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Effects of tetrodotoxin and ouabain on atrial contractions
Pharmacologica/ Research Communications, VoL 1, No.2, 1969
236
EFFECTS
OF TETKODOTOXIN ON ATRIAL
C ONTRAC
AND
OUABAIN
TIONS I
2 O t m a r W a s s e r m a n n and William C. Holland D e p a r t m e n t of P h a r m a c o l o g y and Toxicology University of Mississippi School of Medicine Jackson, Mississippi Received 25 March 1969 It h a s b e e n p r o p o s e d h y P a l m e r
a n d K a v a l i c h (1966) a n d P r u e t t a n d
W o o d s (1967) that s o d i u m (Na) which enters the myocardial cell during depolarization releases calcium (Ca) f r o m binding sites in the endoplasm i c reticulum which in turn initiates contraction.
It has also been pro-
posed on several occasions that the cardiac glycosides increase the force of heart contractions by inducing a further mobilization of C a with each action potential.
It is of interest to note that Lee and Choi (1966)
o b s e r v e d t h a t o u a b a i n i n h i b i t e d t h e u p t a k e of C a b y t h e s a r c o p l a s m i c reticulum, and that the effect was m o r e m a r k e d in N a C I m e d i u m
than
i n a KC1 m e d i u m . The question n o w arises as to whether the ability of digitalis to increase the force of myocardial contraction is the result of a direct effect of the drug
on the C a binding and storage m e c h a n i s m
in the
muscle fiber or is secondary to an increase in N a influx that might be induced by the agent.
A n observed increase in the rate of rise of the
action potential in dog ventricles by Pruett and W o o d s (1967) suggested that such is the case.
However,
these findings in guinea pig atria. I
2
W a s h i z u (1968) was unable to repeat In order to gain further insight into
Supported in part by P H S Grant H E 0 8 6 7 8 . Present address, Dept. of Pharmacology,
Univ. of Kiel, G e r m a n y .
Pharmacologica/ Research Communications, VoL 1, No.2, 1969
237
the matter w e have e x a m i n e d the effects of Tetrodotoxin, a specific inhibitor of the Na-spike generating m e c h a n i s m
on the positive ino-
tropic effect of ouabain. Methods Isolated left atria obtained f r o m rabbits were attached to platinumiridium electrodes.
T h e y were placed in organ baths containing I00 m l
of Ringer-Locke solution of the following composition (raM): N a C I 154.0, K C I 5.4, C a C I Z 1.6, N a H C O 5 6.0, and dextrose ii.0.
The p H was m a i n -
tained at 7.5 and all solutions were vigorously aerated with O Z. The atria were attached to the a r m of z sensitive force displacement transducer and 1 g m tension applied.
T h e y w e r e stimulated at I00
cycles/rain with square w a v e pulses (I0 V, 1.5 m s e c duration).
Con-
tractions were recorded on Sanborn T w i n Visorecorder. T h e drugs used in this study were Tetrodotoxin ("Sankyo" Calbiochem, Los Angeles), ouabain (Nutritional Biochemicals Co., Cleveland) and propranolol hydrochloride (Ayerst Laboratories, N e w York). Results In 80 percent of the atria treated, T T X
exhibited a biphasic effect
on contractile tension {Fig, 1 and Table I). This effect is characterized initially by a small increase in tension of about I0 percent {Fig. 1A) fol~owed by a slight decrease (1Z percent) in contractile force.
Tension
gradually recovered to pretreatment levels over 10-15 rain intervals. Repeated additions of the drug at even higher concentrations (up to 6.3x10-6M) of T T X
caused only a stronger negative inotropic effect. After one hoar or
m o r e treatment with highest concentration (6.3x10-6M) all activity ceased.
Pharmacological Research Communications, Vol. 1, No.2, 1969
238
. L :
.:li!
"' ~."Z ~;
lii:;L.Ll
.... --+
\ : . . . .
-,
I
z,
:?i:!!
:::; :
i ;, ;
; il;il: !i; i ;
: ; ti,:1:
:i#il
Fig. i. Effect of tetrodotoxin (TTX) and ouabain on atrial contractions. Frame
A:
Biphasic effect of 3 . 1 x l 0 - 8 M T T X
t e n s i o n of a d r i v e n (100 c y c l e s / r a i n )
Frame
30 rain); T T X
Table I. Effect of T T X
24
- 15.5
added at a r r o w (3. ixl0-8M).
on isometric tension of left atria.
No. of preparations with positiv e inotropic.effect =
TTX M
3.1
left a t r i u m .
B: A different preparation pretreated with ouabain
(5x10-7M,
N
(arrow) on isometric
x 10 -8
20
No. of preparations with negative inotroPiC ,effect
19
Pharmacologica/ Research Communications, Vol. 1, No.2, 1969 Pretreatment
239
of a t r i a f o r 30 rain with o u a b a i n (5x10-7M) did not i n f l u -
ence the b i p h a s i c e f f e c t of T T X ( F i g . 1B).
Furthermore,
the ~ - a d r e n e r g i c
blocking a g e n t p r o p r a n o l o l in c o n c e n t r a t i o n s (10 -6 - 10"5M) t h a t d e p r e s s contractile tension 10-15 percent also had no effect on the action of T T X .
In T a b l e Z we have s u m m a r i z e d of pretreatment of atria with T T X
s t u d i e s in which we e x a m i n e d the e f f e c t s
concentrations in the range of f r o m
3. I x I 0 - 8 M to 6 . 3 x l O " 6 M on the development of tile positive inotropic (PI) effect of ouabain (5x10-7M).
B e c a u s e there is no statistically significant
difference between the effects of these different concentrations w e present the data obtained with 6 . 3 x 1 0 - 6 M T T X .
T h e data are presented as the percent
increase of contractile amplitude after the addition of ouabain to the bath,
Table II, Influence of T T X
on the onset of ouabain action:
percentage increase
(x + S . E . ) of the contractile amplitude.
Minute s : ,
,
,
, , , I
,,,
5
i0
15
20
40
45
60
7O
6.8 +i.0
14.3 +1.8
ZZ.8 +Z.3
30.2 +3.9
59.9 +9.6
6Z.8 +10.7
74.1 +15.3
79.7 +16.7
3.1 +0.7
9-Z +1.4
15.7 +Z.5
Z2.6 +3.4
45.5 +4.8
48.5 +5.1
64.7 +9.3
74.5 +9.6
,,
Control O u a b a i n ( 5 x i 0 "TM) n = I0
TTX (6.3x10"6M)
+ o u a b a i n --(SxlO'7M) n=
12
<0.05o.1
p -values
~ m l l
~
~
~
~
~
'
,,
T
~ ~
=
~
~
i.......
<0.z y 0.z >o.1 ~
~
~
itI
~
L
~
J
~
Pharmacological Research Communications, VoL 1, No.2, 1969
:240
Daring the first 15 rnin TTX exhibited a small but significant depression of the ouabain induced PI effect. increases
in c o n t r a c t i l e
Thereafter
(up to 70 r a i n ) t h e p e r c e n t a g e
t e n s i o n in t h e p r e s e n c e
of T T X w e r e l e s s ,
but t h e s e
differences w e r e not signlf~lcant. Discussion Sano et el. (1968) using rabbit atrial preparations s h o w e d that T T X concentrations ranging f r o m 10 -7 to 10 -6 M
in
slowed the rate of rise of the
action potential and diminished its ¢%~nplitude. T h e toxin ~%iso terminated acfnitine, acetylcholine and Ca-induced fibrillation. T h e y concluded that TTX
acts to suppress the N a carrier system in the myocardial n~ernbrane. Using similar concentrations w e have shown that T T X
has a biphasic
effect on contractile tension, initially increasing tension followed by a small depression ~,'h'ichw a s either transitory or sustained.
At the present
tin%e there is no clear explanation of these effects on contractile tension. O n e possible explanation for the transitory inotropic effect is that T T X
is
displacing C a f r o m the binding
The
'~ in the myocardial cell m e m b r a n e . ,-~
negative inotropic effect is m o r e influx.
It was noted that T T X
development
and magnitade
than likely related to inhibition of N a
had little influence on the onset, rate of of the positive inotropic effect of ouabain°
This finding supports the view that digitalis-like substances produce their therapeutic actions not by acting on N a influx, but by releasing C a f r o m binding and storage sites in the sarcoplasmic reticulum. Suramary Tetrodotoxin (TTX) an inhibitor of the '5Na spike" exhibited a biphasic effect on isometric tension over the range of concentrations e m p l o y e d
Pharmacological Research Communications, Vol. I, No.,?, I969
( 3 . 1 x 1 0 "8 to 6 . 3 x 1 0 "6 M ) .
It f i r s t c a u s e d a
241
s m a l l t r a n s i t o r y i n c r e a s e in
t e n s i o n f o l l o w e d by a s m a l l e f f e c t on the o n s e t and r a t e of d e v e l o p m e n t of the p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n .
It was c o n c l u d e d t h a t o u a b a i n a c t s
to i n c r e a s e t e n s i o n not by i n c r e a s i n g Na e n t r y , bat by a c t i n g d i r e c t l y on the Ca b i n d i n g m e c h a n i s m in the m u s c l e f i b e r . References L e e , K . S. and C h o i , S. J° (1966):
J. PharmacoL. Exptl. Therap.
153, 114.
Palmer, R. F. and Kavalich, V. P. (1966): Federation Proc. Z5: no. Z, 349. Pruett, J. K. and Woods, E. F. (1967): J. Pharmacol. Exptl. Therap. 157, I. Sano, T., Suzuki, F., Salvo, S° and lida, Y. (1968): Jap. Heart J. 9, 161. Washizu, Y. (1968), personal communication.
236
EFFECTS
OF TETKODOTOXIN ON ATRIAL
C ONTRAC
AND
OUABAIN
TIONS I
2 O t m a r W a s s e r m a n n and William C. Holland D e p a r t m e n t of P h a r m a c o l o g y and Toxicology University of Mississippi School of Medicine Jackson, Mississippi Received 25 March 1969 It h a s b e e n p r o p o s e d h y P a l m e r
a n d K a v a l i c h (1966) a n d P r u e t t a n d
W o o d s (1967) that s o d i u m (Na) which enters the myocardial cell during depolarization releases calcium (Ca) f r o m binding sites in the endoplasm i c reticulum which in turn initiates contraction.
It has also been pro-
posed on several occasions that the cardiac glycosides increase the force of heart contractions by inducing a further mobilization of C a with each action potential.
It is of interest to note that Lee and Choi (1966)
o b s e r v e d t h a t o u a b a i n i n h i b i t e d t h e u p t a k e of C a b y t h e s a r c o p l a s m i c reticulum, and that the effect was m o r e m a r k e d in N a C I m e d i u m
than
i n a KC1 m e d i u m . The question n o w arises as to whether the ability of digitalis to increase the force of myocardial contraction is the result of a direct effect of the drug
on the C a binding and storage m e c h a n i s m
in the
muscle fiber or is secondary to an increase in N a influx that might be induced by the agent.
A n observed increase in the rate of rise of the
action potential in dog ventricles by Pruett and W o o d s (1967) suggested that such is the case.
However,
these findings in guinea pig atria. I
2
W a s h i z u (1968) was unable to repeat In order to gain further insight into
Supported in part by P H S Grant H E 0 8 6 7 8 . Present address, Dept. of Pharmacology,
Univ. of Kiel, G e r m a n y .
Pharmacologica/ Research Communications, VoL 1, No.2, 1969
237
the matter w e have e x a m i n e d the effects of Tetrodotoxin, a specific inhibitor of the Na-spike generating m e c h a n i s m
on the positive ino-
tropic effect of ouabain. Methods Isolated left atria obtained f r o m rabbits were attached to platinumiridium electrodes.
T h e y were placed in organ baths containing I00 m l
of Ringer-Locke solution of the following composition (raM): N a C I 154.0, K C I 5.4, C a C I Z 1.6, N a H C O 5 6.0, and dextrose ii.0.
The p H was m a i n -
tained at 7.5 and all solutions were vigorously aerated with O Z. The atria were attached to the a r m of z sensitive force displacement transducer and 1 g m tension applied.
T h e y w e r e stimulated at I00
cycles/rain with square w a v e pulses (I0 V, 1.5 m s e c duration).
Con-
tractions were recorded on Sanborn T w i n Visorecorder. T h e drugs used in this study were Tetrodotoxin ("Sankyo" Calbiochem, Los Angeles), ouabain (Nutritional Biochemicals Co., Cleveland) and propranolol hydrochloride (Ayerst Laboratories, N e w York). Results In 80 percent of the atria treated, T T X
exhibited a biphasic effect
on contractile tension {Fig, 1 and Table I). This effect is characterized initially by a small increase in tension of about I0 percent {Fig. 1A) fol~owed by a slight decrease (1Z percent) in contractile force.
Tension
gradually recovered to pretreatment levels over 10-15 rain intervals. Repeated additions of the drug at even higher concentrations (up to 6.3x10-6M) of T T X
caused only a stronger negative inotropic effect. After one hoar or
m o r e treatment with highest concentration (6.3x10-6M) all activity ceased.
Pharmacological Research Communications, Vol. 1, No.2, 1969
238
. L :
.:li!
"' ~."Z ~;
lii:;L.Ll
.... --+
\ : . . . .
-,
I
z,
:?i:!!
:::; :
i ;, ;
; il;il: !i; i ;
: ; ti,:1:
:i#il
Fig. i. Effect of tetrodotoxin (TTX) and ouabain on atrial contractions. Frame
A:
Biphasic effect of 3 . 1 x l 0 - 8 M T T X
t e n s i o n of a d r i v e n (100 c y c l e s / r a i n )
Frame
30 rain); T T X
Table I. Effect of T T X
24
- 15.5
added at a r r o w (3. ixl0-8M).
on isometric tension of left atria.
No. of preparations with positiv e inotropic.effect =
TTX M
3.1
left a t r i u m .
B: A different preparation pretreated with ouabain
(5x10-7M,
N
(arrow) on isometric
x 10 -8
20
No. of preparations with negative inotroPiC ,effect
19
Pharmacologica/ Research Communications, Vol. 1, No.2, 1969 Pretreatment
239
of a t r i a f o r 30 rain with o u a b a i n (5x10-7M) did not i n f l u -
ence the b i p h a s i c e f f e c t of T T X ( F i g . 1B).
Furthermore,
the ~ - a d r e n e r g i c
blocking a g e n t p r o p r a n o l o l in c o n c e n t r a t i o n s (10 -6 - 10"5M) t h a t d e p r e s s contractile tension 10-15 percent also had no effect on the action of T T X .
In T a b l e Z we have s u m m a r i z e d of pretreatment of atria with T T X
s t u d i e s in which we e x a m i n e d the e f f e c t s
concentrations in the range of f r o m
3. I x I 0 - 8 M to 6 . 3 x l O " 6 M on the development of tile positive inotropic (PI) effect of ouabain (5x10-7M).
B e c a u s e there is no statistically significant
difference between the effects of these different concentrations w e present the data obtained with 6 . 3 x 1 0 - 6 M T T X .
T h e data are presented as the percent
increase of contractile amplitude after the addition of ouabain to the bath,
Table II, Influence of T T X
on the onset of ouabain action:
percentage increase
(x + S . E . ) of the contractile amplitude.
Minute s : ,
,
,
, , , I
,,,
5
i0
15
20
40
45
60
7O
6.8 +i.0
14.3 +1.8
ZZ.8 +Z.3
30.2 +3.9
59.9 +9.6
6Z.8 +10.7
74.1 +15.3
79.7 +16.7
3.1 +0.7
9-Z +1.4
15.7 +Z.5
Z2.6 +3.4
45.5 +4.8
48.5 +5.1
64.7 +9.3
74.5 +9.6
,,
Control O u a b a i n ( 5 x i 0 "TM) n = I0
TTX (6.3x10"6M)
+ o u a b a i n --(SxlO'7M) n=
12
<0.05
p -values
~ m l l
~
~
~
~
~
'
,,
T
~ ~
=
~
~
i.......
<0.z y 0.z >o.1 ~
~
~
itI
~
L
~
J
~
Pharmacological Research Communications, VoL 1, No.2, 1969
:240
Daring the first 15 rnin TTX exhibited a small but significant depression of the ouabain induced PI effect. increases
in c o n t r a c t i l e
Thereafter
(up to 70 r a i n ) t h e p e r c e n t a g e
t e n s i o n in t h e p r e s e n c e
of T T X w e r e l e s s ,
but t h e s e
differences w e r e not signlf~lcant. Discussion Sano et el. (1968) using rabbit atrial preparations s h o w e d that T T X concentrations ranging f r o m 10 -7 to 10 -6 M
in
slowed the rate of rise of the
action potential and diminished its ¢%~nplitude. T h e toxin ~%iso terminated acfnitine, acetylcholine and Ca-induced fibrillation. T h e y concluded that TTX
acts to suppress the N a carrier system in the myocardial n~ernbrane. Using similar concentrations w e have shown that T T X
has a biphasic
effect on contractile tension, initially increasing tension followed by a small depression ~,'h'ichw a s either transitory or sustained.
At the present
tin%e there is no clear explanation of these effects on contractile tension. O n e possible explanation for the transitory inotropic effect is that T T X
is
displacing C a f r o m the binding
The
'~ in the myocardial cell m e m b r a n e . ,-~
negative inotropic effect is m o r e influx.
It was noted that T T X
development
and magnitade
than likely related to inhibition of N a
had little influence on the onset, rate of of the positive inotropic effect of ouabain°
This finding supports the view that digitalis-like substances produce their therapeutic actions not by acting on N a influx, but by releasing C a f r o m binding and storage sites in the sarcoplasmic reticulum. Suramary Tetrodotoxin (TTX) an inhibitor of the '5Na spike" exhibited a biphasic effect on isometric tension over the range of concentrations e m p l o y e d
Pharmacological Research Communications, Vol. I, No.,?, I969
( 3 . 1 x 1 0 "8 to 6 . 3 x 1 0 "6 M ) .
It f i r s t c a u s e d a
241
s m a l l t r a n s i t o r y i n c r e a s e in
t e n s i o n f o l l o w e d by a s m a l l e f f e c t on the o n s e t and r a t e of d e v e l o p m e n t of the p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n .
It was c o n c l u d e d t h a t o u a b a i n a c t s
to i n c r e a s e t e n s i o n not by i n c r e a s i n g Na e n t r y , bat by a c t i n g d i r e c t l y on the Ca b i n d i n g m e c h a n i s m in the m u s c l e f i b e r . References L e e , K . S. and C h o i , S. J° (1966):
J. PharmacoL. Exptl. Therap.
153, 114.
Palmer, R. F. and Kavalich, V. P. (1966): Federation Proc. Z5: no. Z, 349. Pruett, J. K. and Woods, E. F. (1967): J. Pharmacol. Exptl. Therap. 157, I. Sano, T., Suzuki, F., Salvo, S° and lida, Y. (1968): Jap. Heart J. 9, 161. Washizu, Y. (1968), personal communication.