European Journal of Pharmacology, 32 (1975) 116--119
© North-Holland Publishing Company, Amsterdam - Printed in The Netherlands Short communication ACTIONS OF BILE SALTS AND OF PAPAVERINE AND INTRACELLULAR IN ISOLATED RAT UTERUS
CYCLIC AMP
TSUTOMU URUNO, *ISSEI TAKAYANAGI, KAZUHIKO KUBOTA and *KEIJIRO TAKAGI Department o f Pharmacology, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Ichigaya, Tokyo, Japan
Received 17 December 1974, accepted 7 March 1975
T. URUNO, I. TAKAYANAGI, K. KUBOTA and K. TAKAGI, Actions of bile salts and of papaverine and intracellular cyclic AMP in isolated rat uterus, European J. Pharmacol. 32 (1975) 116--119. Both antioxytocin and antiphosphodiesterase activities of desoxycholate were potentiated by lowering the pH of the medium. The results suggest the possibility that it is the bile salt in the non-ionized form which exerts the antioxytocin and antiphosphodiesterase action. The concentration of tissue cyclic AMP was measured at different times during relaxation of the uterus. The results show a significant increase in tissue cyclic AMP concentration at a time when the muscle was just beginning to relax in response to papaverine, but not in response to chenodesoxycholate. The intracellular level of cyclic AMP during relaxation of the uterus produced by papaverine or chenodesoxycholate was, however, significantly increased when relaxation was about 90% complete. The difference between the mode of action of papaverine and that of the bile salts is discussed. 'Papaverine-like' action
Papaverine
Bile salts
1. I n t r o d u c t i o n The mechanism of smooth muscle relaxation b y ' p a p a v e r i n e - l i k e ' drugs is o b s c u r e . R e c e n t l y it has b e e n s h o w n t h a t t h e r e l a x i n g a c t i o n s o f p a p a v e r i n e o n s m o o t h m u s c l e m i g h t be e x e r t e d t h r o u g h p h o s p h o d i e s t e r a s e (PDE) i n h i b i t i o n a n d c o n s e c u t i v e a c c u m u l a t i o n o f cyclic 3 ' , 5 ' AMP (cyclic AMP) ( T r i n e r et al., 1 9 7 0 ; PSch a n d K u k o v e t z , 1 9 7 1 ; U r u n o et al., 1 9 7 4 a ) . F u r t h e r , m a n y investigators ( T a k a y a n a g i et al., 1 9 7 2 ; A n d e r s s o n , 1 9 7 2 ; U r u n o et al., 1 9 7 3 ; Marshall a n d Kroeger, 1 9 7 3 ; I n a t o m i et al., 1 9 7 4 ) h a v e r e p o r t e d t h a t p a p a v e r i n e increased t h e i n t r a c e l l u l a r level o f cyclic AMP in t h e intestinal a n d u t e r i n e s m o o t h muscle. It has b e e n r e p o r t e d b y s o m e a u t h o r s ( T a k a y a n a g i et * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Cyclic AMP
al., 1 9 7 3 ; U r u n o et al., 1 9 7 3 ; I n a t o m i et al., 1 9 7 4 ; Ferrari, 1 9 7 4 ) t h a t t h e ' p a p a v e r i n e - l i k e ' a c t i o n o f s o m e a n t i s p a s m o d i c s is n o t m e d i a t e d t h r o u g h an increased i n t r a c e l l u l a r cyclic AMP level w h i c h is c a u s e d b y i n h i b i t i o n o f PDE. It has b e e n also r e p o r t e d t h a t bile salts s h o w a n t i c h o l i n e r g i c activities, this on t h e basis o f t h e i r ' p a p a v e r i n e - l i k e ' a c t i o n s o n t h e intestinal s m o o t h m u s c l e ( K u b o t a et al., 1 9 7 0 ) . In o u r p r e v i o u s p a p e r ( U r u n o et al., 1 9 7 4 b ) we have m e n t i o n e d t h a t bile salts i n h i b i t e d PDE and significantly i n c r e a s e d t h e i n t r a c e l l u l a r level o f cyclic AMP in t h e isolated r a t u t e r u s , t h e r e f o r e t h e a n t i o x y t o c i n a c t i o n o f t h e bile salts is likely t o be m e d i a t e d t h r o u g h an increase o f the i n t r a c e l l u l a r level o f cyclic AMP, w h i c h is caused by inhibition of the PDE-catalyzed b r e a k d o w n o f cyclic AMP. Since t h e p K a value o f d e s o x y c h o l a t e is 6.58, this c o m p o u n d is suitable f o r s t u d y o f a n y c h a n g e s in its a n t i o x y t o c i n a n d a n t i - P D E
117
BILE SALTS, PAPAVERINE AND CYCLIC AMP activities caused by changing the pH of the medium. Therefore, relationships between pH in the medium and both activities of desoxycholate were tested. Furthermore, the possibility of an increase in the intracellular level of cyclic AMP during relaxation of the rat uterus in the presence of papaverine or chenodesoxycholate was also investigated.
2. Materials and methods Virgin female Wistar rats weighing 200--250 g were ovariectomized and allowed to recover for more than 5 days before the experiments. An ovariectomized rat was sacrificed by a blow on the neck and the uterine horns were removed. One was suspended in a 10 ml organ bath filled with modified Tyrode solution (pH 7.4 or 6.7, Kimura, 1959), kept at 32°C, and gassed with CO2-free air, which was obtained by bubbling air through 10% NaOH solution. Uterine contractions induced by oxytocin were recorded isotonically on a smoked drum by means of a lever with 0.5 g load. Desoxycholate (sodium salt) was allowed to act for 3 rain and oxytocin was then added to it in cumulative amounts. PDE-activity was assayed according to the method of PSch (1971). The uteri isolated from five ovariectomized rats were homogenized with 10 volumes of a buffer Mg acetate solution (Tris HCI 16 X 10 -2 M, Mg acetate 5 X 10 -3 M; pH 7.5) at 0°C. The homogenate was centrifuged at 2,000 X g for 15 min at 0 ° C and the supernatant was used as an enzyme preparation. Incubation was carried o u t in the medium (Tris HC1, Mg acetate; pH 7.4 or 6.7) at 37°C for 30 min. For tissue cyclic AMP analysis an ovariectomized rat (200--250 g b o d y weight) was stunned and bled, and the uterine horns were removed and suspended in t w o similar baths of 10 ml. The baths were filled with Locke-Ringer solution bubbled with air, and kept at 18°C. At 18°C the latency between the addition o f a drug and the relaxation was prolonged and relaxation was slowed. Responses of the
uterus were isotonically recorded on a smoked drum by means of a lever with a 0.5 g load. Chenodesoxycholate (sodium salt) instead of desoxycholate was used as a bile salt, since the former was more stable in aqueous solution. After the maximal contraction had been induced b y oxytocin (10 -2 units/ml), papaverine (3 X 10 -s g/ml) or chenodesoxycholate (5 X 10 -4 g/ml) was introduced into the organ bath. One horn to serve for cAMP analysis, was then plunged into liquid nitrogen at a time when it was just beginning to relax (about 28 sec for papaverine and a b o u t 43 sec for chenodesoxycholate) or when relaxation was a b o u t 90% complete (about 5.5 min for papaverine and a b o u t 10 min for chenodesoxycholate). The other horn which was treated with oxytocin always served as a control. Measurement of the a m o u n t of cyclic AMP in the tissue was carried o u t according to the method originally described by Gilman (1970).
3. Results In order to examine whether the 'papaverine-like' action of, and inhibition of PDE by desoxycholate are due to its non-ionized molecules, an experiment was done with results as follows. Desoxycholate (2 X 10 -s g/ml) inhibited the oxytocin-induced contraction by 32% at pH 7.4 and by 56% at pH 6.7 in the medium, and the PDE activity by 6% at pH 7.4 and by 36% at pH 6.7 in the medium (fig. 1). Both the antioxytocin and anti-PDE activities were potentiated b y lowering the pH of the medium. Moreover, there was good parallelism b e t w e e n (a) pH-antioxytocin activity, (b) pH-anti-PDE activity and (c) the plot of non-ionized molecules obtained theoretically from pH values of the medium and pKa value (6.58) of desoxycholic acid (fig. 1). The concentration of tissue cyclic AMP at different times during the relaxation produced by papaverine (3 X 10 -s g / m l ) o r chenodesoxycholate (5 X 10 -4 g/ml) is shown in fig. 2. Our results show a significant increase in the tissue cyclic AMP concentration at a time when the
118
T. U R U N O ET AL.
i00-
C o
"13 (D eq -.4
~
v
-4 4J
duced by papaverine or chenodesoxycholate was about 90% complete, the intracellular level of cyclic AMP was significantly increased.
-i00
50-
c.
O
50
4. Discussion
o e~ ¢.
Both antioxytocin and anti-PDE activities of desoxycholate were potentiated by lowering the pH in the medium (fig. 1). These results indicate the possibility that the antioxytocin and anti-PDE actions of desoxycholate are due to its non-ionized molecules. However, the concentrations of desoxycholate which were necessary to relax the uterus are quite different from those needed to inhibit PDE (fig. 1). This fact suggests the possibility that the 'papaverine-like' action of desoxycholate is not directly concerned with inhibition of PDE. In the present study the concentration of tissue cyclic AMP was measured at different times during relaxation. From the results shown in fig. 2, it can be concluded that the antioxytocin action of papaverine at a concentration of 3 X 10 -s g/ml is due to the increaseof intracellular cyclic AMP, while the relaxing
(a) (9 (b)
(c) 0
|
0
|
6.7
7.4 pH
Fig. 1. Changes in a n t i o x y t o c i n and a n t i p h o s p h o d i esterase activities o f d e s o x y c h o l a t e and ratio o f noni o n i z e d m o l e c u l e s o f d e s o x y c h o l a t e p r o d u c e d by changing the pH in the m e d i u m . (a) ~:~©: antio x y t o c i n action ( i n h i b i t i o n %) of d e s o x y c h o l a t e (2 × 10- s g/ml); (b) : a n t i p h o s p h o d i e s t e r a s e action ( i n h i b i t i o n %) o f d e s o x y c h o l a t e (10- 4 g/ml); (c) ; e: ratio o f n o n - i o n i z e d m o l e c u l e s (%), w h i c h was calculated f r o m pH values and the pKa value ( 6 . 5 8 ) o f d e s o x y c h o l i c acid. Each point represents the mean -+ S.E. o f 6 e x p e r i m e n t s .
muscle was just beginning to relax in response to papaverine, but not in response to chenodesoxycholate. However, when the relaxation pro(A)
PAPAVERINE
© North-Holland Publishing Company, Amsterdam - Printed in The Netherlands Short communication ACTIONS OF BILE SALTS AND OF PAPAVERINE AND INTRACELLULAR IN ISOLATED RAT UTERUS
CYCLIC AMP
TSUTOMU URUNO, *ISSEI TAKAYANAGI, KAZUHIKO KUBOTA and *KEIJIRO TAKAGI Department o f Pharmacology, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Ichigaya, Tokyo, Japan
Received 17 December 1974, accepted 7 March 1975
T. URUNO, I. TAKAYANAGI, K. KUBOTA and K. TAKAGI, Actions of bile salts and of papaverine and intracellular cyclic AMP in isolated rat uterus, European J. Pharmacol. 32 (1975) 116--119. Both antioxytocin and antiphosphodiesterase activities of desoxycholate were potentiated by lowering the pH of the medium. The results suggest the possibility that it is the bile salt in the non-ionized form which exerts the antioxytocin and antiphosphodiesterase action. The concentration of tissue cyclic AMP was measured at different times during relaxation of the uterus. The results show a significant increase in tissue cyclic AMP concentration at a time when the muscle was just beginning to relax in response to papaverine, but not in response to chenodesoxycholate. The intracellular level of cyclic AMP during relaxation of the uterus produced by papaverine or chenodesoxycholate was, however, significantly increased when relaxation was about 90% complete. The difference between the mode of action of papaverine and that of the bile salts is discussed. 'Papaverine-like' action
Papaverine
Bile salts
1. I n t r o d u c t i o n The mechanism of smooth muscle relaxation b y ' p a p a v e r i n e - l i k e ' drugs is o b s c u r e . R e c e n t l y it has b e e n s h o w n t h a t t h e r e l a x i n g a c t i o n s o f p a p a v e r i n e o n s m o o t h m u s c l e m i g h t be e x e r t e d t h r o u g h p h o s p h o d i e s t e r a s e (PDE) i n h i b i t i o n a n d c o n s e c u t i v e a c c u m u l a t i o n o f cyclic 3 ' , 5 ' AMP (cyclic AMP) ( T r i n e r et al., 1 9 7 0 ; PSch a n d K u k o v e t z , 1 9 7 1 ; U r u n o et al., 1 9 7 4 a ) . F u r t h e r , m a n y investigators ( T a k a y a n a g i et al., 1 9 7 2 ; A n d e r s s o n , 1 9 7 2 ; U r u n o et al., 1 9 7 3 ; Marshall a n d Kroeger, 1 9 7 3 ; I n a t o m i et al., 1 9 7 4 ) h a v e r e p o r t e d t h a t p a p a v e r i n e increased t h e i n t r a c e l l u l a r level o f cyclic AMP in t h e intestinal a n d u t e r i n e s m o o t h muscle. It has b e e n r e p o r t e d b y s o m e a u t h o r s ( T a k a y a n a g i et * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Cyclic AMP
al., 1 9 7 3 ; U r u n o et al., 1 9 7 3 ; I n a t o m i et al., 1 9 7 4 ; Ferrari, 1 9 7 4 ) t h a t t h e ' p a p a v e r i n e - l i k e ' a c t i o n o f s o m e a n t i s p a s m o d i c s is n o t m e d i a t e d t h r o u g h an increased i n t r a c e l l u l a r cyclic AMP level w h i c h is c a u s e d b y i n h i b i t i o n o f PDE. It has b e e n also r e p o r t e d t h a t bile salts s h o w a n t i c h o l i n e r g i c activities, this on t h e basis o f t h e i r ' p a p a v e r i n e - l i k e ' a c t i o n s o n t h e intestinal s m o o t h m u s c l e ( K u b o t a et al., 1 9 7 0 ) . In o u r p r e v i o u s p a p e r ( U r u n o et al., 1 9 7 4 b ) we have m e n t i o n e d t h a t bile salts i n h i b i t e d PDE and significantly i n c r e a s e d t h e i n t r a c e l l u l a r level o f cyclic AMP in t h e isolated r a t u t e r u s , t h e r e f o r e t h e a n t i o x y t o c i n a c t i o n o f t h e bile salts is likely t o be m e d i a t e d t h r o u g h an increase o f the i n t r a c e l l u l a r level o f cyclic AMP, w h i c h is caused by inhibition of the PDE-catalyzed b r e a k d o w n o f cyclic AMP. Since t h e p K a value o f d e s o x y c h o l a t e is 6.58, this c o m p o u n d is suitable f o r s t u d y o f a n y c h a n g e s in its a n t i o x y t o c i n a n d a n t i - P D E
117
BILE SALTS, PAPAVERINE AND CYCLIC AMP activities caused by changing the pH of the medium. Therefore, relationships between pH in the medium and both activities of desoxycholate were tested. Furthermore, the possibility of an increase in the intracellular level of cyclic AMP during relaxation of the rat uterus in the presence of papaverine or chenodesoxycholate was also investigated.
2. Materials and methods Virgin female Wistar rats weighing 200--250 g were ovariectomized and allowed to recover for more than 5 days before the experiments. An ovariectomized rat was sacrificed by a blow on the neck and the uterine horns were removed. One was suspended in a 10 ml organ bath filled with modified Tyrode solution (pH 7.4 or 6.7, Kimura, 1959), kept at 32°C, and gassed with CO2-free air, which was obtained by bubbling air through 10% NaOH solution. Uterine contractions induced by oxytocin were recorded isotonically on a smoked drum by means of a lever with 0.5 g load. Desoxycholate (sodium salt) was allowed to act for 3 rain and oxytocin was then added to it in cumulative amounts. PDE-activity was assayed according to the method of PSch (1971). The uteri isolated from five ovariectomized rats were homogenized with 10 volumes of a buffer Mg acetate solution (Tris HCI 16 X 10 -2 M, Mg acetate 5 X 10 -3 M; pH 7.5) at 0°C. The homogenate was centrifuged at 2,000 X g for 15 min at 0 ° C and the supernatant was used as an enzyme preparation. Incubation was carried o u t in the medium (Tris HC1, Mg acetate; pH 7.4 or 6.7) at 37°C for 30 min. For tissue cyclic AMP analysis an ovariectomized rat (200--250 g b o d y weight) was stunned and bled, and the uterine horns were removed and suspended in t w o similar baths of 10 ml. The baths were filled with Locke-Ringer solution bubbled with air, and kept at 18°C. At 18°C the latency between the addition o f a drug and the relaxation was prolonged and relaxation was slowed. Responses of the
uterus were isotonically recorded on a smoked drum by means of a lever with a 0.5 g load. Chenodesoxycholate (sodium salt) instead of desoxycholate was used as a bile salt, since the former was more stable in aqueous solution. After the maximal contraction had been induced b y oxytocin (10 -2 units/ml), papaverine (3 X 10 -s g/ml) or chenodesoxycholate (5 X 10 -4 g/ml) was introduced into the organ bath. One horn to serve for cAMP analysis, was then plunged into liquid nitrogen at a time when it was just beginning to relax (about 28 sec for papaverine and a b o u t 43 sec for chenodesoxycholate) or when relaxation was a b o u t 90% complete (about 5.5 min for papaverine and a b o u t 10 min for chenodesoxycholate). The other horn which was treated with oxytocin always served as a control. Measurement of the a m o u n t of cyclic AMP in the tissue was carried o u t according to the method originally described by Gilman (1970).
3. Results In order to examine whether the 'papaverine-like' action of, and inhibition of PDE by desoxycholate are due to its non-ionized molecules, an experiment was done with results as follows. Desoxycholate (2 X 10 -s g/ml) inhibited the oxytocin-induced contraction by 32% at pH 7.4 and by 56% at pH 6.7 in the medium, and the PDE activity by 6% at pH 7.4 and by 36% at pH 6.7 in the medium (fig. 1). Both the antioxytocin and anti-PDE activities were potentiated b y lowering the pH of the medium. Moreover, there was good parallelism b e t w e e n (a) pH-antioxytocin activity, (b) pH-anti-PDE activity and (c) the plot of non-ionized molecules obtained theoretically from pH values of the medium and pKa value (6.58) of desoxycholic acid (fig. 1). The concentration of tissue cyclic AMP at different times during the relaxation produced by papaverine (3 X 10 -s g / m l ) o r chenodesoxycholate (5 X 10 -4 g/ml) is shown in fig. 2. Our results show a significant increase in the tissue cyclic AMP concentration at a time when the
118
T. U R U N O ET AL.
i00-
C o
"13 (D eq -.4
~
v
-4 4J
duced by papaverine or chenodesoxycholate was about 90% complete, the intracellular level of cyclic AMP was significantly increased.
-i00
50-
c.
O
50
4. Discussion
o e~ ¢.
Both antioxytocin and anti-PDE activities of desoxycholate were potentiated by lowering the pH in the medium (fig. 1). These results indicate the possibility that the antioxytocin and anti-PDE actions of desoxycholate are due to its non-ionized molecules. However, the concentrations of desoxycholate which were necessary to relax the uterus are quite different from those needed to inhibit PDE (fig. 1). This fact suggests the possibility that the 'papaverine-like' action of desoxycholate is not directly concerned with inhibition of PDE. In the present study the concentration of tissue cyclic AMP was measured at different times during relaxation. From the results shown in fig. 2, it can be concluded that the antioxytocin action of papaverine at a concentration of 3 X 10 -s g/ml is due to the increaseof intracellular cyclic AMP, while the relaxing
(a) (9 (b)
(c) 0
|
0
|
6.7
7.4 pH
Fig. 1. Changes in a n t i o x y t o c i n and a n t i p h o s p h o d i esterase activities o f d e s o x y c h o l a t e and ratio o f noni o n i z e d m o l e c u l e s o f d e s o x y c h o l a t e p r o d u c e d by changing the pH in the m e d i u m . (a) ~:~©: antio x y t o c i n action ( i n h i b i t i o n %) of d e s o x y c h o l a t e (2 × 10- s g/ml); (b) : a n t i p h o s p h o d i e s t e r a s e action ( i n h i b i t i o n %) o f d e s o x y c h o l a t e (10- 4 g/ml); (c) ; e: ratio o f n o n - i o n i z e d m o l e c u l e s (%), w h i c h was calculated f r o m pH values and the pKa value ( 6 . 5 8 ) o f d e s o x y c h o l i c acid. Each point represents the mean -+ S.E. o f 6 e x p e r i m e n t s .
muscle was just beginning to relax in response to papaverine, but not in response to chenodesoxycholate. However, when the relaxation pro(A)
PAPAVERINE
