Response of rabbit detrusor muscle to bradykinin

Life Sciences, Vol. 28, pp. 603-608 Printed in the U.S.A.

Pergamon Press

RESPONSE OP RABBIT DEmRU~OR MUSCLE mO BRADYKININ J.W. Downie and S. Rouffignac Department of Urology and Department of Pharmacology Queen's University Kingston, Ontario, Canada (Received in final form November 18, 1980) Summar~ The contractile response to bradykinin was studied in isolated longitudinal strips of detrusor muscle from rabbit urinary bladder. Strips responded slowly with contractions w h i c h were comparable in magnitude to acetylcholine but much greater than those produced by arachidonic acid. The bradvkinin dose-response curve was very shallow (Clark's rat{o = 10-5), with an ED50 of 0.2 ~M. Bradykinin-induced contractions were unaffected by 0.4 pM atropine or 0.2 ~M eserine. This suggests, in contrast to reports on rat bladder, that acetvlcholine release does not contribute to the response. However, pretreatment with i0 ~M naproxen antagonized bradvkinininduced contractions without affecting acetylcholine. It is concluded that, as in many other tissues, in the urinary bladder at least part of the response to bradykinin is mediated through prostaqlandins. Bradykinin probably also has a direct action since higher concentrations are less susceptible to naDroxen, and it produces a much greater contraction than the maximum achievable with arachidonic acid. The excitatory innervation of the urinary bladder is accepted as being parasympathetic (i) but there is continuing disagreement over whether it is cholinergic (1-3). An alternate hypothesis which has some experimental support is that ATP might be a transmitter (4-6). Neuropeptides would also be candidates for a noncholinergic excitatory transmitter in the bladder but few studies have explored this possibility (7,8). Bradykinin or a related peptide has been advanced as a candidate for the noncholinergic nonadrenergic inhibitory transmitter in anococcygeus muscle (9). However, it is known in some systems to act in an indirect manner, particularly by release of prostaglandins (e.g. i0). In rat urinary bladder bradykinin may release acetylcholine (7). In the experiments reported here we have assessed the sensitivity of rabbit detrusor to bradykinin ~n vitro and examined to what extent its action is mediate,~ through release of ~r2staHlandins and acetylcholine. Address reprint requests to Dr. J.W. Downie, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. 0024-3205/81/060603-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

604

Bradykinin in Rabbit Detrusor

Materials

Vol. 28, No. 6, 1981

and M e t h o d s

Longitudinally oriented strips from the lateral aspects of the rabbit detrusor muscle were o b t a i n e d as p r e v i o u s l y d e s c r i b e d (3). The tissues were mounted in I0 ml glass organ baths containing Krebs' solution of the following c o m p o s i t i o n (mM): NaCI 118, KCI 4.69, CaCI 2 2.45, MgSO 4 1.18, KH2PO 4 1.18, NaHCO 3 25, g l u c o s e 5.55, bubbled with q5% oxygen and 5% carbon dioxide and maintained at 35°C. Initially the tissue was stretched to 2 g tension and then allowed to e q u i l i b r a t e for one hour with frequent washing. I s o m e t r i c responses were monitored with Grass VT.03c t r a n s d u c e r s coupled to a Grass Dolvgraph. Acetylcholine chloride, atropine sulfate, bradvkinin triacetate and eserine sulfate were o b t a i n e d from Sigma C h e m i c a l Co. (St. Louis, MO). N a p r o x e n was o b t a i n e d from ~yntex L a b o r a t o r i e s (Palo Alto, CA). Stock s o l u t i o n s of a c e t y l c h o l i n e (0.1 mM HCI) and a t r o D i n e sulfate (deionized water) were stored as aliquots at -4°C. S o l u t i o n s of b r a d v k i n i n (saline) eserine (deionized water, then finally in Krebs' solution) and n a p r o x e n (0.I M o h o s ~ h a t e buffer) were p r e p a r e d fresh on the day of the experiment. Because of p r o b l e m s with the s t a b i l i t y of bradvkinin, it was p u r c h a s e d in small lots and used within a week of arrival in the laboratory. The vials were sealed with P a r a f i l m -M (American C a n Company) and stored desiccated at -4°C. Bradvkinin was found to be t a c h y p h y l a c t i c so for most studies the tissue was c h a l l e n q e d at 30 or 60 minute intervals with frequent washing between.

'

~10

4

100

T

T

Bk

ACh

FIG.

I_

R e p r e s e n t a t i v e tracings of the c o n t r a c t i o n p r o d u c e d by bradykinin (Bk) and acetvlcholine (ACh). ~uperimposed b r a d y k i n i n resoonses are in four strips from the same rabbit. Doses on all traces are in ~M. Vertical and h o r i z o n t a l bars represent 2 q tension and 5 min respectively.

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Bradykinin in Rabbit Detrusor

605

Results The rabbit urinary bladder was very sensitive to bradykinin. W i t h a fresh lot, the threshold c o n c e n t r a t i o n was 1 DM or lower, but this appeared to increase with the age of the lot. The time taken to reach the peak of the response to b r a d y k i n i n was shorter at higher concentrations, but even at high c o n c e n t r a t i o n s was longer than for a c e t y l c h o l i n e (Figure I). B r a d y k i n i n p r o d u c e d as g r e a t a c o n t r a c t i o n as a c e t y l c h o l i n e (Figure i).

lO0

Z

o

80

U nZ

60

o

U

E:)

40

X <{

20

oL L

I

1

I

I

r2 I0 8 6 4 -log [-BRADYKININ~(M) FIG.

2

Mean dose-response curve of bradykinin in 8 d e t r u s o r strips from 4 rabbits. ED~0 is 0.2 ~M and the ratio of the cor~=entrations p r o d u c l n g 16% and 84% of m a x i m u m (Clark's ratio, ref. ii) is about 10 -5 . Vertical bars are s t a n d a r d errors of the means. The concentration-response relationship for b r a d y k i n i n was very shallow, s p a n n i n g 8 log units (Figure 2). The ED50 was 0.2 ~M. C o n c e n t r a t i o n s in the m i d - r a n g e of the curve were used to test whether the response had c h o l i n e r g i c or p r o s t a g l a n d i n components. B r a d y k i n i n - i n d u c e d c o n t r a c t i o n s were not s i g n i f i c a n t l y affected by a t r o p i n e or e s e r i n e (Table I). ~retreatment with naproxen antagonized the response to bradykinin, particularl V at low concentrations (Table I). The maximum response achieved with b r a d y k i n i n (0.i n%M) (9.8 ± 0.46 g, n=7) I was c o n s i d e r a b l y greater than that o b t a i n e d at the m a x i m u m of the d o s e - r e s p o n s e curve of a r a c h i d o n i c acid ~I0 ~M) (2.0 ± 0.32 HA n=10). - - Y ( M e a n ± S.E.M., n ~ n u m b e r 5~ 6 5 s e r v a E T o n s i .

606

Bradykinin in Rabbit Detrusor

TABLE Effect

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I

of A t r o p i n e , E s e r i n e and N a p r o x e n on B r a d v k i n i n - i n d u c e d C o n t r a c t i o n s in R a b b i t D e t r u s o r S t r i p s Contraction

Bradykinin (~M)

Control (n=6)

as p e r c e n t

Atropine (0.4~M, n=6)

of

initial

response

Eserine (0.2~M, n=3)

NaDroxen (10~M, n=6)

0.i

i01.2±1.2

90.3+

5.3

i01.7±13.2

25.1-+ 4.4

0.3

100.2-+1.1

116.0-+

7.6

84.4 + g.4

37.0-+ 8.5

1.0

98.6±1.4

107.0±10.0

98.5±13.8

72.7±!1.4

R e s p o n s e s to the three c o n c e n t r a t i o n s of b r a d y k i n i n w e r e d e t e r m i n e d in each strip before and after t r e a t m e n t with the drugs indicated. At least 30 min elapsed b e t w e e n each dose. T h e second r e s p o n s e at each conc e n t r a t i o n is e x p r e s s e d as p e r c e n t of the first. Only one drug treatment was used per strip, and control strips r e c e i v e d no treatment. Drug c o n t a c t times were: atropine, 20 min; e s e r i n e and naproxen, 50 min. Values r e p r e s e n t m e a n s + S.E.M. Discussion Bradykinin has been shown p r e v i o u s l y to be a s t i m u l a n t of b l a d d e r smooth m u s c l e both in v i t r o (7,12) and in v i v o (13). The t h r e s h o l d c o n c e n t r a t i o n found in this study is 1/30 to 1/300 of that r e p o r t e d by F a l c o n e i r i - E r s D a m e r et al. (12). H o w e v e r , for a given lot this value i n c r e a s e d over a week to the leve]s prev i o u s l y reported. B r a d y k i n i n induces t a c h y p h y l a x i s (12), but this c o u l d be m i n i m i z e d by p r o l o n g e d w a s h i n g of the tissue. T h e interdose interval used in the p r e s e n t study was c o n s i d e r a b l y longer than that used by H u k o v i c et al. (7). The shallow concentration-response relationship yielded a C l a r k ' s ratio of about 10 -5 i n d i c a t i n g c o n s i d e r a b l e d e v i a t i o n from a classical bimolecular reaction (theoretical Clark's ratio = 1/27, Ref. ii). This type of a n a l y s i s has been applied to b r a d y k i n i n in a number of tissues (14) and s u b s t a n t i a l tissue d i f f e r e n c e s were noted. T h e q u e s t i o n has been raised w h e t h e r the nonclassical r e l a t i o n s h i p r e p r e s e n t s an indirect a c t i o n in those tissues. H u k o v i c et al. (7) suggested that the a c t i o n of bradykinin on rat bladder was mediated partly through the release of a c e t y l c h o l i n e since the r e s p o n s e s were p o t e n t i a t e d by e s e r i n e and partly a n t a g o n i z e d by atropine. In a p r e v i o u s study in rabbit detrusor 0.2 ~M e s e r i n e was shown to be e f f e c t i v e in p o t e n t i a t i n g r e s p o n s e s to a c e t y l c h o l i n e and 0.4 ~M a t r o p i n e was a p p r o x i m a t e l y the m a x i m u m concentration which was a n t i m u s c a r i n i c and free of n o n s p e c i f i c d e p r e s s a n t a c t i v i t V (3). S i n c e neither of these druqs a f f e c t e d the r e s p o n s e to b r a d y k i n i n , we c o n c l u d e that there is no e v i d e n c e for an a c e t y l c h o l i n e release c o m p o n e n t to the a c t i o n of b r a d y k i n i n in rabbit detrusor.

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Bradykinin in Rabbit Detrusor

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Naproxen (I0 ~M) markedly antagonizes the response to arachidonic acid in rabbit detrusor without altering that to acetylcholine (15). In the present experiments bradykinin-induced responses were susceptible to 10 ~ M naDroxen. Therefore, in agreement with many studies of bradykinin in other tissues (e.g. I0), we have found presumptive evidence for prostaglandin mediation of a portion of bradykinin's effect on the bladder. Since the effect of naproxen was less at higher bradykinin concentrations and the maximum response to bradykinin was much greater than that to arachidonic acid, it is possible that a non-prostaglandin mechanism might be involved particularly at higher concentrations. Although we found no unusual inflection in the dose-response curve which would lend credence to this suggestion, a more detailed analysis of the dose-response relationship might reveal it. Recently, the response to bradvkinin in guinea pig bladder was shown to be indomethacin-sensitive but it was nevertheless interpreted as being primarily prostaglandin-independent (16). The contraction produced by field stimulation in detrusor strips from various species is very brisk (3,4,17) and is m i m i c k e d by ATP but not by acetylcholine or more slowly acting agents such as arachidonic acid or prostaglandins (4,5,15,17). On this basis, prostaglandin is not considered to be a suitable candidate for the noncholinergic excitatory transmitter and bradykinin would also seem to be unlikely based on a similar argument (16, this paper). However, it is evident that prostaglandins play some role in excitatory transmission in the bladder (18,19) and, at least in rabbit detrusor, this role appears to be modulation of the noncholinergic component (15,20). Whether bradykinin or a similar peptide could participate directly or indirectly (through prostaglandins) in a modulatory function in the bladder is unknown. However, its apparently powerful ability to induce prostaglandin release may make it a useful tool for the investigation of neuromodulation in the urinary bladder. ~cknowled@ements The Medical Research Council of Canada provided a grant-inaid for this research and a scholarship for JWD. Naproxen was a gift of Syntex Laboratories. References i.

2. 3. 4. 5. 6. 7. 8. 9.

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203 44 J. ~07 ~4 62

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i0. ii. 12. 13 14. 15 16. 17 18. 19 20.

Bradykinin in Rabbit Detrusor

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J.C. McGIFF, N.A. TERRAGNO, K.U. MALIK and A.J. LONIGRO, Circulation Res. 31 36-43 (1972). A.J. CLARK, General Pharmacoloqz (Handbook of Experimental Pharmacology, Vol. 4), 0.63-64, Springer, Berlin (1937). G. FALCONIERI-ERSPAMER, L. NEGRI and D. PICCINELLI, NaunvnSchmiedebergs Arch. Pharmac. 279 61-74 (1978). S. MATSUMURA, N. TAIRA and K. HASHIMO~O, Tohoku J. Exp. Med. 96 247-258 (1968). J. ~ARABE, W.K. PARK and D. REGOLI, Can. J. Physiol. Pharmac. 53 345-353 (I~75). J.W. DOWNIE and C. LARSSON, Can. J. Physiol. Pharmac. (In press). J.D. FERRERO, T. COCKS and G. BURNSTOCK, Eur. J. Pharmac. 63 295-302 (1980). N. AMBACHE and M.A. ZAR, J. Physiol., Lond. 210 761-783 (1970). L.K. CHO0 and F. MITCHELSON, Prostaqlandins !~ 917-926 (1977). A. JOHNS and D.M. PATON, Prostaqlandins 13 245-254 (1977). D.M. DEAN and J.W. DOWNIE, Prostaqlandins 16 245-251 (1978).