PROSTAG LAN DI NS CONTRACTILE EFFECTS OF 16-METHYLANALOGUES OF PGE 2 ON THE CIRCULAR AND LONGITUDINAL MUSCLES OF THE GUINEA-PIG ISOLATED COLON M.

Ishizawa

Laboratory of Physiology, School of Allied Health Professions, Sapporo Medical College, S-3, W-17, Sapporo, 060, Japan

ABSTKACT The mechanical e f f e c t s of 16-methyl a n a l o g u e s of PGE2, mainly 1 6 , 1 6 - d i m e t h y l PGE2, on c i r c u l a r and l o n g i t u d i n a l muscles of the g u i n e a - p i g i s o l a t e d proximal c o l o n were i n v e s t i g a t e d . In c i r c u l a r muscle s t r i p s , PGE2 100 nM produced an i n i t i a l cont r a c t i o n f o l l o w e d by r e l a x a t i o n , while 16(R)-methyl PGE2 and 16,16 d i m e t h y l PGE2 (1 n M - 1 pM) produced s u s t a i n e d c o n t r a c t i o n s . In l o n g i t u d i n a l muscle s t r i p s , PGE 2 and 16 methyl a n a l o g u e s of PGE2 produced o n l y c o n t r a c t i o n s . The c o n t r a c t i l e r e s p o n s e s of both muscle s r i p s to 1 6 , 1 6 - d i methyl PGE2 were not i n f l u e n c e d by a t r o p i n e or t e t r o d o t o x i n , i n d i c a t ing t h a t t h e s e a n a l o g u e s a c t d i r e c t l y on the m u s c l e s , b u t were e l i m i n a t e d by the o m i s s i o n of e x t r a c e l l u l a r Ca ions or in the p r e s e n c e of 1 mH lanthanum ions. However, v e r a p a m i l , a Ca c h a n n e l b l o c k e r , did not block the c o n t r a c t i l e r e s p o n s e to the methyl a n a l o g u e s in c i r c u l a r muscle s t r i p s , a l t h o u g h i t c o m p l e t e l y i n h i b i t e d the c o n t r a c t i l e r e s p o n s e of l o n g i t u d i n a l muscle s t r i p s . These r e s u l t s s u g g e s t t h a t the c o n t r a c t i l e e f f e c t of 16-methyl a n a l o g u e s of PG~2 on the c i r c u l a r muscle may be due to an i n c r e a s e d i n f l u x of Ca ions mainly v i a r e c e p t o r - s e n s i t i v e and p a r t l y v o l t a g e s e n s i t i v e Ca c h a n n e l s , w h i l e the c o n t r a c t i l e e f f e c t of the a n a l o g u e s on the l o n g i t u d i n a l muscle may be due to an i n c r e a s e in i n f l u x of Ca ions v i a v o l t a g e - s e n s i t i v e Ca c h a n n e l s .

INTRODOCTION In g e n e r a l , l o n g i t u d i n a l muscles of the g a s t r o i n t e s t i n a l t r a c t c o n t r a c t in the p r e s e n c e of E,F and D t y p e s of p r o s t a g l a n d i n s (PGs), but c i r c u l a r muscles a r e i n h i b i t e d by PGs of the E and I t y p e s (1,2, 3). However, i t is a l s o r e p o r t e d t h a t E type of PGs responded with a p h a s i c c o n t r a c t i o n in the c i r c u l a r muscles of g u i n e a - p i g stomach ( 4 ) , c o l o n (5) and human ileum (6). The methyl a n a l o g u e s of PGE2, 1 5 ( S ) - m e t h y l PGE2 and 1 6 , 1 6 - d i methyl PGE2 were 30-50 times more a c t i v e than n a t u r a l l y o c c u r r i n g PGE2 as i n h i b i t o r s of c a n i n e g a s t r i c s e c r e t i o n . However, t h e s e methyl a n a l o g u e s a r e s i m i l a r to PGE2 in t h e i r a c t i o n on i s o l a t e d i n t e s t i n a l

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PROSTAGLANDINS muscles (7). I t has been r e p o r t e d t h a t 1 5 ( S ) - m e t h y l and 1 6 , 1 6 - d i m e t h y l PGE2 were over 30 times as p o t e n t as PGE2 in c a u s i n g i n c r e a s e in t h e i n t r a l u m i n a l p r e s s u r e of t h e r a t i n t e s t i n e (8). I t is a l s o r e p o r t e d t h a t i n t r a v e n o u s i n j e c t i o n of 1 6 , 1 6 - d i m e t h y l PGE~, n o t PGE2, was a b l e to s t i m u l a t e g a s t r i c emptying in r a t s (9) and in man ( l O ) . T h e s e r e p o r t s s u g g e s t t h a t t h e methyl a n a l o g u e s of PGE2 might produce t o n i c c o n t r a c t i o n s of c i r c u l a r muscles as w e l l as l o n g i t u d i n a l muscles of t h e g a s t r o i n t e s t i n a l tract. The p r e s e n t s t u d y was d e s i g n e d to d e t e r m i n e t h e e f f e c t s of 16methyl a n a l o g u e s of PGE2, m a i n l y 1 6 , 1 6 - d i m e t h y l PGE 2, based on m e c h a n i c a l a c t i v i t y in c i r c u l a r and l o n g i t u d i n a l muscles of t h e guinea-pig isolated proximal colon.

NKTHODS Male guinea-pigs weighing 400-600g were stunned and bled. A 3 cm portion of proximal colon was excised about 4 cm from the ileocaecal junction,and circular and longitudinal muscle strips (I0 mm long and I mmwide) were cut parallel to the circular and longitudinal muscle layers respectively. The mucosa and submucosa were mechanically removed from each muscle strip. The muscle strips were then immersed v e r t i c a l l y in an organ bath (I0 ml capacity) containing a modified Locke's solution composed of 125 mMNaCl, 5 mMKCI, 2 mMCaCl2, 0.5 mMMgCI2, 11.5 mM glucose and 5 mM tris-maleate buffer (pH:7.2). The bath solution was kept at 37o C and gassed with 02.

The m e c h a n i c a l a c t i v i t y of t h e muscle s t r i p was r e c o r d e d i s o m e t r i c a l l y with a f o r c e - d i s p l a c e m e n t t r a n s d u c e r and an i n k - w r i t i n g pen r e c o r d e r . The i n i t i a l load on t h e muscle s t r i p was 0 . 3 - 0 . 5 g . The f o l l o w i n g drugs were used: PGE2 ( O n o ) , 1 6 ( R ) - m e t h y l PGE2 ( O n o ) , 1 6 , 1 6 - d i m e t h y l PGE2 (Cayman C h e m . ) , a t r o p i n e s u l p h a t e (Sigma), t e t r o d o t o x i n ( S a n k y o ) , v e r a p a m i l ( E i s a i ) and e t h y l e n e - b i s ( o x y - e t h y l ene-nitrilo) t e t r a a c e t i c a c i d ( E G T h ; S i g m a ) . P r o s t a g l a n d i n s were d i s s o l v e d in e t h a n o l (10 mg/ml) and d i l u t e d in 154 mM N a C l , o t h e r drugs were d i s s o l v e d in 154 mM NaC1. A s m a l l volume (10 pl - 100 ~1) of each drug s o l u t i o n was added to t h e b a t h s o l u t i o n and t h e c o n c e n t r a t i o n s were e x p r e s s e d in molar terms of the b a t h s o l u t i o n . The maximal c o n c e n t r a t i o n of e t h a n o l in the b a t h s o l u t i o n was 0.1 ~. RESULTS

E f f e c t s of PGE2,16(R)-methyl PGE2 and 1 6 , 1 6 - d i m e t h y l PGE2 c o l o n i c c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s . In 6 e x p e r i m e n t s ,

580

PGE2 100 nM produced an i n i t i a l

on

p h a s i c con-

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PROSTAGLANDINS t r a c t i o n followed by r e l a x a t i o n , while 16(R)-methyl PGE 2 and 16, 16dimethyl PGE2 100 nM produced sustained c o n t r a c t i o n in c i r c u l a r muscle s t r i p s . In l o n g i t u d i n a l muscle s t r i p s , PGE2 and the m e t h y l analogues e l i c i t e d c o n t r a c t i o n (Fig. l).

PG( 2

~ec

c

A

I

IOOnM

t

,oo ,,

I

,oo,,

t

16(R)M PGE2

B

1 6 DM PG[ 2

C

Fig. l. E f f e c t s of PGE2 (A), 16(R)-methyl PGE2 (B) and 16,16-dimethyl PGE2 (C) on c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s . C : c i r c u l a r muscle s t r i p , L : l o n g i t u d i n a l muscle s t r i p . Horizontal bars i n d i c a t e the presence of PGs (100 nM).16(R)-methyl PGEz:16(R)M PGE2. 16,16-dimethyl PGEz:16-DM PGE2.Each t r a c e of A,B and C was obtained from d i f f e r e n t p r e p a r a t i o n s .

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PROSTAGLANDINS

lg L ~

I

I

L

lmin Ig L _ _ 1rim

i

I

L I00 nM

lOnM

1 pM

16 DM PGE 2

Fig. 2. Concentration-responses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2. C : c i r c u l a r muscle s t r i p , L : l o n g i t u d i n a l muscle s t r i p . Horizontal bars i n d i c a t e the presence of 16,16-dimethyl PGE2.16,16-dimethyl PGE2: 16-DM PGE 2 (1 nM- 1 pM). i6,16-dimethyl PGE2 (1 nM- 1 pM) c o n c e n t r a t i o n - d e p e n d e n t l y c o n t r a c t e d both c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s (Fig. 2). The c o n t r a c t i l e responses of both muscle s t r i p s to 16,16dimethyl PGEz (100 n~) were not influenced by a t r o p i n e (1 pM) or t e t r o d o t o x i n (1 pM) in 4 experiments. 2 E f f e c t s of external Ca ions on the c o n t r a c t i l e responses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2. The c o n t r a c t i l e responses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2 (100 nH) were eliminated by the omission of Ca ions from the bathing s o l u t i o n , and with 1 mM EGTA in 3 experiments (Fig. 3). They were also eliminated in the presence of 1 mM La ions in 4 experiments (Fig. 4). The Ca channel blocker, verapamil (1 pM), completely i n h i b i t e d c o n t r a c t i l e response of l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2 (100 nM) (Fig. 5). I t did not block the response of c i r c u l a r muscle s t r i p s to the lower c o n c e n t r a t i o n s (1 and 10 nH) of the methyl analogue, but higher c o n c e n t r a t i o n s (100 nM and 1 pM) were s l i g h t l y suppressed by 1 pM verapamil (Fig. 6).

DISCUSSION In the present study, PGE2 100 nM produced a biphasic response in the c i r c u l a r muscle of guinea-pig proximal colon which c o n s i s t e d of an i n i t i a l c o n t r a c t i o n followed by r e l a x a t i o n . However, 16(R)-

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PROSTAGLAN DINS methyl PGE2 and 16,16 d i m e t h y l PGE2 produced s u s t a i n e d c o n t r a c t i o n s of the c i r c u l a r muscle. In l o n g i t u d i n a l muscle, a l l PGs produced a contraction. Since the c o n t r a c t i l e r e s p o n s e s of both muscles to 16, 1 6 - d i m e t h y l PGE2 were n o t i n f l u e n c e d by a t r o p i n e or t e t r o d o t o x i n , the a n a l o g u e seems to a c t d i r e c t l y on the muscles.

Cl(-)

EGTA lmld

L m [

L

--

Im---~n

"%"s"t~~- ~ lg I 16 DM PG[ 2

lOOnM

I lOOnM

Fig. 3. E f f e c t of removing e x t e r n a l Ca ions on the c o n t r a c t i l e r e sponses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 1 6 , 1 6 - d i m e t h y l PGE2 . C : c i r c u l a r muscle s t r i p , L : l o n g i t u d i n a l muscle s t r i p . A : c o n t r o l ( i n the p r e s e n c e of 2 mM Ca i o n s ) , B : i n the a b s e n c e of Ca ions with EGTA (1 m H ) . 1 6 , 1 6 - d i m e t h y l P G E 2 : 1 6 DM PGE2 (100 nM). The muscle c o n t r a c t i o n may depend on an i n c r e a s e in the i n t r a c e l l u l a r f r e e Ca ion c o n c e n t r a t i o n . This i n c r e a s e may r e s u l t from the i n f l u x of e x t r a c e l l u l a r Ca ions which e n t e r the muscle c e l l through r e c e p t o r - s e n s i t i v e or v o l t a g e - s e n s i t i v e Ca c h a n n e l s , or from the r e l e a s e of Ca ions from i n t r a c e l l u l a r Ca s t o r e s (11). In the p r e s e n t s t u d y , the c o n t r a c t i l e e f f e c t s of 1 6 , 1 6 - d i m e t h y l PGE2 on both c i r c u l a r and l o n g i t u d i n a l muscles were e l i m i n a t e d by o m i t t i n g Ca ions from the e x t e r n a l s o l u t i o n , or by a d d i n g La ions which p r e v e n t Ca ion f l u x e s through the smooth muscle membrane (12). These r e s u l t s i n d i c a t e t h a t the c o n t r a c t i l e e f f e c t s of the methyl a n a l o g u e s on both muscle l a y e r s may be due to an i n c r e a s e in i n f l u x of Ca ions through the muscle membrane.

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PROSTAGLANDINS

LoCI~ 1 mM

0.Sg

_

n

1 ~'nin

L I 16 DM PGE2

0.5g

lOOnM lOOnM

Fig. 4. E f f e c t of La ions on the c o n t r a c t i l e responses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2. C : c i c u l a r muscle s t r i p , L : l o n g i t u d i n a l muscle s t r i p . A:control, B:in the presence of LaC13 (1 mH).16,]6-dimethyl PGE2:16-DH PGE2 (100 nH)

lg

I 16 DMPG[2 lOOnH

i Verspamil lpi

F--

I

lOOnM lmin

L

I 16 DHPGE2 IO0,1i

lg

~

.,

i lOOdl

Fig. 5. E f f e c t of verapamil on the c o n t r a c t i l e responses of c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s to 16,16-dimethyl PGE2. C : c i r c u l a r muscle s t r i p , L : l o n g i t u d i n a l muscle s t r i p . A : c o n t r o l , B : i n the presence of verapamil (1 pM). 16,16-dimethyl PGE2:16-DH PGE2 (100 nH).

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PROSTAGLAN DINS 100 80

C_,

6O 40 ~0 ns I

0.1 n

I

In

I

1On

I

lOOn

I

lp

16 D M PGE2(M) Fig. 6. E f f e c t of v e r a p a m i l on c o n c e n t r a t i o n - r e s p o n s e c u r v e to 1 6 , 1 6 d i m e t h y l PGE 2 in c i r c u l a r muscle s t r i p . 0 0 : c o n t r o l (n=lO), 0------------0 : in t h e p r e s e n c e of 1 pM v e r a p a m i l (n=lO). 1 6 , 1 6 - d i m e t h y l PGE2:16-DM PGE2. The maximal r e s p o n s e to 16-DH PGE2 (1 pM) in c o n t r o l was t a k e n as 100 ~. Each p o i n t r e p r e s e n t s t h e mean + SEn shown by v e r t i c a l b a r s . : p < 0 . 0 5 compared w i t h c o n t r o l ( t h e s t a t i s t i c a l significance was a n a l y s e d by S t u d e n t ' s t - t e s t f o r p a i r e d d a t a ) , ns: p>O. 1. In c i r c u l a r muscle, however, t h e c o n t r a c t i l e r e s p o n s e to t h e methyl a n a l o g u e was s l i g h t l y r e d u c e d by v e r a p a m i l which i s a v o l t a g e s e n s i t i v e Ca channel b l o c k e r (11). Perhaps in t h e c i r c u l a r muscle a t low c o n c e n t r a t i o n s t h e methyl a n a l o g u e a c t s on r e c e p t o r - s e n s i t i v e Ca c h a n n e l s , and a t c o n c e n t r a t i o n s h i g h e r than 10 n~ i t may a c t l a r g e l y on r e c e p t o r - s e n s i t i v e Ca c h a n n e l s and p a r t l y on v o l t a g e s e n s i t i v e Ca c h a n n e l s . By c o n t r a s t , ! n l o n g i t u d i n a l muscle t h e methyl a n a l o g u e may a c t o n l y on v o l t a g e - s e n s i t i v e Ca c h a n n e l s , s i n c e t h e c o n t r a c t i l e e f f e c t of the a n a l o g u e was c o m p l e t e l y i n h i b i t e d by v e r p a m i l . A p r e v i o u s s t u d y (5) and the p r e s e n t o b s e r v a t i o n s s u g g e s t t h a t PGE2 has two o p p o s i t e a c t i o n s , c o n t r a c t i o n and r e l a x a t i o n , on p r o x i m a l c i r c u l a r muscles. However, in t h i s s t u d y 16-methyl a n a l o g u e s of PGE 2 produced s u s t a i n e d c o n t r a c t i o n w i t h o u t showing any r e l a x a t i o n . I t i s of i n t e r e s t t h a t t h e r e i s a d i f f e r e n c e in m e c h a n i c a l e f f e c t between PGE2 and i t s methyl a n a l o g u e s on c i r c u l a r m u s c l e s . However, t h e r e a s o n f o r t h i s d i f f e r e n c e was not c l a r i f i e d in t h i s s t u d y . One p o s s i b l e e x p l a n a t i o n f o r t h e s u s t a i n e d c o n t r a c t i o n induced by 16-methyl a n a l o g u e s of PGE2 in c o l o n i c c i r c u l a r muscles may depend on t h e i r l a c k of d e g r a d a t i o n by C-15 PG d e h y d r o g e n a s e , and a n o t h e r may depend on a d i r e c t a c t i o n of C-16 m e t h y l a t i o n of PGE2 on t h e muscle c e l l s .

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PROSTAGLANDINS ACKNOWLEDGENENTS The a u t h e r would l i k e to express a p p r e c i a t i o n to Ono Pharmac. Co.,(Osaka, Japan) f o r d o n a t i o n of PGE 2 and 16(R)-methyl PGE2.

RgFER~NCgS l. Bennett, A. and Fleshler, B. Prostaglandins and gastrointestinal tract. Gastroenterology. 59:790-800,1970. 2. Ishizawa, M. E f f e c t s of p r o s t a c y c l i n (PGI2) on the smooth muscles of the g u i n e a - p i g stomach. Arch. i n t . Pharmacodyn. Ther. 253:80-89,1981 3. Ishizawa, M. C o n t r a c t i l e e f f e c t s of PGE2 and PGF2a on mechanical and e l e c t r i c a l a c t i v i t i e s in l o n g i t u d i n a l and c i r c u l a r muscles of the g u i n e a - p i g colon. P r o s t a g l a n d i n s . 26:175-186,1983. 4. Milenov, K.,Pakovska, A. and Chernaeva, L. E f f e c t s of p r o s t a g l a n d i n E1 and E2 on the mechanical a c t i v i t y of i s o l a t e d c i r c u l a r and l o n g i t u d i n a l muscle s t r i p s of g u i n e a - p i g stomach. Arch. i n t . Pharmacodyn. Ther. 247:320-328,1976. 5. Ishizawa, M. Biphasic r e s p o n s e s of c i r c u l a r muscles to p r o s t a g l a n d i n E2 in g u i n e a - p i g colon. Arch. i n t . Pharmacodyn. Ther. 295: 282-290,1988. 6, Bennett, A . , E l e y , K.G. and Scholes, G.B. E f f e c t s of p r o s t a g l a n d i n E1 and E2 on human, g u i n e a - p i g and r a t i s o l a t e d small i n t e s t i n e s . B r i t . J. Pharmacol.34:630-638,1968.

7. Robert, A. and Magerlein, B.J. 15-methyl PGE2 and 16,16-dimethyl PGE2:potent inhibitor of gastric secretion. Advances in the Bioscience. 9:247-253,1972. 8. Main, I.H.M. and W h i t t l e , B.J.R. Potency and s e l e c t i v i t y of methyl analogues of p r o s t a g l a n d i n E2 on r a t g a s t r o i n t e s t i n a l f u n c t i o n . B r i t . J. Pharmacol.54:309-317,1975. 9. Ruwart, M . J . , K l e p p e r , M.S. and Rush, B.D. The b e n e f i c a l e f f e c t s of p r o s t a g l a n d i n s in p o s t - o p e r a t i v e i l e u s . G a s t r o e n t e r o l o g y . 74:1088, 1978. lO. Nylander, B. and Mattsson, D. E f f e c t of 16,16-dimethyl PGE2 on g a s t r i c emptying and i n t e s t i n a l t r a n s i t of barium-food t e s t meal in man. Scand. J. G a s t r o e n t e r o l o g y . lO:289-292,1975.

ll. Bolton, T.B. Mechanism of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59:606-718,1979. 12. Weiss, G.B. and Goodman, F.R. E f f e c t of Lanthanum on c o n t r a c t i o n , calcium d i s t r i b u t i o n and 45Ca movement in i n t e s t i n a l smooth muscle. J. Pharmacol. Exp, Ther. 169:46,1969. Editor: A. Bennett

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Received:

5-14-91

Accepted: 10-4-91

DECEMBER 1991 VOL. 42 NO. 6

Contractile effects of 16-methyl analogues of PGE2 on the circular and longitudinal muscles of the guinea-pig isolated colon.

The mechanical effects of 16-methyl analogues of PGE2, mainly 16,16-dimethyl PGE2, on circular and longitudinal muscles of the guinea-pig isolated pro...
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