J. Auton. Phartnacol. ( I 992). 12, 2 15-222
Effect of [D-Phe6] bornbesin (6-1 3) methylester, a bombesin receptor antagonist, towards bornbesin-induced contractions in the guinea-pig and rat isolated urinary bladder Carlo Alberto Maggi, David H. Coy" & Sandro Giuliani Pharmacology Department, A . Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, 1taI.v and *Peptide Research Laboratories, Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana, USA
1 The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guineapig and rat isolated urinary bladder.
2. Bombesin (0. I n M - I 0 p ~ produced ) a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCI ( 8 0 m ~ )The . response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-1 3) OMe (0.3-lOp~). ~ ratios=2.4,9 and 39 Degree of antagonism was concentration-dependent between 0.3 and 3 p (dose in the presence of 0.3, I , 3pM of the antagonist). However, a larger concentration (10pM) of the antagonist was not more effective (dose ratio= 36) than 3 p M .
3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3pM each) or by atropine, indomethacin, chlorpheniramine and desensitization of Pzx purinoceptors by a,B methylene ATP. 4 The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder
produced by the NK-I tachykinin receptor-selective agonist, [Sar9]substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3pM). a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5 The bombesin antagonist ( I - 1 0 , ~ had ~ ) no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation. Likewise, the ~ )not affect the contraction produced by capsaicin ( IOpM) on muscle bombesin antagonist ( 1 0 ~ did strips from the dome of the guinea-pig urinary bladder. 6 Bombesin ( I nM-1 p ~ produced ) concentration-dependent contraction of the rat isolated bladder
which was unaffected by [D-Phe6] bombesin (6-13) OMe ( l O p ~ ) ,which alone produced a contraction of the isolated rat bladder, suggesting partial agonist activity.
7 These findings indicate that [D-Phe*] bombesin (6-13) OMe is a suitable antagonist for establishing the putative role of bombesin in the guinea-pig urinary bladder, although the nature of its action at this level cannot be explained by simple competition of one bombesin receptor only. The failure of [D-Phe6] bombesin (6-1 3) OMe to antagonize the action of bombesin in the rat urinary bladder suggests that different mechanisms (receptors?) mediate the response to this peptide in the urinary bladder of different species. 8 These findings fail to reveal any role for a bombesin-like peptide as excitatory transmitter in the guinea-pig urinary bladder nor indicate a role for bombesin-like peptides as mediators of the efferent function of capsaicin-sensitive primary afferents. Correspondence: Carlo Alberto Maggi.
216
C . A . MAGGI e t a / .
Introduction Bombesin is a tetradecapeptide of amphibian origin isolated from frog skin (Anastasi, Erspamer & Bucci, 197 1 ). Bombesin-like immunoreactivity (bombesin-LI) is present in the mammalian nervous system and in peripheral tissues as well, and several bombesin-like peptides have been identified in mammals (see Erspamer, 1988, for review; Domin, Polak & Bloom, 1988; Minamino, Kangawa & Matsuo. 1988;Spindel & Krane, 1988). Bombesin-Ll has been detected in the rat urinary bladder. equally present in the dome and trigone region (Ghatei. Gu, Allen, Polak & Bloom. 1985). Bombesin itself markedly affects urinary bladder motility in mammals by acting at both central (Porreca, Burks & Sheldon. 1988) and peripheral sites. Bombesin is a powerful spasmogen of the isolated detrusor muscle from several species such as guinea-pig or rats (Falconieri Erspamer el a/.. 1973, 1988: Abdel-Hakim, Rioux & Elhilali, I98 1 : Mizrahi. Dion. D'Orleans-Juste & Regoli. 1985: Regoli ef al., 1988). In recent years much advance has been made in the development of specific bombesin antagonists (Coy et d.. 1988, 1990a,b). Availability of potent and selective bombesin antagonists is important to define the physiological role of peptides of this family. In this study we have assessed the effect of [D-Phe6] bombesin (6-13) methylester (OMe). a potent and selective bombesin antagonist (Coy el al.. 1990a; Varga et a/., 1991) against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. Methods Male albino guinea pigs (250-300 g b.w.) or Wistar rats (300-34Og)were killed by a blow on the back of the head and exsanguination. The whole urinary bladder was removed and placed in oxygenated (96% 0, and 4% CO,) Krebs solution (pH 7.4 at 37°C) of the following composition (mM): NaCl 119; NaHCO, 25,KCI 4.7,MgS04 1.2,CaCI, 2.5, KH,PO, I .2 and glucose 1 1 . Longitudinal strips (about 1 cm long and 3 mm wide) of the detrusor muscle were excised and placed in 5-ml organ baths. The mucosal layer was
gently removed from the guinea-pig bladder before excision of the strips. The strips were connected to isometric force transducers (load IOmN) and mechanical activity was recorded by means of a B a d e 7050 Unirecord. In some experiments electrical field stimulation of intramural nerves was made by means of two wire platinum electrodes placed at the top and the bottom of the organ bath, connected to a Grass S88 stimulator. Trains of pulses were delivered automatically for 5 s every 90 s at a frequency of 20 Hz (pulse duration 0.25 ms, maximal voltage). For experiments involving the effect of bombesin against contractions produced by capsaicin, two parallel strips were excised from the dome of the guinea-pig urinary bladder (cf. Maggi el al., 1988)one of which received the antagonist while the other served as control. All experiments commenced after a 60min equilibration period. At this stage, the response to KCI (80 mM) was recorded and used as an internal standard to normalize the response to bombesin. For experiments involving antagonism of the response to bombesin, two strips were excised from each bladder, one of which served as control while the other received the stated concentration of the antagonist. This was done because the bombesin-induced contraction was very resistant to wash-out, making multiple curves difficult to obtain on the same preparation. Cumulative concentration-response curves to bombesin were constructed by adding increasing concentration of the peptide (either in presence or absence of the antagonist). The next concentration was added when the effect of the preceding one had produced a maximal effect. All responses were expressed as Yo of contraction produced by KCI (80mM). Some experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 3 p each, ~ 15min before) or in the presence of atropine (3p~), indomethacin ( 5 p ~ and ) chlorpheniramine ( 3 p ~and ) after desensitization of PZxpurinoceptors. Atropine, indomethacin and chlorpheniramine were present in the Krebs solution from the beginning of the experiments. Desensitization of Pzx purinoceptors was achieved by repeated addition of the stable
A N T A G O N I S M OF BOMBESIN RESPONSE I N THE U R I N A R Y BLADDER
217
-
10 rnin
ATP analogue, a,/Imethylene ATP ( 1 0 ~ ~ three times at 10-min intervals). Starisrical analysis Each value in the text and Figures is the mean 2 SEM. Statistical analysis was made by means of Student’s I-test for paired or unpaired data or by means of analysis of variance when applicable. Regression analysis was made by the least square method: EC,, and 95% confidence limits were calculated accordingly. Schild plot analysis was performed using a computer program for Apple Ile as described by Tallarida & Murray ( I98 I).
t 1 1 1
Results Guinea-pig urinary bladder Bombesin produced concentration-dependent contraction of the guinea-pig bladder strips (Fig. I). As already noted by AbdelHakim el al. ( I 98 I ) and Mizrahi el al. ( 1 985), the effect of bombesin was difficult to remove by washing and the same occurred for the rat isolated bladder. The maximal response to bombesin averaged 65 f 4 mN or 99 -e 6% of the response to KCI ( n = 12). The effect of [D-Phe6] bombesin (6-13) OMe toward bombesin-induced contraction was studied at concentrations ranging from 0.3 to 1 0 ~[D-Phe6] ~ . bombesin (6-13) OMe
t
t t t t t
t
1 10 loo ~ o o o ~ o o o o BOMBESIN (nM)
z
Drugs Drugs used were: atropine HCI (Serva), chlorpheniramine, captopril. bestatin, thiorphan, indomethacin, a, p methylene ATP (Sigma), bombesin and [Sar9] substance P sulphone (Peninsula). L 668, 169 or cyclo (Gln-DTrp- (NMe)Phe(R)Gly [Anc-2)Leu Met) was from Cambridge Research Biochemicals. [D-Phe6] bombesin (6-1 3) OMe was synthesized as described previously (Wang et al., 1990) and dissolved ( I0 mM) i n bidistilled water, L 668, 169 ( I mM) was dissolved in DMSO. Concentrations of L 668, 169 higher than 3 p were ~ not tested because of precipitation in the bath. The solvent was without effect on responses under study.
t
ai
a1
1 10 100 1000 ioooo
BOMBESIN (nM)
I
ID-Phe61-BN (6-13) OMe
Fig. 1. Typical tracings showing the contractile effect of bombesin on the guinea-pig isolated urinary bladder and its antagonism by [D-Phe6) bombesin (6-13) OMe. I O ~ M ,added 15min before. The effect of bombesin in the absence and presence of the antagonist was studied on paired strips from the same bladder.
had no consistent effect on tone and spontaneous activity of the guinea-pig bladder: either an increase or decrease in tone or no effect was observed at the concentrations used. [D-Phe6] bombesin (6-1 3 ) OMe antagonized the response to bombesin. As shown in Figs I and 2, the response to low bombesin concentrations was abolished by the antagonist and even the lowest concentration tested ( 0 . 3 ~ ~produced ) a significant rightward shift of the curve to the agonist. Although a concentration-dependent shift was observed at 0.3, 1 and 3 p concentration ~ of the antagonist (dose ratios 2.4. 9 and 39. respectively), the largest concentration tested ( I O ~ M dose , ratio 36) was not more effective than 3 p (Fig. 2). Schild plot analysis performed using the dose-ratios obtained at antagonist concentralions of 0.3, 1 and 3 1 1 ~ yielded a slope significantly different from unity ( - 1.43, 95% confidence limits 1.33-1.54). An apparent pA, value of 6.62 (6.57-6.68) was obtained.
218
C. A . MAGGI e r a / [D-Phes] 100
B N (6-13) OMe
L-668,169
-
80. 60.
w
u)
z
g
40.
20.
u)
0.1
1
10
100 1000 10000 0.1 BOMBESIN (nM)
[D-Phes] BN (6-13) OMe
z
1
10
100 1000
L-668,169
100,
LL
80.
8 60. 40.
20.
0-
oi
i
io
.
m
. 1000
a1
1
[Sargl-SUBSTANCE P sulfone
10
100 1000
(nM)
Fig. 2. Concentration-dependent contraction of the guinea-pig isolated urinary bladder by bombesin (upper panels) or the NK- 1 tachykinin receptor-selective agonist [Sar9] substance P sulphone (lower panels) and effect of [D-Phe6]bombesin (6-1 3) OMe and L 668, 169. Left panels: [D-Phe6]bombesin ( 6 - 1 3 ) O M e 0 . 3 , ~ 0 ~ ,, 1 PM A , 3 p ~0, and 10,m M. Right panels: control. 0; 688. 169 3 p ~ 0, . Each value is mean+SEM of at least four experiments. control. 0:
The question was raised as to whether the response t o bombesin might involve an indirect mechanism or that the activity of bombesin and the antagonist might have been influenced by peptidases. Either one of these two mechanisms could have a bearing on the unusual behaviour of the antagonist towards bombesin-induced contractions. Neither the maximal response nor the concentration-response curve to bombesin was modified in the presence of peptidase inhibitors (bestatin, captopril and thiorphan 3 p each) ~ or in bladder strips exposed to atropine ( 3 p ) , chlorpheniramine ( 3 1 1 ~ )and indomethacin ( 5 1 1 ~and ) subjected to PZxreceptor desensitization by repeated administration of the stable ATP analogue, a,/I methylene ATP (Fig. 3). In the combined presence of three peptidase inhibitors (n=4), a 3-PM concentration of the antagonist shifted the bombesin concentration-response curve to the same extent as observed in control strips
(dose ratio= 34). Similar results were obtained in the presence of atropine, chlorpheniramine, indomethacin and a, /I methylene ATP (dose-ratio= 39, n = 4). To check the selectivity of antagonism by [D-Phe6] bombesin (6-1 3) OMe, the effect of this antagonist ( I O p ) was investigated against contractions produced by [Sar9] substance P sulphone, a selective NK-I tachykinin receptor agonist (Drapeau el a/., 1987). Data in Fig. 2 show that the bombesin antagonist had no effect against the response to the NK-I receptor-selective agonist. The concentration-response curve for [SarP]substance P sulphone was shifted to the right in the presence of the NK-1 tachykinin receptor antagonist, L 668, 169 ( 3 p ~ (Fig. ) 2), which had no effect against bombesin-induced contraction (Fig. 2, 3 p ~ n=5). , To assess whether a bombesin-like peptide may be involved in the non-adrenergic noncholinergic response to nerve stimulation,
100
-
Rat urinary bladder
W
2
) concentraBombesin ( I nM-1 p ~ produced tion-dependent contraction of the rat isolated bladder. The maximal response to bombesin averaged 66 ? 8% of that to KCI or 4 9 ? 4 m ~(n=4). [D-Phe6] bombesin (6-13) OMe ( l o p ) alone produced a contraction of the rat isolated bladder (29 +- 7% of the KCl response or 25 +- 5 mN, n =4). In the presence of [D-Phe6] bombesin (6-13) OMe, bombesin produced a maximal response (34 f 6% of
80-
v)
60J
a
405 a
8
20-
z 0-
and of bombesin ( I p ~ averaged ) a maximum (63 f 8% of KCI, 56 f 6 mN) not significantly of the guinea-pig isolated bladder by bombesin in control strips (0)and in the presence of different from the maximal response to bompeptidase inhibitors (bestatin, captopril and besin in the absence of the putative antagonthiorphan, 3pM each, 15min before, A ) or in ist. As shown in Fig. 4, the concentration, the presence of atropine ( ~ P M ) indomethacin response curve to bombesin obtained in the ( S ~ M )and chlorpheniramine ( 3 , u ~ and ) after rat urinary bladder in the presence of [Ddesensitization of PZxpurinoceptors by a, /3 Phe6] bombesin (6-1 3) OMe was not signifimethylene ATP (total dose administered ~ O P M , cantly different from that of bombesin alone 0).Each value is mean -t SEM of at least four when expressed as O/o of their respective experiments. maximal effect ( n= 4). Fig. 3. Concentration-dependent contraction
the effect of [D-Phe6] bombesin (6-13) OMe was investigated against neurogenic contractions produced by electrical stimulation (20Hz. 0.25ms pulse width, maximal voltage, trains of 5 s every 90 s) in the presence of 3 , u ~atropine. This produced regular phasic bladder contractions which were suppressed by tetrodotoxin ( 1 p ) .The bombesin antagonist ( 1-1 O ~ M )had no significant inhibitory effect on the atropine-resistant response (n=4), which averaged 58 +- 4% of the KCI response. Similar negative results were obtained after Pzxreceptor desensitization by a,/3 methylene ATP (n=4). In the presence of a,p methylene ATP (30,m), the response to electrical stimulation averaged 26 ~t_3% of the response to KCL. The possibility that endogenous bombesinlike peptides may be involved in the capsaicin-induced contraction of the dome of the guinea-pig bladder (cf. Maggi et al.. 1988) was also investigated. In control strips, capsaicin ( 1 0 ~ produced ~ ) a rapid phasic contraction averaging 15 6%of the response to KCI. In the presence of the bombesin antagonist ( l o p ) , the response to capsaicin averaged 1 7 f 5% of the KCI response (n = 4).
w
100 -
fn 2
2
80-
fn W
K
-I
a
60-
z
5
s
40-
1
10
100
1000
BOMBESIN (nM)
Fig. 4. Concentration-dependent contraction of the rat isolated urinary bladder by bombesin in the absence (0)or presence (0) of I O p of
the bombesin antagonist, [D-Phe6] bombesin SEM of at least four experiments.
(6-1 3) OMe. Each value is mean
*
*
Discussion The present findings indicate that [D-Phe6]
220
C. A . MAGGI e r a / .
bombesin (6-13) OMe acts as a selective bombesin antagonist i n the guinea-pig isolated urinary bladder. The question has been raised as to whether bombesin may act, in certain tissues, by stimulating NK- I tachykinin receptors (Regoli ef al., 1988). The present data seem to exclude this possibility, at least for the guinea-pig urinary bladder, inasmuch as the response to bombesin was unaffected by L 668, 169, a cyclic peptide which acts as a selective NK-I receptor antagonist (Williams ef al., 1988). Conversely. antagonism by [D-Phe6] bombesin (6-1 3) OMe seems specific because this bombesin analogue failed to affect the response to [Sar9] substance P sulphone, a potent and selective NK- 1 receptor agonist (Drapeau ef al., 1987). The contraction produced by bombesin in the rat or guinea-pig isolated bladder was unaffected by atropine, indomethacin, tetrodotoxin. antihistaminics, adrenoceptor blockers. methysergide, and angiotensin or bradykinin antagonists and is thought therefore to involve direct activation of bombesin receptors on smooth muscle cells (AbdelHakim ct al.. 1981; Mizrahi et al.. 1985). In confirmation of these previous observations we found that the effect of bombesin on the guinea-pig bladder is unchanged by atropine, diphenydramine, indomethacin and by desensitization of P,, purinoceptors, achieved by repeated administrations of the stable .4TP analogue, a, p methylene ATP. The latter seems important in view of the role of ATP as non-cholinergic excitatory transmitter in the guinea-pig urinary bladder. Although direct evidence (i.e. binding or autoradiographic data) for the expression of specific bombesin receptors on smooth muscle cells in the urinary bladder is not available. the evidence discussed above excludes a variety of indirect mechanisms in the action of this peptide in the rat and guinea-pig urinary bladder muscle. The differential antagonism showed by [D-Phe6] bombesin (6-13) OMe in the guinea-pig vs rat bladder smooth muscle could be explained by the presence, in the two preparations. of different types of bombesin receptors. Indeed. the antagonist we have used has been shown to possess high affinity for one class of bombesin receptors only (e.g. those
expressed in rat pancreatic acini, which yield a PA, of 8.89 from Varga ef al.. 1991) while being virtually inactive at others (e.g. those expressed on rat oesophageal smooth muscle, see Jensen & Coy, I99 I ). It should be noted that [D-Phe6] bombesin (6-13) OMe is devoid of any affinity for this second class of bombesin receptors, which recognize neuromedin B with high affinity (Jensen & Coy, 199 I ) and this mechanism cannot explain its agonist activity in the rat isolated urinary bladder, which deserves further investigation. That the bombesin receptor present in vesical smooth muscle from rats and guineapigs may not be identical had already been suggested by Falconieri-Erspamer ef al. ( 1988) on the basis of the different rank order of potency of natural bombesin-like peptides and synthetic agonists, and more recently, by Rouissi et al. (1 99 1 ) on the basis of antagonist affinities. The latter authors proposed that BBS, receptors are present in the guinea-pig urinary bladder while BBS2 receptors mediate the contractile response of the rat urinary bladder (Rouissi ef al., 1991). In the case of the guinea-pig urinary bladder, however, the action of [D-Phe6] bombesin (6-13) OMe cannot be explained through a competitive interaction with a single class of bombesin receptors. I n fact the shifts produced by the antagonist at 3 and 1 0 p were ~ superimposable. Furthermore, the slope of the Schild plot calculated for shifts of the bombesin curve obtained in the presence of 0 . 3 - 3 p of the antagonist was significantly different from unity. The apparent PA, value calculated for the antagonist (6.62) is about two orders of magnitude lower than that measured by Varga et al. (1991) in rat pancreas. This means that high concentrations of the agonist produce bladder contraction through a mechanism which is not susceptible of antagonism by [D-Phe6]bombesin (6- 13) OMe. This behaviour suggests that at least two distinct mechanisms determine the response to bombesin in the guinea-pig bladder and further studies on this topic are warranted. The present findings also demonstrate that [D-Phe6] bombesin (6-1 3) OMe, at a concentration effective in antagonizing exogenously administered bombesin, did not affect the non-adrenergic-non-cholinergic contraction
A N T A G O N I S M O F BOMBESIN RESPONSE IN THE URINARY BLADDER
produced by electrical field stimulation. This provides evidence against a role of endogenous bombesin as excitatory transmitter in the guinea-pig urinary bladder. Bombesinlike peptides are also expressed by certain primary afferent neurons which are capsaicin-sensitive a n d exert a n ‘efferent’ function through peptide release in the periphery (see M a g i & Meli, 1988 for review). The capsaicin-induced contraction of the dome o f the guinea-pig bladder has been shown t o be blocked by a tachykinin antagonist (Maggi el al., 1988) but the possible involvement of bombesin-like peptides could not be ruled out because some tachykinin antagonists of the first generation also act as bombesin antagonists. T h e present findings indicate that a specific bombesin antagonist failed to affect t h e capsaicin-induced contraction of the guinea-pig bladder. Therefore, as shown previously for the rat urinary bladder (Maggi, Patacchini, Santicioli & Meli, 1991), there is no evidence for involvement o f bombesinlike peptides in the ‘efferent’ function of capsaicin-sensitive primary afferents in the guinea-pig urinary bladder. References ABDELHAKIM, A., Rloux, F. & ELHILALI, M. ( 1 98 I). The contractile effect of bombesin on the rat isolated urinary bladder. Eur. J. Pharmacol., 70, 167-1 73. ANASTASI,A., ERSPAMER, V . & BUCCI, M. ( 1 97 1 ). Isolation and structure of bombesin and alytesin two analogous peptides from the skin of the European amphibians, Bombina and Alytes. Experienria. 27, 166- 170. COY. D.H.. HEINZ-ERIAN. P., JIANG,N . Y . , TAYLOR,J., MOREAU,J.P., GARDNER, J.D. & JENSEN,R.T. (1988). Progress in the development of competitive bombesin antagonists. Ann. N. Y. Acad. Sci., 547, 150-157. COY, D.H., TAYLOR,J.E., JIANG, N . Y . , WANG, L.H., HUANG,S.C., QIAN,J.M., MOREAU,J.P. & JENSEN,R.T. (1990a). Developing receptor antagonists of neuropeptides-the bombesin/GRP and substance P systems. In: Neuropeplides and Their Receptors. A. Benzon. Symposium 29, pp. 376-385. Eds T.W. Schwartz, L.M. Hilsted & J.F. Rehfeld. Munksgaard, Copenhagen. COY, D.H.. WANG, L.H., JIANG, N . Y . &
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JENSEN,R.T. ( 1 990b). Short chain bombesin neuropeptides with potent bombesin receptor antagonist activity in rat and guinea-pig pancreatic acinar cells. Eiu. J. Pharmacol., 190, 31-38. DOMIN, N., POLAK,J.M. & BLOOM,S.R. ( I 988). The distribution and biological effects of neuromedins B and U. Ann. N.Y. Acad. Sci.. 547, 391-403. DRAPEAU, G., D’ORLEANS-JUSTE, P., DION. S., RHALEB,N.E., ROUISSI,N. & REGOLI, D. (1987). Selective agonists for substance P and neurokinin receptors. Neuropeprides, 10, 43-54. ERSPAMER, V. (1988). Discovery, isolation and characterization of bombesin-like peptides. Ann. N.Y. Acad. Sci., 547, 3-9. FALCONIERI-ERSPAMER, G., NEGRI,L. & PICCINELLI, D. ( 1 973). The use of preparations of urinary bladder smooth muscle for bioassay of and discrimination between polypeptides. Naunyn-Schmiedeberg’s Arch. PharmaC O ~279, . , 61-74. FALCONIERI-ERSPAMER, G., SEVERINI, C., ERSPAMER, v., MELCHIORRI, P., DELLEFAVE, G. & NAKAJIMA, T. ( 1 988). Parallel bioassay of 27 bornbesin-like peptides on 9 smooth muscle preparations. Structure activity relationships and bombesin receptor subtypes. Regul. Peptides, 21, 1-1 1. GHATEI,M.A., G u , J., ALLEN,J.M., POLAK, J.M. & BLOOM,S.R. (1985). Bombesin-like immunoreactivity in female rat genitourinary tract. Neurosci. Letlers, 54, 13- 19. JENSEN. R.T. & COY,D.H. (1991). Progress in the development of potent bombesin receptor antagonists. Trends Pharmacol. Sci., 12, 13-19. MAGGI,C.A. & MELI,A. (1988). The sensoryefferent function of capsaicin-sensitive sensory neurons. Gen. Pharmacol., 19, 1-43. MAGGI,C.A., PATACCHINI, R., SANTICIOLI, P. & MELI,A. (1991). Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission. Hr. J. Pharmacol., 103, 1545-1541. MAGGI,C.A., SANTICIOLI, P., PATACCHINI, R., GEPPETTI,P., GIULIANI, S., ASTOLFI,M.. BALDI, E., PARLANI, M., THEODORSSON, E. & MELI,A . (1988). Regional differences in the motor response to capsaicin in the guinea-pig urinary bladder: relative role of pre-
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releasing peptide, human neuromedin B and and postjunctional factors related to neuroamphibian ranatensin. Ann. N . Y. Acad. Sci.. pcptide containing sensory nerves. Neurosci547, 10-20. CttCP, 27, 675-688. R.J. & MURRAY,R.B. (1981). MINAMINO, N., KANGAWA.K. & MATSUO.H. TALLARIDA, Manual of Pharrnacologic Calculations with ( I 988). Neuromedin B and neuromedin C; Cotnputer Programs, Springer Verlag, New two mammalian bombesin-like peptides York. identified in porcine spinal cord and brain. :inti. N . Y..&ad. Sei.. 547. 373-390. VARGA, G.. REIDELBERGER, R.D., LIEHR, R.M., USSJAEGER, L.J., COY,D.H. & SOLOMIZRAHI.J.. DION.S., D’ORLEANS-JUSTE, P. M O N , T.E. (1991). Effect of potent bombesin & REGOLI.D. ( 1 985). Activities and antagoantagonist on exocrine pancreatic secretion nism of bombesin on urinary smooth muscles. Eur. J . Pharrnacol.. 1 1 1 , 339-346. in rats. Peptides.12, 493-497. R.J. WANG, L.H., COY, D.H., TAYLOR,J.E.. PORRECA, F.. BURKS. T.F. & SHELDON, JIANG, N.Y., MOREAU, J.P., HUANG,S.C., ( 1988). Central and peripheral visceral effects of bombesin. .-lrin. N . Y. k a d . Sci.. 5477, FRUCHT, H., HAFFAR,B.M. & JENSEN, R.T. ( 1990). Des-Met carboxy-terminally modified 194-203. REGOLI. D.. DION.S., RHALEB.N.E., DRAanalogues of bombesin function as potent PEAU. G.. Rouissi, N . & D’ORLEANS-JUSTE. bombesin receptor antagonists, partial agonists or agonists. J. Biol. Chern., 265, P. ( I 988). Rcccptors for neurokinins, tachyk15695-15703. inins and bombesin: a pharmacological study. .-inn. N . Y. .-lead. SCI..547, 158- 173. WILLIAMS, B.J., CURTIS,N.R.. MCKNIGHT, ROUISSI. N.. RHALEB. N.E., NANTEL. F.. A.T., MAGUIRE, J . , FOSTER,A. & TRIDDION. S.. DRAPEAU.G. & REGOLI, D. GETT, R . (1988). Development of NK-2 ( I99 I ). Characterization of bombesin recepselective antagonists. Regul. Pepiides. 22, 189. tors in peripheral contractile organs. Br. J. f’/~arrnaco/.. 101, 1141-1 147. SPINDEL. E . R . & KRANE.I.M.(1988). Molecu(Received 13 September 1991 lar biology of bombesin-like peptides: comRevised 14 November 1991 parison of cDNAs encoding human gastrin .4ccepted 2 December 1991)
Effect of [D-Phe6] bornbesin (6-1 3) methylester, a bombesin receptor antagonist, towards bornbesin-induced contractions in the guinea-pig and rat isolated urinary bladder Carlo Alberto Maggi, David H. Coy" & Sandro Giuliani Pharmacology Department, A . Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, 1taI.v and *Peptide Research Laboratories, Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana, USA
1 The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guineapig and rat isolated urinary bladder.
2. Bombesin (0. I n M - I 0 p ~ produced ) a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCI ( 8 0 m ~ )The . response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-1 3) OMe (0.3-lOp~). ~ ratios=2.4,9 and 39 Degree of antagonism was concentration-dependent between 0.3 and 3 p (dose in the presence of 0.3, I , 3pM of the antagonist). However, a larger concentration (10pM) of the antagonist was not more effective (dose ratio= 36) than 3 p M .
3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3pM each) or by atropine, indomethacin, chlorpheniramine and desensitization of Pzx purinoceptors by a,B methylene ATP. 4 The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder
produced by the NK-I tachykinin receptor-selective agonist, [Sar9]substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3pM). a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5 The bombesin antagonist ( I - 1 0 , ~ had ~ ) no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation. Likewise, the ~ )not affect the contraction produced by capsaicin ( IOpM) on muscle bombesin antagonist ( 1 0 ~ did strips from the dome of the guinea-pig urinary bladder. 6 Bombesin ( I nM-1 p ~ produced ) concentration-dependent contraction of the rat isolated bladder
which was unaffected by [D-Phe6] bombesin (6-13) OMe ( l O p ~ ) ,which alone produced a contraction of the isolated rat bladder, suggesting partial agonist activity.
7 These findings indicate that [D-Phe*] bombesin (6-13) OMe is a suitable antagonist for establishing the putative role of bombesin in the guinea-pig urinary bladder, although the nature of its action at this level cannot be explained by simple competition of one bombesin receptor only. The failure of [D-Phe6] bombesin (6-1 3) OMe to antagonize the action of bombesin in the rat urinary bladder suggests that different mechanisms (receptors?) mediate the response to this peptide in the urinary bladder of different species. 8 These findings fail to reveal any role for a bombesin-like peptide as excitatory transmitter in the guinea-pig urinary bladder nor indicate a role for bombesin-like peptides as mediators of the efferent function of capsaicin-sensitive primary afferents. Correspondence: Carlo Alberto Maggi.
216
C . A . MAGGI e t a / .
Introduction Bombesin is a tetradecapeptide of amphibian origin isolated from frog skin (Anastasi, Erspamer & Bucci, 197 1 ). Bombesin-like immunoreactivity (bombesin-LI) is present in the mammalian nervous system and in peripheral tissues as well, and several bombesin-like peptides have been identified in mammals (see Erspamer, 1988, for review; Domin, Polak & Bloom, 1988; Minamino, Kangawa & Matsuo. 1988;Spindel & Krane, 1988). Bombesin-Ll has been detected in the rat urinary bladder. equally present in the dome and trigone region (Ghatei. Gu, Allen, Polak & Bloom. 1985). Bombesin itself markedly affects urinary bladder motility in mammals by acting at both central (Porreca, Burks & Sheldon. 1988) and peripheral sites. Bombesin is a powerful spasmogen of the isolated detrusor muscle from several species such as guinea-pig or rats (Falconieri Erspamer el a/.. 1973, 1988: Abdel-Hakim, Rioux & Elhilali, I98 1 : Mizrahi. Dion. D'Orleans-Juste & Regoli. 1985: Regoli ef al., 1988). In recent years much advance has been made in the development of specific bombesin antagonists (Coy et d.. 1988, 1990a,b). Availability of potent and selective bombesin antagonists is important to define the physiological role of peptides of this family. In this study we have assessed the effect of [D-Phe6] bombesin (6-13) methylester (OMe). a potent and selective bombesin antagonist (Coy el al.. 1990a; Varga et a/., 1991) against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. Methods Male albino guinea pigs (250-300 g b.w.) or Wistar rats (300-34Og)were killed by a blow on the back of the head and exsanguination. The whole urinary bladder was removed and placed in oxygenated (96% 0, and 4% CO,) Krebs solution (pH 7.4 at 37°C) of the following composition (mM): NaCl 119; NaHCO, 25,KCI 4.7,MgS04 1.2,CaCI, 2.5, KH,PO, I .2 and glucose 1 1 . Longitudinal strips (about 1 cm long and 3 mm wide) of the detrusor muscle were excised and placed in 5-ml organ baths. The mucosal layer was
gently removed from the guinea-pig bladder before excision of the strips. The strips were connected to isometric force transducers (load IOmN) and mechanical activity was recorded by means of a B a d e 7050 Unirecord. In some experiments electrical field stimulation of intramural nerves was made by means of two wire platinum electrodes placed at the top and the bottom of the organ bath, connected to a Grass S88 stimulator. Trains of pulses were delivered automatically for 5 s every 90 s at a frequency of 20 Hz (pulse duration 0.25 ms, maximal voltage). For experiments involving the effect of bombesin against contractions produced by capsaicin, two parallel strips were excised from the dome of the guinea-pig urinary bladder (cf. Maggi el al., 1988)one of which received the antagonist while the other served as control. All experiments commenced after a 60min equilibration period. At this stage, the response to KCI (80 mM) was recorded and used as an internal standard to normalize the response to bombesin. For experiments involving antagonism of the response to bombesin, two strips were excised from each bladder, one of which served as control while the other received the stated concentration of the antagonist. This was done because the bombesin-induced contraction was very resistant to wash-out, making multiple curves difficult to obtain on the same preparation. Cumulative concentration-response curves to bombesin were constructed by adding increasing concentration of the peptide (either in presence or absence of the antagonist). The next concentration was added when the effect of the preceding one had produced a maximal effect. All responses were expressed as Yo of contraction produced by KCI (80mM). Some experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 3 p each, ~ 15min before) or in the presence of atropine (3p~), indomethacin ( 5 p ~ and ) chlorpheniramine ( 3 p ~and ) after desensitization of PZxpurinoceptors. Atropine, indomethacin and chlorpheniramine were present in the Krebs solution from the beginning of the experiments. Desensitization of Pzx purinoceptors was achieved by repeated addition of the stable
A N T A G O N I S M OF BOMBESIN RESPONSE I N THE U R I N A R Y BLADDER
217
-
10 rnin
ATP analogue, a,/Imethylene ATP ( 1 0 ~ ~ three times at 10-min intervals). Starisrical analysis Each value in the text and Figures is the mean 2 SEM. Statistical analysis was made by means of Student’s I-test for paired or unpaired data or by means of analysis of variance when applicable. Regression analysis was made by the least square method: EC,, and 95% confidence limits were calculated accordingly. Schild plot analysis was performed using a computer program for Apple Ile as described by Tallarida & Murray ( I98 I).
t 1 1 1
Results Guinea-pig urinary bladder Bombesin produced concentration-dependent contraction of the guinea-pig bladder strips (Fig. I). As already noted by AbdelHakim el al. ( I 98 I ) and Mizrahi el al. ( 1 985), the effect of bombesin was difficult to remove by washing and the same occurred for the rat isolated bladder. The maximal response to bombesin averaged 65 f 4 mN or 99 -e 6% of the response to KCI ( n = 12). The effect of [D-Phe6] bombesin (6-13) OMe toward bombesin-induced contraction was studied at concentrations ranging from 0.3 to 1 0 ~[D-Phe6] ~ . bombesin (6-13) OMe
t
t t t t t
t
1 10 loo ~ o o o ~ o o o o BOMBESIN (nM)
z
Drugs Drugs used were: atropine HCI (Serva), chlorpheniramine, captopril. bestatin, thiorphan, indomethacin, a, p methylene ATP (Sigma), bombesin and [Sar9] substance P sulphone (Peninsula). L 668, 169 or cyclo (Gln-DTrp- (NMe)Phe(R)Gly [Anc-2)Leu Met) was from Cambridge Research Biochemicals. [D-Phe6] bombesin (6-1 3) OMe was synthesized as described previously (Wang et al., 1990) and dissolved ( I0 mM) i n bidistilled water, L 668, 169 ( I mM) was dissolved in DMSO. Concentrations of L 668, 169 higher than 3 p were ~ not tested because of precipitation in the bath. The solvent was without effect on responses under study.
t
ai
a1
1 10 100 1000 ioooo
BOMBESIN (nM)
I
ID-Phe61-BN (6-13) OMe
Fig. 1. Typical tracings showing the contractile effect of bombesin on the guinea-pig isolated urinary bladder and its antagonism by [D-Phe6) bombesin (6-13) OMe. I O ~ M ,added 15min before. The effect of bombesin in the absence and presence of the antagonist was studied on paired strips from the same bladder.
had no consistent effect on tone and spontaneous activity of the guinea-pig bladder: either an increase or decrease in tone or no effect was observed at the concentrations used. [D-Phe6] bombesin (6-1 3 ) OMe antagonized the response to bombesin. As shown in Figs I and 2, the response to low bombesin concentrations was abolished by the antagonist and even the lowest concentration tested ( 0 . 3 ~ ~produced ) a significant rightward shift of the curve to the agonist. Although a concentration-dependent shift was observed at 0.3, 1 and 3 p concentration ~ of the antagonist (dose ratios 2.4. 9 and 39. respectively), the largest concentration tested ( I O ~ M dose , ratio 36) was not more effective than 3 p (Fig. 2). Schild plot analysis performed using the dose-ratios obtained at antagonist concentralions of 0.3, 1 and 3 1 1 ~ yielded a slope significantly different from unity ( - 1.43, 95% confidence limits 1.33-1.54). An apparent pA, value of 6.62 (6.57-6.68) was obtained.
218
C. A . MAGGI e r a / [D-Phes] 100
B N (6-13) OMe
L-668,169
-
80. 60.
w
u)
z
g
40.
20.
u)
0.1
1
10
100 1000 10000 0.1 BOMBESIN (nM)
[D-Phes] BN (6-13) OMe
z
1
10
100 1000
L-668,169
100,
LL
80.
8 60. 40.
20.
0-
oi
i
io
.
m
. 1000
a1
1
[Sargl-SUBSTANCE P sulfone
10
100 1000
(nM)
Fig. 2. Concentration-dependent contraction of the guinea-pig isolated urinary bladder by bombesin (upper panels) or the NK- 1 tachykinin receptor-selective agonist [Sar9] substance P sulphone (lower panels) and effect of [D-Phe6]bombesin (6-1 3) OMe and L 668, 169. Left panels: [D-Phe6]bombesin ( 6 - 1 3 ) O M e 0 . 3 , ~ 0 ~ ,, 1 PM A , 3 p ~0, and 10,m M. Right panels: control. 0; 688. 169 3 p ~ 0, . Each value is mean+SEM of at least four experiments. control. 0:
The question was raised as to whether the response t o bombesin might involve an indirect mechanism or that the activity of bombesin and the antagonist might have been influenced by peptidases. Either one of these two mechanisms could have a bearing on the unusual behaviour of the antagonist towards bombesin-induced contractions. Neither the maximal response nor the concentration-response curve to bombesin was modified in the presence of peptidase inhibitors (bestatin, captopril and thiorphan 3 p each) ~ or in bladder strips exposed to atropine ( 3 p ) , chlorpheniramine ( 3 1 1 ~ )and indomethacin ( 5 1 1 ~and ) subjected to PZxreceptor desensitization by repeated administration of the stable ATP analogue, a,/I methylene ATP (Fig. 3). In the combined presence of three peptidase inhibitors (n=4), a 3-PM concentration of the antagonist shifted the bombesin concentration-response curve to the same extent as observed in control strips
(dose ratio= 34). Similar results were obtained in the presence of atropine, chlorpheniramine, indomethacin and a, /I methylene ATP (dose-ratio= 39, n = 4). To check the selectivity of antagonism by [D-Phe6] bombesin (6-1 3) OMe, the effect of this antagonist ( I O p ) was investigated against contractions produced by [Sar9] substance P sulphone, a selective NK-I tachykinin receptor agonist (Drapeau el a/., 1987). Data in Fig. 2 show that the bombesin antagonist had no effect against the response to the NK-I receptor-selective agonist. The concentration-response curve for [SarP]substance P sulphone was shifted to the right in the presence of the NK-1 tachykinin receptor antagonist, L 668, 169 ( 3 p ~ (Fig. ) 2), which had no effect against bombesin-induced contraction (Fig. 2, 3 p ~ n=5). , To assess whether a bombesin-like peptide may be involved in the non-adrenergic noncholinergic response to nerve stimulation,
100
-
Rat urinary bladder
W
2
) concentraBombesin ( I nM-1 p ~ produced tion-dependent contraction of the rat isolated bladder. The maximal response to bombesin averaged 66 ? 8% of that to KCI or 4 9 ? 4 m ~(n=4). [D-Phe6] bombesin (6-13) OMe ( l o p ) alone produced a contraction of the rat isolated bladder (29 +- 7% of the KCl response or 25 +- 5 mN, n =4). In the presence of [D-Phe6] bombesin (6-13) OMe, bombesin produced a maximal response (34 f 6% of
80-
v)
60J
a
405 a
8
20-
z 0-
and of bombesin ( I p ~ averaged ) a maximum (63 f 8% of KCI, 56 f 6 mN) not significantly of the guinea-pig isolated bladder by bombesin in control strips (0)and in the presence of different from the maximal response to bompeptidase inhibitors (bestatin, captopril and besin in the absence of the putative antagonthiorphan, 3pM each, 15min before, A ) or in ist. As shown in Fig. 4, the concentration, the presence of atropine ( ~ P M ) indomethacin response curve to bombesin obtained in the ( S ~ M )and chlorpheniramine ( 3 , u ~ and ) after rat urinary bladder in the presence of [Ddesensitization of PZxpurinoceptors by a, /3 Phe6] bombesin (6-1 3) OMe was not signifimethylene ATP (total dose administered ~ O P M , cantly different from that of bombesin alone 0).Each value is mean -t SEM of at least four when expressed as O/o of their respective experiments. maximal effect ( n= 4). Fig. 3. Concentration-dependent contraction
the effect of [D-Phe6] bombesin (6-13) OMe was investigated against neurogenic contractions produced by electrical stimulation (20Hz. 0.25ms pulse width, maximal voltage, trains of 5 s every 90 s) in the presence of 3 , u ~atropine. This produced regular phasic bladder contractions which were suppressed by tetrodotoxin ( 1 p ) .The bombesin antagonist ( 1-1 O ~ M )had no significant inhibitory effect on the atropine-resistant response (n=4), which averaged 58 +- 4% of the KCI response. Similar negative results were obtained after Pzxreceptor desensitization by a,/3 methylene ATP (n=4). In the presence of a,p methylene ATP (30,m), the response to electrical stimulation averaged 26 ~t_3% of the response to KCL. The possibility that endogenous bombesinlike peptides may be involved in the capsaicin-induced contraction of the dome of the guinea-pig bladder (cf. Maggi et al.. 1988) was also investigated. In control strips, capsaicin ( 1 0 ~ produced ~ ) a rapid phasic contraction averaging 15 6%of the response to KCI. In the presence of the bombesin antagonist ( l o p ) , the response to capsaicin averaged 1 7 f 5% of the KCI response (n = 4).
w
100 -
fn 2
2
80-
fn W
K
-I
a
60-
z
5
s
40-
1
10
100
1000
BOMBESIN (nM)
Fig. 4. Concentration-dependent contraction of the rat isolated urinary bladder by bombesin in the absence (0)or presence (0) of I O p of
the bombesin antagonist, [D-Phe6] bombesin SEM of at least four experiments.
(6-1 3) OMe. Each value is mean
*
*
Discussion The present findings indicate that [D-Phe6]
220
C. A . MAGGI e r a / .
bombesin (6-13) OMe acts as a selective bombesin antagonist i n the guinea-pig isolated urinary bladder. The question has been raised as to whether bombesin may act, in certain tissues, by stimulating NK- I tachykinin receptors (Regoli ef al., 1988). The present data seem to exclude this possibility, at least for the guinea-pig urinary bladder, inasmuch as the response to bombesin was unaffected by L 668, 169, a cyclic peptide which acts as a selective NK-I receptor antagonist (Williams ef al., 1988). Conversely. antagonism by [D-Phe6] bombesin (6-1 3) OMe seems specific because this bombesin analogue failed to affect the response to [Sar9] substance P sulphone, a potent and selective NK- 1 receptor agonist (Drapeau ef al., 1987). The contraction produced by bombesin in the rat or guinea-pig isolated bladder was unaffected by atropine, indomethacin, tetrodotoxin. antihistaminics, adrenoceptor blockers. methysergide, and angiotensin or bradykinin antagonists and is thought therefore to involve direct activation of bombesin receptors on smooth muscle cells (AbdelHakim ct al.. 1981; Mizrahi et al.. 1985). In confirmation of these previous observations we found that the effect of bombesin on the guinea-pig bladder is unchanged by atropine, diphenydramine, indomethacin and by desensitization of P,, purinoceptors, achieved by repeated administrations of the stable .4TP analogue, a, p methylene ATP. The latter seems important in view of the role of ATP as non-cholinergic excitatory transmitter in the guinea-pig urinary bladder. Although direct evidence (i.e. binding or autoradiographic data) for the expression of specific bombesin receptors on smooth muscle cells in the urinary bladder is not available. the evidence discussed above excludes a variety of indirect mechanisms in the action of this peptide in the rat and guinea-pig urinary bladder muscle. The differential antagonism showed by [D-Phe6] bombesin (6-13) OMe in the guinea-pig vs rat bladder smooth muscle could be explained by the presence, in the two preparations. of different types of bombesin receptors. Indeed. the antagonist we have used has been shown to possess high affinity for one class of bombesin receptors only (e.g. those
expressed in rat pancreatic acini, which yield a PA, of 8.89 from Varga ef al.. 1991) while being virtually inactive at others (e.g. those expressed on rat oesophageal smooth muscle, see Jensen & Coy, I99 I ). It should be noted that [D-Phe6] bombesin (6-13) OMe is devoid of any affinity for this second class of bombesin receptors, which recognize neuromedin B with high affinity (Jensen & Coy, 199 I ) and this mechanism cannot explain its agonist activity in the rat isolated urinary bladder, which deserves further investigation. That the bombesin receptor present in vesical smooth muscle from rats and guineapigs may not be identical had already been suggested by Falconieri-Erspamer ef al. ( 1988) on the basis of the different rank order of potency of natural bombesin-like peptides and synthetic agonists, and more recently, by Rouissi et al. (1 99 1 ) on the basis of antagonist affinities. The latter authors proposed that BBS, receptors are present in the guinea-pig urinary bladder while BBS2 receptors mediate the contractile response of the rat urinary bladder (Rouissi ef al., 1991). In the case of the guinea-pig urinary bladder, however, the action of [D-Phe6] bombesin (6-13) OMe cannot be explained through a competitive interaction with a single class of bombesin receptors. I n fact the shifts produced by the antagonist at 3 and 1 0 p were ~ superimposable. Furthermore, the slope of the Schild plot calculated for shifts of the bombesin curve obtained in the presence of 0 . 3 - 3 p of the antagonist was significantly different from unity. The apparent PA, value calculated for the antagonist (6.62) is about two orders of magnitude lower than that measured by Varga et al. (1991) in rat pancreas. This means that high concentrations of the agonist produce bladder contraction through a mechanism which is not susceptible of antagonism by [D-Phe6]bombesin (6- 13) OMe. This behaviour suggests that at least two distinct mechanisms determine the response to bombesin in the guinea-pig bladder and further studies on this topic are warranted. The present findings also demonstrate that [D-Phe6] bombesin (6-1 3) OMe, at a concentration effective in antagonizing exogenously administered bombesin, did not affect the non-adrenergic-non-cholinergic contraction
A N T A G O N I S M O F BOMBESIN RESPONSE IN THE URINARY BLADDER
produced by electrical field stimulation. This provides evidence against a role of endogenous bombesin as excitatory transmitter in the guinea-pig urinary bladder. Bombesinlike peptides are also expressed by certain primary afferent neurons which are capsaicin-sensitive a n d exert a n ‘efferent’ function through peptide release in the periphery (see M a g i & Meli, 1988 for review). The capsaicin-induced contraction of the dome o f the guinea-pig bladder has been shown t o be blocked by a tachykinin antagonist (Maggi el al., 1988) but the possible involvement of bombesin-like peptides could not be ruled out because some tachykinin antagonists of the first generation also act as bombesin antagonists. T h e present findings indicate that a specific bombesin antagonist failed to affect t h e capsaicin-induced contraction of the guinea-pig bladder. Therefore, as shown previously for the rat urinary bladder (Maggi, Patacchini, Santicioli & Meli, 1991), there is no evidence for involvement o f bombesinlike peptides in the ‘efferent’ function of capsaicin-sensitive primary afferents in the guinea-pig urinary bladder. References ABDELHAKIM, A., Rloux, F. & ELHILALI, M. ( 1 98 I). The contractile effect of bombesin on the rat isolated urinary bladder. Eur. J. Pharmacol., 70, 167-1 73. ANASTASI,A., ERSPAMER, V . & BUCCI, M. ( 1 97 1 ). Isolation and structure of bombesin and alytesin two analogous peptides from the skin of the European amphibians, Bombina and Alytes. Experienria. 27, 166- 170. COY. D.H.. HEINZ-ERIAN. P., JIANG,N . Y . , TAYLOR,J., MOREAU,J.P., GARDNER, J.D. & JENSEN,R.T. (1988). Progress in the development of competitive bombesin antagonists. Ann. N. Y. Acad. Sci., 547, 150-157. COY, D.H., TAYLOR,J.E., JIANG, N . Y . , WANG, L.H., HUANG,S.C., QIAN,J.M., MOREAU,J.P. & JENSEN,R.T. (1990a). Developing receptor antagonists of neuropeptides-the bombesin/GRP and substance P systems. In: Neuropeplides and Their Receptors. A. Benzon. Symposium 29, pp. 376-385. Eds T.W. Schwartz, L.M. Hilsted & J.F. Rehfeld. Munksgaard, Copenhagen. COY, D.H.. WANG, L.H., JIANG, N . Y . &
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JENSEN,R.T. ( 1 990b). Short chain bombesin neuropeptides with potent bombesin receptor antagonist activity in rat and guinea-pig pancreatic acinar cells. Eiu. J. Pharmacol., 190, 31-38. DOMIN, N., POLAK,J.M. & BLOOM,S.R. ( I 988). The distribution and biological effects of neuromedins B and U. Ann. N.Y. Acad. Sci.. 547, 391-403. DRAPEAU, G., D’ORLEANS-JUSTE, P., DION. S., RHALEB,N.E., ROUISSI,N. & REGOLI, D. (1987). Selective agonists for substance P and neurokinin receptors. Neuropeprides, 10, 43-54. ERSPAMER, V. (1988). Discovery, isolation and characterization of bombesin-like peptides. Ann. N.Y. Acad. Sci., 547, 3-9. FALCONIERI-ERSPAMER, G., NEGRI,L. & PICCINELLI, D. ( 1 973). The use of preparations of urinary bladder smooth muscle for bioassay of and discrimination between polypeptides. Naunyn-Schmiedeberg’s Arch. PharmaC O ~279, . , 61-74. FALCONIERI-ERSPAMER, G., SEVERINI, C., ERSPAMER, v., MELCHIORRI, P., DELLEFAVE, G. & NAKAJIMA, T. ( 1 988). Parallel bioassay of 27 bornbesin-like peptides on 9 smooth muscle preparations. Structure activity relationships and bombesin receptor subtypes. Regul. Peptides, 21, 1-1 1. GHATEI,M.A., G u , J., ALLEN,J.M., POLAK, J.M. & BLOOM,S.R. (1985). Bombesin-like immunoreactivity in female rat genitourinary tract. Neurosci. Letlers, 54, 13- 19. JENSEN. R.T. & COY,D.H. (1991). Progress in the development of potent bombesin receptor antagonists. Trends Pharmacol. Sci., 12, 13-19. MAGGI,C.A. & MELI,A. (1988). The sensoryefferent function of capsaicin-sensitive sensory neurons. Gen. Pharmacol., 19, 1-43. MAGGI,C.A., PATACCHINI, R., SANTICIOLI, P. & MELI,A. (1991). Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission. Hr. J. Pharmacol., 103, 1545-1541. MAGGI,C.A., SANTICIOLI, P., PATACCHINI, R., GEPPETTI,P., GIULIANI, S., ASTOLFI,M.. BALDI, E., PARLANI, M., THEODORSSON, E. & MELI,A . (1988). Regional differences in the motor response to capsaicin in the guinea-pig urinary bladder: relative role of pre-
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releasing peptide, human neuromedin B and and postjunctional factors related to neuroamphibian ranatensin. Ann. N . Y. Acad. Sci.. pcptide containing sensory nerves. Neurosci547, 10-20. CttCP, 27, 675-688. R.J. & MURRAY,R.B. (1981). MINAMINO, N., KANGAWA.K. & MATSUO.H. TALLARIDA, Manual of Pharrnacologic Calculations with ( I 988). Neuromedin B and neuromedin C; Cotnputer Programs, Springer Verlag, New two mammalian bombesin-like peptides York. identified in porcine spinal cord and brain. :inti. N . Y..&ad. Sei.. 547. 373-390. VARGA, G.. REIDELBERGER, R.D., LIEHR, R.M., USSJAEGER, L.J., COY,D.H. & SOLOMIZRAHI.J.. DION.S., D’ORLEANS-JUSTE, P. M O N , T.E. (1991). Effect of potent bombesin & REGOLI.D. ( 1 985). Activities and antagoantagonist on exocrine pancreatic secretion nism of bombesin on urinary smooth muscles. Eur. J . Pharrnacol.. 1 1 1 , 339-346. in rats. Peptides.12, 493-497. R.J. WANG, L.H., COY, D.H., TAYLOR,J.E.. PORRECA, F.. BURKS. T.F. & SHELDON, JIANG, N.Y., MOREAU, J.P., HUANG,S.C., ( 1988). Central and peripheral visceral effects of bombesin. .-lrin. N . Y. k a d . Sci.. 5477, FRUCHT, H., HAFFAR,B.M. & JENSEN, R.T. ( 1990). Des-Met carboxy-terminally modified 194-203. REGOLI. D.. DION.S., RHALEB.N.E., DRAanalogues of bombesin function as potent PEAU. G.. Rouissi, N . & D’ORLEANS-JUSTE. bombesin receptor antagonists, partial agonists or agonists. J. Biol. Chern., 265, P. ( I 988). Rcccptors for neurokinins, tachyk15695-15703. inins and bombesin: a pharmacological study. .-inn. N . Y. .-lead. SCI..547, 158- 173. WILLIAMS, B.J., CURTIS,N.R.. MCKNIGHT, ROUISSI. N.. RHALEB. N.E., NANTEL. F.. A.T., MAGUIRE, J . , FOSTER,A. & TRIDDION. S.. DRAPEAU.G. & REGOLI, D. GETT, R . (1988). Development of NK-2 ( I99 I ). Characterization of bombesin recepselective antagonists. Regul. Pepiides. 22, 189. tors in peripheral contractile organs. Br. J. f’/~arrnaco/.. 101, 1141-1 147. SPINDEL. E . R . & KRANE.I.M.(1988). Molecu(Received 13 September 1991 lar biology of bombesin-like peptides: comRevised 14 November 1991 parison of cDNAs encoding human gastrin .4ccepted 2 December 1991)
