
and reaction
mM
(3H-yohimbine).
each
of
SHRSP.
16-20
resulting
of per
at
of
The
50
Na/K
4•Ž
difference
Tissue
we
that
mM
from
binding
of
a Polytron was centrifuged
The
report
the
in
at
buffer.
stroke-prone
decapitation,
removed.
equivalent used
SHRSP
with
min.
now
brainstem
by
(pons+medulla nized in 40 (pH 7.7) homogenate
we
and
50
min
tracted
selective
of ƒ¿2-adrenoceptor
lower
were
of
3H-clonidine lower brainstem
(WKY)
and
hypertension,
binding
3H-prazosin, in the
Kyoto
significance
Sciences,
(3H-prazosin
of
alter-
brainstem
the in
examined
radioligands, 3H-yohimbine
the
of
HAYASHI,
25•Ž
in
glass washed
However,
regarding
in ƒ¿-adrenoceptors
hypertensive
is
(SHR).
available
spon-
(1984)
Rats
radioligand
absence have of
in
Brainstem
Pharmaceutical
binding
cardiovascular others (7)
468
Japan
at
120
the
35,
1984
and
experi-
enhancement
rats
information
25,
min
in
-adrenoceptors
of
422,
each
hypothalamus
of and
significant
hypothalamic ƒ¿1
indicate
implicated
regulation 5). We (6)
(4,
May
Central ƒ¿-
in the
brainstem,
Accepted
of
(1-3).
Pharmacol.
HAYASHI
College
Shizuoka
to
Lower
Machiko
Eiichi
Oshika,
catecholaminergic
pathogenesis
hypertension
and
Shizuoka
2-2-1
There
ISHIMA,
TOMITA
Pharmacology,
J.
Hypertensive
Tsuyoshi
Takako
possible
in
Spontaneously
Shizuo
a
Japan.
for
much
brain
yohimbine,
3Han
Short Communications Table 1. Inhibition of specific binding of {3H]prazosin, drugs in the lower brainstem of WKY
The
degree
of
inhibition
[3H]yohimbine concentrations for
of
displacing
value
of
(0.48 these
50%
represents
specific
nM)
the
binding
of
adrenergic
drugs.
of
the
by
The
specific
1050
from
was
values
binding
mean•}S.E.
of
3 to
which
each
of
value
represents
WKY
were
expressed
±3.2
fmol/mg
prazosin, in
the
on
ligand
of
in
prazosin while
of
of WKY
antagonist.
and
competing
Thus labels ƒ¿1 3H-clonidine
protein
with and these
On
clonidine the
specific
6
data
suggest
to
10
molar
were
by
nM)
ligand of
log
by adrenergic
(1.38
each
concentrations
calculated
animals. was
concentration
and
with
4 to
unlabled
probit
6
drugs
analysis.
fmol/mg and
SHRSP
the
other
were
more
in
WKY
Each
SHRSP
There
(WKY=62•}5,
than 3H-
selectively, label
α2-adrenoceptors.
The specific binding of each radioligand in the lower brainstem of WKY and SHRSP was measured using five or six concentrations of 3H-clonidine (0.34-4.13 nM), 3H-yohimbine (0 .19-1.89 nM) and 3Hprazosin (0.08-1.68 nM). The maximal binding sites (Bmax) and apparent dissociation constant (Kd) were determined by Scatchard analysis of the saturation isotherms. As shown in Table 2, the analysis demonstrated a significant reduction (23-29%) in the Bmax values for both 3H-clonidine and 3H-
from when
no
protein;
difference
SHRSP=61•}5
value values
SHRSP=60.3
fmol/mg
was
the Bmax
WKY=79.9•}3.5,
SHRSP=30.6•}1.6
protein).
and [3H]prazosin
different and
([3H]clonidine,
binding
that
[3H]yohimbine
*,**Significantly seen
WKY=39.9•}2.9,
3H-yohimbine
-adrenoceptors and 3H-yohimbine
the
[3H]clonidine
incubating
of [3H]clonidine,
significance
[3H]yohimbine,
brainstem
3H-clonidine
prazosin.
basis
nM), by
are
and [3H]yohimbine
5 animals.
from Similar
SHRSP=126•}7
lower
yohimbine
potent
the
protein;
α2-adrenoceptor
mean•}S.E.
**P<0.01).
WKY=133•}8,
protein
hand,
the
(*P<0.05,
(0.32
determined
Table 2. The B. and Ka for specific binding in the lower brainstem of WKY and SHRSP
Each
[3Hjclonidine
[3H]prazosin
drugs
Japan. J. Pharmacol. 35, 469 (1984)
in
mg/g
the
[3H]amount
tissue,
of
n=10).
yohimbine in SHRSP as compared to WKY, on the bases of both tissue weight and protein concentration. Since the Kd values for 3H-clonidine and 3H-yohimbine were not different from each control value, the decrease in specific binding of these radioligands in SHRSP brainstem is due mainly to a significant alteration in the density of α2-adrenoceptors
the
affinity.
Kd values the
On for
brain
with
those
of
the
and
rat
brain
were
and in
identical
of
induced an
by
it
of
binding
has
been
central ƒ¿1-
regulated
catechol6-hydroxy-
enhancement
3H-clonidine
of is
in
Bmax binding
SHRSP
destruction
(9),
number
noceptors
change
the
WKY.
causes
WB4101
a
3H-prazosin
neurons
dopamine
the
hand,
of
the
aminergic
than
other
specific
region
Because
rather
the
by
suggested and ƒ¿2-adre-
catecholaminergic
3H-
sites
in that
Short Communications neuronal
activity.
was
a
In
decrease
in
α1-adrenoceptors
The
its
are
a
in
reflects
in
adrenergic
two
(4)
an
that
on
and
in
have
the
being
baroreceptor of
clonidine
terms
of
its
noceptors provides that
the a
lower
and
reduction brainstem
is of
10 in
these ƒ¿-adrepresent
evidence
study to
possibly
involved
spontaneous
11
suggest
in ƒ¿2-adrenoceptors
pathogenesis
in of
explained
of
9
Stimu-
hypotensive
be
the
biochemical
the
activation
the
might
Thus,
two
located
an
stimulation (4).
that
pressors.
mimics
8
and
exist in depressors
probably
reflex,
effect
of
Bousquet
being
7
are
inhibition
suggested
brainstem,
an and
regions
and
of ƒ¿-adrenoceptors, lower
and
these
of ƒ¿-adrenoceptors the ƒ¿2-receptors
the
having functions,
Recently,
the ƒ¿1-receptors
lation
exist
pathway,
activation
(5)
populations rat brain:
there
hypothalamic
outflow.
Schwartz
nor-
6 that
adrenergic
both
sympathetic
5
(down
systems
a-adrenoceptors in
is
SHRSP
increased
cardiovascular
bulbar
involved
it
of
assumed
excitatory
inhibitory
SHR
of ƒ¿2-adre-
an
adrenergic
effects
namely,
to
4
activity. has
central
opposing
the
changes
response
neuronal
Hauesler
in
brainstem
compensatory
regulation)
of
and
therefore, loss
lower
not
brainstem
and
specific
the
there but
noradrenaline
reduced
13),
that
study, of ƒ¿2-
lower
of
reportedly (12,
noceptors
the
synthesis
brainstem unlikely
present
number
in
SHRSP. level
the
the
in
the
in
the
hypertension
in
SHRSP.
12 References 1
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J.:
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415s-421s amines
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of 469-
(1975)
Saavedra,
J.M.,
Grobecker,
H.
and
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J.:
13
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