Life Sciences, Vol. 29, pp. 1803-1809 Pergamon Press Printed in the U.S.A.

THE EFFECT OF INDOMETHACIN AND SUBSTANCE P ON THE GUINEA PIG URINARY BLADDER

A. Johns

Depar tmen t of O b s t e t r i c s and Gyneco logy , The U n i v e r s i t y of Texas Health Science C e n t e r at San Anton io , San Anton io , Texas 78284

(Received in final form August 27, 1981)

Summary

The effects of indomethacin on the responses of the guinea pig urinary bladder to nerve stimulation, acetylcholine, adenosine 5' triphosphate and Substance P have been investigated. Indomethacin alone had no significant effect on responses of the bladder to nerve stimulation but did significantly reduce the atropine-resistant contractions. Responses of the tissue to acetylcholine were unaffected by indomethacin but responses to Substance P were significantly reduced. Only the highest dose of ATP (10 -3 M) was significantly reduced by indomethacin. The possibility that Substance P is the transmitter responsible for the atropine-resistant contractions of the urinary bladder to nerve stimulation is discussed.

Stimulation of the parasympathetic nerves to the bladder results in a contraction and release of acetylcholine (1). The nerve induced contraction is only partially antagonized by atropine at concentrations that antagonize responses to exogenous acetylcholine (2). This atropine-resistance of nerve induced contractions has led a number of authors to suggest that the parasympathetic innervation is partially non-cholinergic (2,3,4).

The atropine-resistant contractions of the bladder to nerve stimulation are resistant to antagonists of noradrenaline, 5-hydroxytryptamine, and histamine and the guinea pig bladder does not respond to most amino acids (3). Ambache and Zar (3) first pointed out the similarity between responses of the bladder to nerve stimulation and responses to exogenous ATP and Dumsday (4) and Burnstock, Dumsday and Smythe (5) suggested that the atropine-resistant con- tractions were mediated by ATP released from purinergic nerves in the bladder. Ambache and Zar (3) showed tachyphylaxis to ATP without tachyphylaxis to nerve stimulation and Ambache et al. (6) showed that the adenosine uptake inhibitor dipyridamole was unable to potentiate the atropine-resistant contrac- tions suggesting that ATP was not the transmitter. Burnstock, Dumsday and Smythe (5) reported that quinidine selectively inhibited responses of the bladder to both ATP and atropine-resistant contractions, although responses to acetylcholine were also inhibited. A specific ATP receptor blocker 2-2'-pyridyl- isatogen tosylate (PIT) has been shown not to be effective as an antagonist of the atropine-resistant contractions (7) as has theophylline (8).

In rabbit and monkey bladder, it has been suggested that prostaglandins may mediate the atropine-resistant contractions (9,10,ii), however, in rat urinary bladder, Choo and Mitchelson (12) showed indomethacin only marginally effective in inhibiting atropine-resistant contractions suggesting that in this species prostaglandins do not mediate the atropine-resistant contractions of the bladder to nerve stimulation.

oo24-32o5/81/1718o3-o7502.0o/o Copyright (c) 1981 Pergamon Press Ltd.

1804 Indomethacin and Bladder Vol. 29, No. 17, 1981

The p u r p o s e of the p r e s e n t s t u d y was to i n v e s t i g a t e the effects of indomethac in on r e s p o n s e s of the gu inea pig u r i n a r y b l a dde r to n e r v e s t imula t ion and o the r poss ib le t r a n s m i t t e r s in the b l a d d e r .

Methods

The u r i n a r y b l a d d e r was removed from female gu inea p igs ( 2 5 0 - 350 g body we igh t ) in to cooled, modified K r e b s - H e n s e l e i t t ype so lu t ion . S t r ips of d e t r u s o r muscle were p r e p a r e d and mounted ve r t i ca l ly in 5 ml o rgan ba ths c o n t a i n i n g the modified Krebs so lu t ion at 37°C. Isometr ic con t r ac t ions were r eco rded u s i n g force d i sp lacement t r a n s d u c e r s (Grass FTO3C) and d i sp layed on a p o l y g r a p h (E and M I n s t r u m e n t s Model DMP 4A). All t i s sues were main ta ined at 1.0 g r e s t i n g t ens ion t h r o u g h o u t the expe r imen t and were allowed to r ecove r from the d i s sec t ion p r o c e d u r e for 60 rain before r e s p o n s e s were de t e rmined .

Con t r ac t i ons were el ic i ted e i the r by t r a n s m u r a l s t imula t ion or by add i t ion of va r ious d r u g s . T r a n s m u r a l s t imula t ion was ach ieved by u s i n g a Grass s t imula tor (Model SD9) t ha t supp l i ed monophasic pu l ses of I msec d u r a t i o n and supramaximal vol tage for 30 sec be tween two p la t inum e lec t rodes s i t ua t ed at the top and bottom of the o rgan ba th . Cont rac t i l e r e s p o n s e s were e x p r e s s e d as a p e r c e n t a g e of the t i s s u e ' s r e s p o n s e to t r a n s m u r a l s t imula t ion at a f r e q u e n c y of 20 Hz (maximum con t r ac t i on to t r a n s m u r a l s t imula t ion) de t e rmined in the p r e s e n c e of only g u a n e t h i d i n e (4 x 10 -s M). D u r i n g t r a n s m u r a l s t imula t ion , the ba th f luid was c o n t i n u o u s l y changed by overf low at a r a t e of 1 - 2 ml/min.

The modified K r e b s - H e n s e l e i t t ype so lu t ion had the following composit ion (raM): NaC1, 116; KC1, 5 .4 ; CaCI2, 2 .5 ; MgC12, 1 .2 ; NaH2PO4, 1 .2 ; NaHCO3, 22.0; D glucose 11.2. This so lu t ion when e q u i l i b r a t e d with 95% 02:5% CO 2 had a pH of 7 .4. In o r d e r to p r e v e n t re lease of n o r a d r e n a l i n e d u r i n g t r a n s m u r a l s t imula t ion , the Krebs con ta ined 4 x 10 -s M g u a n e t h i d i n e .

A s tock solut ion of indomethac in was f r e s h l y p r e p a r e d for each expe r imen t and used wi th in t h r ee h o u r s . This so lu t ion con ta ined indomethac in 10 mg and a n h y d r o u s sodium c a r b o n a t e 5 mg d i sso lved in 10 ml of d is t i l led wate r . This s tock so lu t ion was added to the Krebs so lu t ion d u r i n g p r e p a r a t i o n to ob ta in the r e q u i r e d c o n c e n t r a t i o n of indomethac in . Indomethac in , ace ty lcho l ine , S u b s t a n c e P, adenos ine 5' t r i p h o s p h a t e disodium sal t and a t rop ine were ob ta ined from Sigma Chemical Company and g u a n e t h i d i n e monosu lpha te was dona ted by C iba -Ge igy Canada Ltd. The r e s u l t s are e x p r e s s e d as the mean +_ S.E. of the mean. The s ign i f i cance of r e s u l t s was de t e rmined u s i n g S t u d e n t ' s T t e s t and r e s u l t s were cons ide red as s i gn i f i c an t when P <0.05.

Resu l t s

The effect of a t rop ine and indomethac in on the r e s p o n s e s to t r a n s m u r a l s t imula t ion is shown in Fig. 1.

The f i r s t and second de t e rmina t i ons of the f r e q u e n c y / r e s p o n s e c u r v e s of gu inea pig u r i n a r y b l a d d e r did not d i f fer s i gn i f i can t ly a l though the second de t e rmina t ion was s l igh t ly lower t h a n the f i r s t . Indomethac in (2 .8 x 10 -6 M) had no s i g n i f i c a n t effect on r e s p o n s e s to t r a n s m u r a l s t imula t ion , whereas a t rop ine (2 .9 x 10 -5 M) s i gn i f i c an t l y i nh ib i t ed r e s p o n s e s of the t i s sue to t r a n s m u r a l s t imula t ion . The combinat ion of a t rop ine (2.9 x 10 ' s M) and indomethac in (2 .8 x 10 -6 M) was no t only s ign i f i can t ly d i f f e r e n t to the control b u t also s i gn i f i c an t l y d i f f e r e n t to both a t rop ine alone or indomethac in alone.

F igu re 2 shows the effect of indomethac in on the r e s p o n s e s to added ace ty lcho l ine . It can be seen tha t indomethac in (2 .8 x 10 -e M) had no s i gn i f i c an t effect on the r e s p o n s e s of the gu inea pig u r i n a r y b l a dde r to added

Vol. 29, No. 17, 1981 Indomethacin and Bladder 1805

80

7O

N ' I "

6O

o

~ 5o c 0

4O

w,I m Z o 3o a . if)

2O

10

I I I I | I I I I 1

1 2 3 4 5 6 7 8 9 10

STIMULUS FREQUENCY (Hz )

F IG . 1

T h e e f f e c t o f i n d o m e t h a c i n on t h e r e s p o n s e s o f g u i n e a p i g b l a d d e r to n e r v e s t i m u l a t i o n in t h e p r e s e n c e o f g u a n e t h i d i n e (4 x 10 -s M ) . e - e c o n t r o l , o - o 2 . 8 x 10 -6 M i n d o m e t h a c i n ; ~ - o 2 . 9 x 10 -s M a t r o p i n e ; A-A 2 . 8 x 10 -6 M i n d o m e t h a c i n ; 2 . 9 x 10 ' s M a t r o p i n e . T h e r e s u l t s s h o w n a r e t h e mean + S . E . M . o f 8 e x p e r i m e n t s .

a c e t y l c h o l i n e . The e f f ec t of indomethac in on the r e s p o n s e s to adenos ine 5' t r i p h o s p h a t e is shown in Fig. 3; a l t h o u g h 2.8 x 10 -6 M indornethacin inh ib i t ed the r e s p o n s e s of the d e t r u s o r to added ATP, the only s i gn i f i c an t d e p r e s s i o n in r e s p o n s e was at 10 -3 M ATP. The e f f ec t of indomethac in on the r e s p o n s e s to S u b s t a n c e P is shown in Fig. 4. Indomethac in (2 .8 x 10 -6 M) s i gn i f i c an t l y r e d u c e d the r e s p o n s e s of the u r i n a r y b l a d d e r to S u b s t a n c e P at all the c o n c e n t r a t i o n s i n v e s t i g a t e d .

Discuss ion

The a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s of the u r i n a r y b l a d d e r to p a r a - s y m p a t h e t i c n e r v e s t imula t ion a re t h o u g h t to be n o n - c h o l i n e r g i c s ince t h e y a re not p o t e n t i a t e d by a n t i c h o l i n e s t e r a s e d r u g s ( 1 3 , 1 4 , 3 ) . The t r a n s m i t t e r r e s p o n s i b l e for c o n t r a c t i o n is not an amino acid s ince con t r ac t i l e r e s p o n s e s could not be e l ic i ted a t r e l a t i v e l y h igh c o n c e n t r a t i o n s . The a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s a re no t i nh ib i t ed by a n t a g o n i s t s of 5 - h y d r o x y t r y p t a m i n e , h is tamine or ca t echo lamines . Ambache and Zar (3) also s u g g e s t e d t h a t in the gu inea pig b l a d d e r , the t r a n s m i t t e r was not a p r o s t a g l a n d i n s ince they fai led to el ici t c o n t r a c t i l e r e s p o n s e s even at r e l a t i v e l y h igh c o n c e n t r a t i o n s of both p r o s t a g l a n d i n E (PGE) and p r o s t a g l a n d i n F2~ (PGF2~) .

1806 Indomethacin and Bladder Vol. 29, No. 17, 1981

100

90

80

~:Tj R "~ 60

O ~ 50

N 40 Z o

m 301 w Q~

2O

!

10

T

I ,,I 10-6 lO-S 10~4 10-3 10-2

C O N C E N T R A T I O N OF A C E T Y L C H O L I N E ( M )

FIG. 2

The e f fec t of indomethac in on the responses of the gu inea p ig u r i n a r y b l a d d e r to ace t y l cho l i ne in the p resence of g u a n e t h i d i n e (4 x l0 -s IVl). I - i c o n t r o l ; o -o 2.8 x 10 6 M indomethac in . The resu l t s shown are the mean -+ S . E . M . of 8 e x p e r i m e n t s .

In r a b b i t and monkey u r i n a r y b l a d d e r , the a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s to n e r v e s t imula t ion are g r ea t l y i nh ib i t ed by indomethac in s u g g e s t i n g t ha t in these species the a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s are mediated by a p r o s t a g l a n d i n ( 9 , 1 0 , 1 1 ) . In the r a t and gu inea pig u r i n a r y b l a d d e r , a l t hough indomethac in does i n h i b i t the a t r o p i n e r e s i s t a n t c o n t r a c t i o n s , i ts ef fects are less dramat ic (12, p r e s e n t s t u d y ) . Alkondon and G a ngu l y (15) s u g g e s t tha t indomethac in has no effect on the mechanical r e s p o n s e s of the gu inea pig b l a d d e r , however , no ev idence is p r e s e n t e d as to what s t imulus f r e q u e n c y was u sed .

Ambache and Zar (3) r e p o r t e d t ha t the a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s of the gu inea pig b l a d d e r r e sembled r e s p o n s e s of the t i s s u e to added ATP a nd Dumsday (4) and B u r n s t o c k , Dumsday and Smythe (5) s u g g e s t e d t ha t the motor t r a n s m i t t e r in the u r i n a r y b l a d d e r was ATP s ince it was b locked by q u i n i d i n e and t a c h y p h y l a x i s to ATP was assoc ia ted with r e d u c e d r e s p o n s e s to n e r v e s t imula t ion . This c ross t a c h y p h y l a x i s , however , could no t be d e m o n s t r a t e d by Ambache and Zar (3 ) , and Ambache et al. (6) showed tha t d ipyr idamol had no

Volo 29, No. 17, 1981 Indomethacin and Bladder 1807

30

2: O

O * , 2 0

0

u~ 111 cD Z O a. cD u l

I I I I 10-6 10-5 I0-4 10-3

CONCENTRATION OF ATP (M) FIG. 3

The e f fec t of indomethac in on the responses of the gu inea p ig u r i n a r y b l a d d e r to ATP in the p resence of a t r o p i n e (2 .9 x I0 -s M) and g u a n e t h i d i n e (4 x 10 -s M). e -e c o n t r o l ; o -o 2.8 x I0 -6 M indomethac in . The resu l t s shown are the mean + S . E . M . of 8 e x p e r i m e n t s . Values t ha t are s i g n i f i c a n t l y d i f f e r e n t (p <0.05) are shown *

effect on the a t r o p i n e - r e s i s t a n t con t r ac t i ons a l though th is compound has been shown to po ten t i a t e r e s p o n s e s to adenos ine and not ATP. The ATP a n t a g o n i s t 2-2 ' p y r i d y l i s a t o g e n tosy la te (P IT) also has no effect on the a t r o p i n e - r e s i s t a n t c o n t r a c t i o n s (7 ,16) and n e i t h e r does t heophy l l i ne (8) .

The i n h i b i t o r y effect of indomethac in on the a t rop ine r e s i s t a n t con t r ac t i ons of the d e t r u s o r to n e r v e s t imula t ion could be exp la ined by the fact tha t r e s p o n s e s of r a b b i t and cat b l a d d e r s to ATP are r e d u c e d by i n h i b i t o r s of p r o s t a g l a n d i n s y n t h e s i s (17 ,18 ,19) . The r e s p o n s e to ATP cons i s t s of an ini t ia l phas ic r e s p o n s e and a main ta ined toxic r e s p o n s e , and only the toxic r e s p o n s e is r e d u c e d by indomethac in (20) . Indomethac in i nh ib i t s both the tonic and phas ic r e s p o n s e to n e r v e s t imula t ion , which would s u g g e s t tha t the phas ic r e s p o n s e is not media ted by ATP (20) .

A n o n - c h o l i n e r g i c mechanism tha t has been p roposed to expla in a t rop ine r e s i s t a n c e in o the r cho l ine rg ica l ly i n n e r v a t e d t i s sue s is the re lease of po lypep t ide t r a n s m i t t e r s (21 ,22) . Hukovic , Rand and Vanov (23) have i n v e s t i g a t e d the r e s p o n s e s of the r a t and r a b b i t b l a dde r to b r a d y k i n i n . The r e s p o n s e was r e p o r t e d as slow in onse t and long in d u r a t i o n un l ike n e r v e s t imula t ion , and these a u t h o r s d i s coun ted b r a d y k i n i n as a poss ib le t r a n s m i t t e r .

1808 Indomethacin and Bladder Vol. 29, No. 17, 198l

40

N 30 "1-

W C 0

~ 20 ~ ,

il

I I I I

10 100 1000 10000

CONCENTRATION OF SUBSTANCE P(ng/ml )

FIG. 4

T h e e f f e c t o f i n d o m e t h a c i n on t h e r e s p o n s e s o f g u i n e a p ig b l a d d e r to S u b s t a n c e P in t h e p r e s e n c e o f a t r o p i n e ( 2 , 9 x 10 -s M) and g u a n e t h i d i n e (4 x 10 -s M ) . e -e c o n t r o l ( n : 12) ; o - o 2 . 8 x 10 -6 M i n d o m e t h a c i n (n = 6 ) . T h e r e s u l t s s h o w n a r e t h e mean + S . E . M . o f t h e sa id n u m b e r o f e x p e r i m e n t s . S i g n i f i c a n t l y d i f f e r e n t ( p <0 .05 ) r e s p o n s e s a re s h o w n *

A similar conc lus ion was r eached by Downie and Rouff ignac (24) on the act ion of b r a d y k i n i n on the r a b b i t d e t r u s o r and they r e p o r t e d some of the act ion of b r a d y k i n i n was due to p r o s t a g l a n d i n re lease . Falconier i E r spamer , Negri and Piccinell i (25) r e p o r t e d tha t the d e t r u s o r from e ight d i f f e r e n t species of mammal exh ib i t ed " in t ense t a c h y p h y l a x i s " to a n g i o t e n s i o n . Responses of the d e t r u s o r to p a r a s y m p a t h e t i c n e r v e s t imula t ion r a r e l y show t a c h y p h y l a x i s (3) . Al though c rossed t a c h y p h y l a x i s to n e r v e s t imula t ion and a n g i o t e n s i o n has no t been i n v e s t i g a t e d in the b l a d d e r , it is un l ike ly t ha t this po lypep t ide is the pa r a - sympa the t i c t r a n s m i t t e r . The po lypep t ide S u b s t a n c e P f i r s t d e s c r i b e d by Euler and Gaddum (26) was r e p o r t e d to be in r e l a t ive ly h igh c o n c e n t r a t i o n s in the u r i n a r y b l a d d e r of the horse and has been d e m o n s t r a t e d in the b l a d d e r of both the dog and mouse (27) . Recen t ly , Alto et al. (28) have de sc r i be d n e r v e s con t a in ing S u b s t a n c e P in the u r i n a r y b l adde r as forming a n e t - l i k e p lexus a r o u n d the smooth muscle cel ls . Falconier i Erspamer , Negri and Piccinelli (25) d e s c r i b e d S u b s t a n c e P as be ing 4-8 times more p o t e n t t han ace ty lcho l ine in c aus ing c o n t r a c t i o n s of r a t , gu inea pig and hams te r u r i n a r y b l a d d e r . The con t rac t i l e r e s p o n s e in the b l a d d e r to S u b s t a n c e P cons i s t s of an ini t ia l spike followed by a ma in ta ined con t r ac t i on and in the p r e s e n c e of indomethac in , both r e s p o n s e s are r e d u c e d . Response to S u b s t a n c e P in o the r t i s s u e s , however , are una f f ec t ed by indomethac in (29) s u g g e s t i n g tha t S u b s t a n c e P is not i tself

Vol. 29, No. 17, 1981 Indomethacin and Bladder 1809

a n t a g o n i z e d by indomethac in . The p r e s e n t s t u d y would ind ica te , however , t ha t in the b l a d d e r , a t leas t some of the ac t ion of S u b s t a n c e P is media ted by a p r o s t a g l a n d i n .

Since indomethac in in the gu inea pig b l a d d e r i nh ib i t s the a t r o p i n e - r e s i s t a n t con t r ac t i on to n e r v e s t imula t ion and r e s p o n s e s to S u b s t a n c e P, b u t no t to ATP, and s ince S u b s t a n c e P is known to be con ta ined wi th in p e p t i d e r g i c n e r v e s i n n e r v a t i n g the b l a d d e r , the n o n - a d r e n e r g i c , n o n - c h o l i n e r g i c t r a n s m i t t e r in the gu inea pig b l a d d e r could be S u b s t a n c e P.

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