Br.
J.
Pharmac.
(1986),
89,
367-375
Inhibition
of
calcium
channels
by
harmaline
and
other
harmala
alkaloids
in
vascular
and
intestinal
smooth
muscles
H.
Karaki',
T.
Kishimoto2,
H.
Ozaki,
K.
Sakata,
H.
Umeno3
&
N.
Urakawa
Department
of
Veterinary
Pharmacology,
Faculty
of
Agriculture,
The
University
of
Tokyo,
Bunkyo-ku,
Tokyo
113,
Japan
1
Effects
of
harmaline
and
other
harmala
alkaloids
on
the
contractions
induced
in
the
vascular
smooth
muscle
of
rabbit
aorta
and
intestinal
smooth
muscle
of
taenia
isolated
from
guinea-pig
caecum
were
examined.
2
In
rabbit
isolated
aorta,
harmaline
inhibited
the
sustained
contraction
induced
by
65.4
mM
K'
with
an
ICo
(concentration
needed
for
50%
inhibition)
of
4.6
x
10-5
M.
This
inhibitory
effect
on
high
K+-
induced
contraction
was
antagonized
by
raising
the
concentration
of
external
Ca2'
but
not
by
Bay
K
8644,
a
Ca2+
channel
facilitator.
Harmaline
also
inhibited
the
sustained
contraction
induced
by
noradrenaline
(10-6
M)
with
an
ICm
of
7.6
x
10-5
M.
The
inhibitory
effects
on
noradrenaline-induced
contractions
were
not
antagonized
by
raising
the
external
Ca2+
concentrations
or
by
Bay
K
8644.
3
In
guinea-pig
taenia,
harmaline
inhibited
the
45.4mM
K+-induced
contraction
with
an
ICso
of
6.8
x
IO-I
M
and
the
carbachol
(106
M)-induced
contraction
with
an
ICE,
of
7.0
x
10-5M.
The
inhibitory
effects
on
both
high
K+-
and
carbachol-induced
contractions
were
antagonized
by
raising
the
external
Ca2+
concentrations
but
not
by
Bay
K
8644.
4
Harmaline,
at
the
concentrations
needed
to
inhibit
the
muscle
contraction,
inhibited
the
increase
in
45Ca2+
uptake
induced
by
high
K',
noradrenaline
and
carbachol
in
aorta
and
taenia.
5
Harmaline
did
not
change
the
cellular
Na+
and
ATP
contents
in
resting
and
high
K+
stimulated
taenia.
6
Other
harmala
alkaloids
also
inhibited
the
contractions
in
these
smooth
muscles.
The
order
of
the
inhibitory
potency
was
6-methoxyharman
=
harmine
>
harmaline
=
2-methylharmine
=
har-
mane
>
6-methoxyharmalan
>
harmalol
=
harmol
for
the
contractions
induced
by
high
K+
in
aorta
and
taenia
and
by
carbachol
in
taenia,
and
2-methylharmine
>
6-methoxyharman
>
6-methoxyhar-
malan
=
harmol
=
harmalol
=
harmane
>
harmine
>
harmaline
for
the
contraction
induced
by
noradrenaline
in
aorta.
7
These
results
suggest
that
harmaline
inhibits
the
contractile
response
of
rabbit
aorta
and
guinea-pig
taenia
by
inhibiting
different
types
of
Ca2`
channel.
The
structure-activity
relationship
indicates
that
the
potency
and
selectivity
of
the
inhibitory
effects
on
these
channels
are
varied
by
modification
of
the
structure
of
this
alkaloid.
Introduction
Harmaline,
an
alkaloid
extracted
from
the
plant
(Canessa
et
al.,
1973).
Further,
harmaline
and
other
Peganum
harmala,
is
a
hallucinogen
(Schultes,
1969).
related
harmala
alkaloids
are
known
to
inhibit
con-
It
also
has
an
inhibitory
effect
on
the
Na+,K+-ATPase
tractile
responses
induced
by
ouabain
and
acetyl-
choline
in
guinea-pig
ileal
smooth
muscle
(Hider
et
al.,
198
1a,b).
However,
the
mechanism
of
the
relaxant
action
in
smooth
muscle
has
not
been
clarified.
'Author
for
correspondence.
I
'Present
address:
Teijin
Co.
Ltd.,
Asahigaoka,
Hino-shi,
In
the
present
study,
we
have
examined
the
mechan-
Tokyo
191,
Japan.
ism
of
the
inhibitory
action
of
harmaline
on
vascular
'Present
address:
Asahi
Chemical
Industry
Co.
Ltd.,
and
intestinal
smooth
muscles.
It
was
found
that
Samejima,
Fuji-shi,
Shizuoka
416,
Japan.
harmaline
has
unique
inhibitory
effects
on
the
dif-
Q
The
Macmillan
Press
Ltd
1986
368
H.
KARAKI
el
al.
ferent
types
of
Ca2"
channel
in
these
smooth
muscles.
The
potency
and
selectivity
of
the
inhibitory
effects
are
varied
by
modifications
of
the
structure
of
the
alk-
aloid.
Some
of
these
experiments
have
been
breifly
reported
at
a
Japanese
Pharmacological
Society
meet-
ing
(Umeno
et
al.,
1985).
Methods
Tissue
preparations
The
thoracic
aorta
was
isolated
from
male
rabbits
(2-3
kg)
and
cut
into
spiral
strips
of
3-4
mm
width
and
about
15mm
length.
The
adventitial
layer
was
removed
from
the
strip
as
described
by
Karaki
&
Urakawa
(1977)
in
order
to
avoid
the
possible
release
of
endogenous
catecholamines
(Karaki
et
al.,
1984a).
Segments
of
taenia,
approximately
10mm
in
length
were
removed
from
the
caecum
of
male
white
guinea-
pigs
weighing
250-300
g.
Solutions
Normal
physiological
salt
solution
contained
(mM):
NaCl
136.9,
KCl
5.4,
CaCl2
1.5,
MgCl2
1.0,
NaHCO3
23.8
and
glucose
5.5.
High
K+
solution
was
made
by
replacing
60
mM
NaCl
with
equimolar
KCI
for
aorta
or
by
adding
40
mM
KCI
to
normal
solution
for
taenia.
The
concentration
of
CaCl2
was
changed
to
0.3
mM
or
7.5
mm
in
some
experiments.
The
solution
was
contin-
uously
bubbled
with
a
95%
02
and
5%
CO2
mixture
at
370C.
The
pH
of
the
solution
was
7.3
±
0.1.
Muscle
tension
Muscle
strips
were
suspended
in
an
organ
bath
(20
ml)
under
resting
tension
of
1
g
for
aorta
and
0.5
g
for
taenia
and
the
contractions
were
recorded
isometrical-
ly
with
a
strain
gauge
transducer
(Nihon-Koden).
The
muscles
were
equilibrated
in
normal
solution
for
at
least
2
h
until
the
contractile
response
to
the
high
K+
solution
became
stable.
After
equilibration,
the
con-
centration
of
external
Ca2+
was
changed
in
some
experiments
and
30
min
later,
muscle
strips
were
stimulated
by
65.4mM
K+
or
1O-6
M
noradrenaline
in
aorta
or
by
45.4
mM
K+
or
10-6
M
carbachol
in
taenia.
Muscle
contraction
reached
a
steady
level
within
30-40min
and
then
harmaline
or
related
alkaloids
were
cumulatively
added
at
15-20min
intervals,
as
shown
in
Figure
1.
In
some
experiments,
a
single
concentration
(2
x
10-4M)
of
harmaline
was
added
during
the
sustained
contraction
to
induce
almost
complete
relaxation
followed
by
an
addition
of
7.5
mM
Ca2+
or
10-7
M
Bay
K
8644,
and
recovery
of
muscle
contraction
was
observed,
as
described
by
Spedding
&
Berg
(1984).
Na+
content
For
the
measurement
of
tissue
Na+
content,
muscle
strips
were
weighed,
tied
to
a
glass
rod
with
silk
string
and
equilibrated
in
normal
solution
for
a
minimum
of
2
h
before
the
start
of
experimental
procedures.
After
incubation
with
a
test
solution,
the
muscles
were
blotted
on
filter
paper
and
transferred
to
a
quartz
test-
tube
containing
0.5
ml
of
a
mixture
containing
equal
volumes
of
HNO3
(61%)
and
HCIO4
(60%)
and
heated
overnight
at
180-240C.
Dried
samples
were
dissolved
in
0.01
N
HCI
shortly
before
the
determina-
tion
of
Na+.
CsCl
(I
g
I-)
was
added
to
the
standard
and
diluted
solutions.
The
Na+
concentration
of
the
diluted
samples
was
measured
with
a
flame
photometer
(Hitachi,
Type
208).
45Ca2+
uptake
Cellular
45Ca2`
content
was
measured
by
a
cold
lanthanum
wash
method
described
by
Karaki
&
Weiss
(1979).
After
a
5
min
incubation
with
a
solution
labelled
with
45Ca2`
(0.5
1Ci
ml-'),
the
muscles
were
washed
with
a
solution
containing
La3+
(LaC13
73.8
mm,
glucose
5.5
mM
and
Tris
23.8
mM
adjusted
to
pH
6.9
with
maleic
acid
at
0°C)
for
30
min
in
order
to
remove
extracellular
bound
45Ca2+.
The
muscles
were
then
placed
in
scintillation
vials
containing
0.5
ml
of
tissue
solubilizer
(Lumasolve,
Lumac)
and
were
diges-
ted
overnight
at
50-60C.
The
solubilized
samples
were
then
mixed
with
5
ml
scintillator
(Lumagel,
Lumac)
and
radioactivity
was
determined
in
a
liquid
scintillation
spectrometer
(Tri-Carb
3380,
Packard).
ATP
content
The
ATP
content
of
the
muscle
was
measured
by
the
method
originally
described
by
Strehler
&
McElroy
(1957)
and
modified
by
Karaki
et
al.
(1982).
After
incubation
in
the
chosen
medium,
muscle
strips
were
boiled
for
5min
in
2
ml
hot
water
and
ATP
was
extracted
for
5
min.
The
extract
was
cooled
to
0°C
and
the
amount
of
ATP
in
the
extract
was
determined
with
a
photon
counter
(Lumac
Biometer)
with
luciferine-
luciferase
reagent.
Saponin-skinned
muscle
Skinned
smooth
muscle
was
prepared
by
the
methods
described
by
Saida
&
Nonomura
(1978).
A
thin
muscle
strip,
approximately
0.1
mm
wide
and
2
mm
long,
was
treated
with
saponin
(100
jgml-')
for
60min
in
a
relaxing
solution
of
the
following
composition:
KCI
136.9
mM,
MgCl2
5
mM,
Tris-maleate
20
mM,
ATP-Na2
5
mM,
EGTA
2mM,
pH
6.8
at
22-24°C.
Ca2+
concen-
tration
was
calculated
using
binding
constant
for
EGTA
with
Ca2'
of
106M-1
(Harafuji
&
Ogawa,
1980).
HARMALA
ALKALOIDS
ON
SMOOTH
MUSCLE
369
Table
1
Structure
of
harmala
alkaloids
CH3
N
9
N
~~3
2
3-4
6
6-Methoxyharman
Harmine
Harmnaline
2-Methylharmine
Harmane
6-Methoxyharmalan
Harmalol
Harmol
=
-OCH3
-H
-H
-OCH3
-
-H
-OCH3
-CH3
=
-H
-OCH3
-H
-H
-
-OCH3
-H
-
-H
-OH
-H
-OH
'Relative
concentration
needed
for
50%
inhibition
(IC50)
of
the
high
K+-induced
contraction
in
rabbit
aorta
taking
the
IC,.
of
harmaline
as
1.00.
2Ratio
of
IC"
value
for
high
K+-induced
contraction
to
that
for
noradrenaline-induced
contraction
in
rabbit
aorta.
Data
are
taken
from
Table
3.
Drugs
and
chemicals
Harmaline
hydrochloride
(Nakarai
Chemicals),
har-
mine
hydrochloride
(Aldrich
Chemical),
harmol
hy-
drochloride
(Sigma
Chemical),
harmalol
hydro-
chloride
(Sigma),
harmane
hydrochloride
(Sigma),
2-
methylharmine
(Sigma),
6-methoxyharman
(Sigma),
6-methoxyharmalan
(Sigma),
saponin
(ICN),
(-)-
noradrenaline
bitartrate
(Wako
Pure
Chemicals),
his-
tamine
hydrochloride
(Wako),
Bay
K
8644
(Schramm
et
al.,
1983;
donated
by
Bayer
AG),
ATP-Na2
(Sigma)
and
45CaC12
(New
England
Nuclear)
were
used.
The
chemical
structures
of
harmala
alkaloids
used
in
the
present
experiments
are
shown
in
Table
1.
Statistics
Results
of
the
experiments
are
expressed
as
mean-
±
s.e.mean.
Student's
t
test
was
used
for
statistical
analysis
and
P
<0.01
was
taken
as
significant.
Results
High
K+
-induced
contractions
Cumulative
application
of
harmaline
inhibited
the
sustained
contractions
induced
by
high-K+
in
rabbit
aorta
and
guinea-pig
taenia
(Figure
1).
Figure
2
shows
the
concentration-response
relationships
for
the
in-
hibitory
effect
of
harmaline
at
three
different
concen-
trations
of
external
Ca2".
The
concentration-response
curves
were
shifted
to
the
right
by
raising
the
concen-
tration
of
external
Ca2"
from
0.3
to
1.5
or
7.5
mM
in
both
aorta
and
taenia.
Interaction
between
harmaline
and
Bay
K
8644,
a
Ca2"
channel
facilitator
(Schramm
et
al.,
1983),
was
examined
as
described
by
Spedding
&
Berg
(1984).
In
both
aorta
and
taenia,
addition
of
2
x
10-4
M
har-
maline
almost
completely
inhibited
the
high
K+-in-
duced
contractions
and
addition
of
7.5mM
Ca2"
recovered
the
muscle
tension.
However,
addition
of
10-7
M
Bay
K
8644
did
not
change
the
muscle
tension
inhibited
by
harmaline
(data
not
shown).
Noradrenaline-
and
carbachol-induced
contractions
As
shown
in
Figure
1,
harmaline
inhibited
the
10-6
M
noradrenaline-induced
contraction
of
rabbit
aorta
and
the
10-6
M
carbachol-induced
contraction
of
taenia.
A
similar
effect
was
obtained
on
the
contrac-
tion
of
the
aorta
induced
by
histamine
(10-
5
M)
(data
not
shown).
Figure
3
shows
the
concentration-res-
ponse
relationship
for
the
inhibitory
effect
of
har-
maline
at
three
different
concentrations
of
external
Ca2+.
The
inhibitory
effect
of
harmaline
on
the
noradrenaline-induced
contraction
in
the
aorta
was
not
modified
by
raising
the
external
Ca2+
concentra-
tion
from
1.5
to
7.5
mM
whereas
the
inhibitory
effect
was
slightly
augmented
by
decreasing
the
Ca2+
con-
centration
to
0.3
mM.
In
contrast,
the
inhibitory
effect
on
the
carbachol-induced
contraction
in
taenia
was
IC50
for
KV-induced
7
contraction'
0.28
0.48
1.00
1.17
1.39
1.80
3.48
4.78
Selectivity'
0.46
0.37
0.61
3.60
1.42
2.24
4.21
5.79
370
H.
KARAKI
el
al.
Rabbit
aorta
10
min
KCI
Noradrenaline
Guinea-pig
taenia
1
2
3
I~
~~~~~~
KCI
1
2
5
Carbachol
Figure
1
Inhibitory
effect
of
harmaline
on
the
contractions
induced
by
65.4mM
K+
and
10-6
M
noradrenaline
in
rabbit
aorta
(left)
and
45.4
mm
K+
and
10-6
M
carbachol
in
guinea-pig
taenia
(right).
Harmaline
was
added
cumulatively
during
the
sustained
contractions
in
these
muscles
(shown
by
dots).
Concentration
of
harmaline
was
as
follows.
(1)
5
x
10
6M,
(2)
10
Nm,
(3)
2
x
105M,
(4)
5
x
10
Nm,
(5)
104M,
(6)
2
x
10-4M
and
(7)
5
x
10
M.
Horizontal
bar
indicates
10
min
and
vertical
bar
indicates
I
g
tension
for
aorta
and
5
g
tension
for
taenia.
Harmaline
(-log
M)
Figure
2
Inhibitory
effects
of
harmaline
on
the
contrac-
tions
induced
by
65.4
mm
K+
in
rabbit
aorta
(a)
and
45.4
mm
K+
in
guinea-pig
taenia
(b)
in
the
presence
of
0.3mM
Ca24
(U),
1.5mM
Ca24
(-)
and
7.5mM
Ca24
(A).
Muscle
strips
were
incubated
with
different
concen-
trations
of
Ca24
for
30
min
before
the
addition
of
stimulant.
Harmaline
was
cumulatively
applied
during
sustained
contraction
induced
by
stimulant,
as
shown
in
Figure
1.
100%
represents
the
muscle
tension
before
the
addition
of
harmaline.
Each
point
represents
mean
of
4
to
6
experiments
and
s.e.mean
is
shown
by
vertical
bar
when
it
is
greater
than
the
symbol.
*Significantly
different
(P
<0.01)
from
the
value
in
1.5
mm
Ca24
solution.
-0
.
Co
c
0
.-_
4C
w
50
F
OL
HARMALA
ALKALOIDS
ON
SMOOTH
MUSCLE
371
Table 2
Changes
in
Na4
and
ATP
content
of
guinea-pig
taenia
50
.
to
10
C
0
4_
0)
cr:
4
Na+
A
TP
(mmol
kg-'
wet
weight)
5
50F
0
.*
Harmaline
(-log
M)
Figure
3
Inhibitory
effects
of
harmaline
on
the
contrac-
tions
induced
by
10-6
M
noradrenaline
in
rabbit
aorta
(a)
and
10-6
M
carbachol
in
guinea-pig
taenia
(b)
in
the
presence
of
0.3mM
Ca24
(U),
1.5
mM
Ca24
(0)
and
7.5
mm
Ca24
(A).
For
further
details,
see
Figure
2.
Control
Ouabain
10-5M
Harmaline
1o-4
M
45.4mM
K+
45.4mM
K+
+
harmaline
10-4
M
7.8
±
0.3
(6)
1.35
±
0.09
(6)
48.1
±
2.5
(6)
-
7.5
±
1.2
(6)
1.14
±
0.08
(6)
-
1.05
±
0.07
(6)
-
1.14±0.10
(6)
Muscle
strips
were
incubated
with
the
high
K+
and/
or
inhibitors
for
60
min.
-:
not
determined.
45Ci+
uptake
As
shown
in
Figure
4,
high
K+
and
noradrenaline
increased
the
45Ca2+
uptake
during
a
5
min
45Ca2+
incubation
period
in
rabbit
aorta.
In
guinea-pig
taenia
coli,
high
K+
and
carbachol
also
increased
the
45Ca2`
uptake.
Although
the
resting
45Ca2+
uptake
was
not
changed
by
5
x
10-4
M
harmaline
in
aorta
and
by
2
x
10-4
M
harmaline
in
taenia.
The
increments
in-
duced
by
high
K+,
noradrenaline
and
carbachol
were
completely
inhibited
by
these
concentrations
of
har-
maline.
Saponin-treated
muscle
In
the
saponin-treated
taenia,
10-6
M
Ca24
induced
a
sustained
contraction.
Addition
of
2
x
10-4
M
har-
maline
during
this
sustained
contraction
decreased
muscle
tension
by
3.6%
and
2
x
I0-3
M
harmaline
by
14.3%
(n=
2).
augmented
by
the
decrease
and
reduced
by
the
in-
crease
in
external
Ca2+
concentrations.
Both
the
noradrenaline-induced
contraction
in
aorta
and
the
carbachol-induced
contraction
in
taenia
were
almost
completely
inhibited
by
2
x
10-4
M
har-
maline.
Addition
of
7.5
mm
Ca2`
recovered
muscle
tension
in
taenia
but
not
in
aorta.
In
contrast,
addition
of
10-7M
Bay
K
8644
did
not
reverse
the
inhibitory
effect
of
harmaline
in
aorta
or
taenia
(data
not
shown).
Na+
and
A
TP
contents
As
shown
in
Table
2,
10-4
M
harmaline
did
not
change
the
tissue
Na+
content,
although
10-M
ouabain,
a
specific
inhibitor
of
Na4,K4-ATPase,
increased
the
Na+
content
in
taenia.
Table
2
also
shows
that
10-4
M
harmaline
had
no
effect
on
the
ATP
content
of
taenia
in
normal
and
high
K+
solutions.
Efjects
of
other
harmala
alkaloids
Cumulative
application
of
harmala
alkaloids,
i.e.,
6-
methoxyharman,
harmine,
2-methylharmine,
har-
mane,
6-methoxyharmalan,
harmalol
and
harmol,
inhibited
the
sustained
contractions
in
aorta
and
taenia.
Concentrations
of
these
alkaloids
needed
to
induce
50%
inhibition
of
the
contractions
(ICO)
in
aorta
and
taenia
are
listed
in
Table
3.
The
order
of
the
inhibitory
potency
for
the
contraction
induced
by
high
K+
in
aorta
was
6-methoxyharman
=
harmine>har-
maline
=
2-methylharmine
=
harmane
>
6-methoxy-
harmalan
>
harmalol
=
harmol.
The
order
of
the
inhibitory
potency
for
the
contraction
of
taenia
in-
duced
by
either
high
K+
or
carbachol
was
almost
the
same.
On
the
noradrenaline-induced
contraction
in
aorta,
in
contrast,
these
alkaloids
showed
a
different
order
of
inhibitory
potency;
2-methylharmine
>6-
methoxyharman
>
6-methoxyharmalan
=
harmol
=
harmalol
=
harmane
>
harmine
>
harmaline.
a
372
H.
KARAKI
et
al.
45Ca
uptake
(nmol
g-1
wt.)
Rabbit
aorta
Control
Harmaline
K4
K+
+
harmaline
Noradrenaline
Noradrenaline
+
harmaline
Guinea-pig
taenia
Control
Harmaline
K
+
_
K+
+
harmaline
_
Carbachol
Carbachol
+
harmaline
-
0
50
100
1
5C
I
i
I
I
I
z~~z3
zIIIzj
-F
+H
+-
+H
IH-
1-
Figure
4
Effects
of
harmaline
on
45Ca2+
uptake
in
rabbit
aorta
and
guinea-pig
taenia.
Harmaline
at
concentrations
of
5
x
0-4
M
and
2
x
10-4
M
was
used
for
aorta
and
taenia,
respectively.
Harmaline
was
added
30
min
before
the
addition
of
45Ca2+
with
or
without
stimulant.
Each
bar
represents
mean
of
6
experiments
and
s.e.mean
is
shown
by
horizontal
error
bar.
*Significantly
greater
(P
<0.01)
than
the
respective
control.
Table
3
Concentrations
of
harmala
alkaloids
needed
to
induce
50%
inhibition
of
the
contraction
induced
in
rabbit
aorta
and
guinea-pig
taenia
6-Methoxyharman
Harmine
Harmaline
2-Methylharmine
Harmane
6-Methoxyharmalan
Harmol
Harmalol
Contractions
induced
by
noradrenaline
high
K+
in
aorta
in
taenia
Harmala
alkaloids
were
cumulatively
added
during
the
sustained
contraction
induced
by
65.4mM
K+
or
10-6
M
noradrenaline
in
aorta
or
45.4
mM
K+
or
10-6
M
carbachol
in
taenia.
Values
are
the
mean
of
6
to
11
experiments.
S.e.means,
which
were
less
than
10%
of
the
respective
means,
are
not
shown.
high
K+
in
aorta
(X
10-5M)
1.3
2.2
4.6
5.4
6.4
8.3
16.0
22.0
2.8
6.0
7.6
1.5
4.5
3.7
3.8
3.8
carbachol
in
taenia
1.3
2.3
7.0
9.0
4.9
6.0
41.0
43.0
0.6
2.3
6.8
6.4
5.0
5.5
31.0
22.0
0
HARMALA
ALKALOIDS
ON
SMOOTH
MUSCLE
373
Discussion
Harmaline
inhibited
all
the
contractions
examined
in
rabbit
aorta
and
guinea-pig
taenia.
Such
nonspecific
effects
suggest
that
harmaline
does
not
inhibit
a
specific
receptor
for
an
agonist.
Further,
harmaline
inhibited
only
slightly
the
Ca2"-induced
contraction
of
the
saponin-treated
taenia,
suggesting
that
the
inhibitory
effect
of
harmaline
is
not
attributable
to
inhibition
of
the
contractile
proteins
in
smooth
mus-
cle.
Harmaline
inhibits
glucose
uptake
in
intestinal
epithelial
cells
by
interfering
with
the
Na'-dependent
sugar
transport
(Sepulveda
et
al.,
1977).
Inhibition
of
glucose
transport
results
in
an
ATP-deficiency
and
inhibition
of
contraction
in
smooth
muscle
(Suzuki
et
al.,
1980;
Karaki
et
al.,
1982).
However,
this
possibility
is
not
likely
because
the
ATP
content
of
taenia
coli
was
not
affected
by
harmaline.
Harmaline
also
inhibits
Na',
K+-ATPase
in
various
tissues
(Canessa
et
al.,
1973;
Becker
&
Willis,
1983).
In
smooth
muscle,
inhibition
of
Na',
K+-ATPase
leads
to
accumulation
of
cellular
Na'
and
inhibition
of
contraction
(Bose,
1975;
Kishimoto
et
al.,
1980).
However,
harmaline
did
not
change
the
Na'
content.
Further
examination
showed
that
the
inhibitory
effects
of
harmaline
on
high
K+-induced
contractions
in
aorta
and
taenia
and
carbachol-induced
contraction
in
taenia
were
antagonized
by
the
external
Ca2+
concentrations
whereas
the
inhibition
of
noradren-
aline-induced
contraction
in
aorta
was
not.
During
these
contractions,
45Ca2+
uptake
of
the
muscle
in-
creased
and
harmaline
inhibited
the increase
in
45Ca2+
uptake
in
these
smooth
muscles.
It
has
been
suggested
that
sustained
contractions
in
smooth
muscle
is
largely
dependent
on
the
influx
of
external
Ca2+
(Cauvin
et
al.,
1983;
Karaki
&
Weiss,
1984),
and
only
initial
transient
contractions
induced
by
noradrenaline
in
rabbit
aorta
(Deth
&
van
Breemen,
1977;
Karaki
et
al.,
1979)
and
by
carbachol
in
guinea-pig
taenia
(Ohashi
et
al.,
1974;
Brading
&
Sneddon,
1980)
are
attributable
to
release
of
cellular
Ca2
.
Inhibition
by
harmaline
of
sustained
contraction
and
associated
45Ca2"
influx
suggest
that
harmaline
inhibits
the
pathways
of
Ca2+
influx
which
are
activated
by
high
K+
and
receptor
agonists.
In
smooth
muscle,
there
are
two
types
of
Ca2+
channels,
voltage-dependent
and
receptor-linked
channels
(Bolton,
1979;
van
Breemen
et
al.,
1979).
In
rabbit
aorta,
these
Ca2+
channels
have
unique
charac-
teristics
in
that
the
voltage-dependent
channels
are
selectively
inhibited
by
the
organic
Ca2+
antagonists
like
verapamil
whereas
the
receptor-linked
channels
are
not
sensitive
to
Ca2+
antagonists
but
are
inhibited
by
sodium
nitroprusside.
In
contrast,
both
types
of
Ca2+
channel
in
guinea-pig
taenia
are
inhibited
by
the
organic
Ca2+
antagonists
(although
the
voltage-de-
pendent
channels
are
more
strongly
inhibited
than
the
receptor
linked
channels)
but
not
by
sodium
nitroprusside.
The
inhibitory
effects
of
the
organic
Ca2"
antagonists
but
not
sodium
nitroprusside
may
be
antagonized
by
raising
the
external
Ca2+
concentra-
tions
(Karaki
&
Weiss,
1984;
Karaki
et
al.,
1984b).
Comparing
these
and
the
present
results,
harmaline
has
inhibitory
effects
similar
to
those
of
the
organic
Ca2+
antagonists;
it
inhibited
high
K+-induced
con-
tractions
in
aorta
and
taenia
and
carbachol-induced
contraction
in
taenia
and
the
effects
were
antagonized
by
external
Ca2
.
In
addition,
harmaline
inhibited
the
noradrenaline-induced
contraction
which
is
not
affec-
ted
by
external
Ca2+
concentrations.
Thus,
harmaline
seems
to
inhibit
both
the
Ca2+
antagonist-sensitive
and
insensitive
(but
sodium
nitroprusside-sensitive)
Ca2+
channels
in
smooth
muscle.
The
inhibitory
effect
of
harmaline
was
not
reversed
by
Bay
K
8644,
a
dihydropyridine
which
stimulates
Ca2'
entry
through
Ca2+
channel
(Schramm
et
al.,
1983).
Spedding
&
Berg
(1984)
found
that
the
in-
hibitory
effect
of
verapamil,
diltiazem
and
dihydropyridine
Ca2+
antagonists
were
antagonized
by
Bay
K
8644
whereas
the
inhibitory
effects
of
diphenyl
alkylamine
Ca2+
antagonists
were
not,
and
proposed
the
use
of
Bay
K
8644
for
classification
of
Ca2+
antagonists.
Thus,
the
site
of
action
of
harmaline
on
Ca2+
channels
seems
to
be
different
from
that
of
verapamil,
diltiazem
and
dihydropyridines.
Although
harmaline
inhibited
these
Ca2+-channels
nonselectively,
some
of
the
harmala
alkaloids
seem
to
have
more
selective
effects
on
one
of
these
two
types
of
Ca2+
channels.
6-Methoxyharman
and
harmine
in-
hibited
the
Ca2+
antagonist-sensitive
contraction
re-
latively
selectively
whereas
harmol
and
harmalol
had
less
effect
on
this
type
of
contraction
(Tables
1
and
3).
These
results
support
the idea
that
the
pathways
of
Ca2+
influx
for
high
K+-
and
noradrenaline-induced
contractions
are
different.
Comparing
the
chemical
structures
and
the
in-
hibitory
effects
on
smooth
muscle
contractions
of
the
harmala
alkaloids
(Tables
1
and
3),
it
was
concluded
that;
(1)
the
presence
of
6-
or
7-methoxy
group
increases
the
inhibitory
effect
on
the
Ca2+
antagonist-
sensitive
channels
but
not
on
the
Ca2+
antagonist-
insensitive
channels;
(2)
7-OH
group
decreases
the
inhibitory
potency
on
the
Ca2`
antagonist-sensitive
channels;
(3)
3-4
double
bond
selectively
increases
the
inhibitory
potency
on
the
Ca2'
antagonist-sensitive
Ca2+
channels
although
this
effect
is
not
seen
in
the
presence
of
7-OH
group;
and
(4)
2-methyl
group
seems
to
increase
the
inhibitory
potency
on
the
Ca2+
antagonist-insensitive
channels.
The
lower
inhibitory
potency
of
the
compounds
with
7-OH
group
on
the
Ca2+
antagonist-sensitive
channels
may
be
due
to
the
decreased
lipophilicity.
However,
since
the
inhibitory
potency
on
the
Ca2+
antagonist-insensitive
Ca2+
channels
is
not
decreased
by
the
7-OH
group,
the
role
374
H.
KARAKI
et
al.
of
the
7-OH
group
may
not
be
explained
solely
by
the
change
in
lipophilicity.
Although
Ca2+
antagonists
are
lipophilic
in
nature,
their
pharmacological
potency
does
not
necessarily
parallel
their
lipophilicity
(Sped-
ding,
1985).
In
conclusion,
it
is
suggested
that
harmaline
inhibits
the
contractile
responses
of
rabbit
aorta
and
guinea-
pig
taenia
by
inhibiting
different
types
of
Ca2+
channel.
The
structure-activity
relationship
indicated
that
the
potency
and
selectivity
of
the
inhibitory
effects
on
these
channels
are
varied
by
modifications
of
the
structure
of
this
alkaloid.
This
work
is
a
part
of
the
M.Sc.
thesis
by
H.U.
This
work
was
supported
by
a
research
grant
(No.
60850048)
from
the
Ministry
of
Education,
Science
and
Culture
of
Japan.
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