Antagonism of the effects of calcitonin gene-related peptide and of capsaicin on the guinea-pig isolated ileum by human α-calcitonin gene-related peptide(8–37)

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Neuroscience Letters, 129 (1991) 156-159 © 1991 ElsevierScientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100432H

NSL 07945

Antagonism of the effects of calcitonin gene-related peptide and of capsaicin on the guinea-pig isolated ileum by human 0c-calcitonin gene-related peptide(8-37) L. B a r t h 6 I, G . K6cz~m 1, P. H o l z e r 2, C.A. M a g g i 3 a n d J. Szolcs~nyi 1 1Departments of Pharmacology, The Medical School of P~cs, Pbcs (Hungary), 2The University of Graz, Graz (Austria) and 3A. Menarini Pharmaceuticals, Florence (Italy)

(Received 3 April 1991; Revisedversion received 7 May 1991; Accepted 13 May 1991) Key words. Enteric; Capsaicin; Calcitonin gene-relatedpeptide; CGRP antagonist

The possible mediating role of calcitonin gene-related peptide (CGRP) in the effects of capsaicin in the guinea-pig ileum has been investigated by means of the CGRP antagonist hCGRP(8-37). Submaximallongitudinal muscle relaxation of the histamine-precontracted ileum evoked by rat CGRP (3 nM) or capsaicin (1/tM) was reversed by hCGRP(8-37) (1.5/~M), while that due to adrenaline or neurotensin was not affected. Inhibition of spontaneous circular muscle movements by capsaicin (100 nM) was also reversed by hCGRP(8-37). The CGRP antagonist had no effect on electrical stimulation-evoked ileal contractions or on the longitudinal or circular muscle tone. It is concluded that (a) hCGRP(8-37) is a specific CGRP antagonist in the ileum, apparently devoid of intrinsic activity or any effect not related to CGRP; (b) the inhibitory actions of capsaicin on the longitudinal and circular muscles of the ileum are mediated, at least in part, by CGRP.

The neuropeptide calcitonin gene-related peptide (CGRP) is present in the guinea-pig ileum, where it is associated with extrinsic (probably spinal afferent) neurons [5]. C G R P has been reported to relax the longitudinal [2, 9] and circular muscles of the guinea-pig ileum [1]. The sensory stimulant capsaicin, which is known to release C G R P and tachykinins from afferents in various preparations, causes both excitatory and inhibitory effects in the guinea-pig ileum; by using C G R P desensitization [3, 11] or immunoblockade [9] evidence has been presented for the involvement of C G R P in the inhibitory component of the action of capsaicin or antidromic activation of capsaicin-sensitive neurons on the longitudinal muscle. However, definitive evidence for a participation of C G R P in capsaicin-induced local m o t o r responses of the ileum is not yet available. Moreover, C G R P desensitization failed to block the inhibitory effect of capsaicin on the circular muscle [12]. Recently, the C-terminal fragment of human ~C G R P , hCGRP(8-37), has been demonstrated to antagonize the effect of C G R P in various tissues [4, 6, 8]. This substance was also able to inhibit capsaicin-sensitive responses in the guinea-pig heart and rat vas deferens [8]. The present study aimed at investigating Correspondence: L. Barth6, Department of Pharmacology, The Medical School of Prcs, Prcs, Hungary.

whether hCGRP(8-37) could act as a specific antagonist of C G R P in the guinea-pig ileum, as well as at assessing the possible involvement of endogenous C G R P in the action of capsaicin in this tissue. Male guinea-pigs of 330-400 g were killed by a blow to the head and bled. Approximately 2-cm segments of the ileum were set up in Tyrode solution of 37°C, gassed with 02, for isotonic recording of longitudinal movements. The load on the tissue was 5 m N (0.5 g). At most 4 preparations were used from each animal. In most experiments, the relaxant effect of rat (r)CGRP or of capsaicin was studied on the atropine-treated, histamineprecontracted ileum, as described earlier [2, 3]. Submaximal longitudinal contraction was elicited by histamine (300 nM); r C G R P , capsaicin, adrenaline or neurotensin were added to the bath 10-15 min later, when a stable plateau of contraction had been reached. Capsaicin was applied only once to each preparation. Atropine (1 ~tM) was present throughout, since both capsaicin and r C G R P have a cholinergic excitatory component in their actions [2, 3]. In a separate set of experiments ileal segments were arranged in a horizontal position for recording circular muscle contractions [1, 7]. Only those segments showing spontaneous circular muscle contractions were used. In this series we utilized Krebs solution because more preparations exhibited spontaneous movements in this

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medium than in Tyrode solution. For avoiding the excitatory effects of rCGRP or capsaicin (and thus unmasking inhibition) tetrodotoxin (1 #M) was added to the bath, which did not abolish spontaneous activity. rCGRP (3 nM) caused marked, but not maximal, longitudinal relaxation of the histamine-precontracted ileum; this action was reversed by 1.5 #M hCGRP(8-37) within 2 min (n---5; Fig. 1). After the effect of hCGRP(8-37) had developed, 30 and 100 nM of CGRP were administered in a cumulative manner (n =4; not shown in Fig. 1). Interpolation revealed that approximately 60 nM of CGRP was necessary to restore the relaxation originally produced by 3 nM of the drug. Approximately half-maximal relaxation elicited by adrenaline (250 nM) was not changed by 1.5 pM hCGRP(8-37) (n = 5; Fig. 1), but was partly reversed by propranolol (1 #M, n = 3). The effect of neurotensin (50 nM), which consisted of initial relaxation (approx. 30% of the maxi-

mum) followed by contraction, was not modified by 1.5 #M hCGRP(8-37) (n = 3). In the absence of histamine and atropine, cholinergic twitch responses of the ileum evoked by single pulses of electrical field stimulation (supramaximal voltage, 0.1 ms pulse width, 0.05 Hz frequency) were not influenced by hCGRP(8-37) (1.5 #M, n=5; 3 #M, n=2); in the presence of atropine (1 #M) hCGRP(8-37) (1.5 #M) did not inhibit contractions due to electrical stimulation (10 Hz for 10 s; n=4). As reported earlier [3] capsaicin (1 #M) caused, after a weak initial contraction, a moderate relaxation of the histamine-precontracted ileum. This latter action was also partly reversed or prevented by 1.5 #M hCGRP(837) (Fig. 1). In the experiments where hCGRP(8-37) was added to the bath before rCGRP, the antagonist by itself caused no change in smooth muscle motility or tone. In 5 experiments, higher concentrations of hCGRP(8-37)

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Fig. 1. Effects of hCGRP(8-37) (1.5 #M) on relaxations induced by rCGRP (3 nM), adrenaline (250 nM) or capsaicin (1 pM) in the guinea-pig ileum, precontracted with histamine (300 nM), in the presence of atropine (1 pM). Relaxation is expressed in~ of the maximal relaxation to the baseline, while initial contraction due to capsaicin (Contr) in ~ of the maximal spasm evoked by histamine (5 pM). hCGRP(8-37) was added to the bath 3 min after the relaxants ( O - - O ) , when their actions had practically developed, as indicated by the control curves ( A - - A ) . In D, hCGRP(8-37) was administered 5 min before capsaicin ( D - - r q ) . Mean + S.E.M. are given. Number of experiments, n = 5 in all groups receiving hCGRP(8-37); in control groups n = 6 in A and C, n = 8 in B. Asterisks indicate statistically significant differences ( P < 0.05 or less); min 3 vs win 5 (A and C, paired t-test); control group vs pretreated group (D; unpaired t-test).

158 were used (3 pM, n = 3 and 6 pM, n = 2); full reversal of the capsaicin-induced relaxation was never observed. On the other hand, capsaicin was ineffective on ileal segments pretreated with resiniferatoxin (100 nM for 15 min, one hour before capsaicin administration; n = 5). Spontaneous circular muscle contractions of the ileum incubated with tetrodotoxin (1 /tM) were partially or fully inhibited by capsaicin (100 nM or 1 pM, see ref. 1). Duration of the inhibition was longer than 10 min (n--6 for each concentration), hCGRP(8-37) (1.5/zM, added to the bath 5 min after capsaicin) was not able to reverse the action of 1/~M ofcapsaicin, which is in line with data of the literature obtained with C G R P desensitization [12]. The effect of 100 nM capsaicin was, however, reversed in 2 out of 4 preparations by 1.5 p M of hCGRP(8-37). A higher concentration of the C G R P antagonist (3/tM) reversed the inhibition caused by 100 nM of capsaicin in 4 out of 5 preparations tested (Fig. 2). hCGRP(8-37) by itself did not modify the spontaneous activity of the circular muscle (3/~M; n--3). These data show that hCGRP(8-37) is a specific antagonist of C G R P in the guinea-pig ileum and is a suitable tool for studying the involvement of this peptide in neu-

rally mediated responses. Possible drawbacks of using C G R P desensitization or immunoneutralization for this purpose, e.g. the long time of incubation, can be avoided by using hCGRP(8-37), which has a quick onset of action. Furthermore, hCGRP(8-37) seems to be devoid of intrinsic activity or smooth muscle stimulant or depressant, as well as of anti-histamine effects. Lack of action of hCGRP(8-37) on the stimulated ileum also implies that the peptide does not modify cholinergic neurotransmission at a prejunctional site, by inhibiting neuronal conduction or transmitter release. The same holds true for tachykinin-mediated non-cholinergic contractions due to field stimulation. Low concentrations of capsaicin have been reported to relax the atropinized ileum probably by acting on extrinsic neurons [3, 9], while higher concentrations have non-specific actions as well [3]. The same holds true for capsaicin-induced inhibition of spontaneous or evoked circular muscle contractions [1, 12], where non-specific effects appear above 1/zM capsaicin [1]. Afferent neuronal mediation of capsaicin-induced relaxation was confirmed by its prevention by resiniferatoxin, a highly potent blocking agent of capsaicin-sensitive neurons (see

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Fig. 2. Inhibitory action of capsaicin (100 nM) and its reversalby 3 pM of hCGRP(8-37) on spontaneous circular musclecontractions, in the presence of tetrodotoxin (1 pM). Top: tracing of an original experiment. Calibration (top right), 1 min. Horizontal arrow (top right) indicates the possible maximal amplitude of the circular spasm (total obstruction of the lumen, evoked by 10 #M carbachol at the end of the experiment). Note that, once added to the bath, capsaicin and hCGRP(8--37) were present throughout the experiment. Bottom: quantitative data obtained from 7 preparations treated with capsaicin only (left panel) or 5 segments receivinghCGRP(8-37), 5 min after capsaicin administration right panel). Motility was expressed in circular muscle units (CU) for each minute, 1 CU being equal to the amplitude of the possible maximal spasm from the given baseline (only those contractions exceeding0.1 CU were evaluated). Mean + S.E.M. Two-minute periods of steady state (a, b and c were compared statistically (Quade test for several related samples). "Significantlydifferent from a; "Zsignificantlydifferent from both a and b (P < 0.05 or less).

159 a m o n g o t h e r s ref. 10). R e v e r s a l o r p r e v e n t i o n o f the inhib i t o r y a c t i o n s o f c a p s a i c i n in the ileum b y h C G R P ( 8 - 3 7 ) clearly indicates t h a t they are m e d i a t e d , at least in p a r t , b y e n d o g e n o u s C G R P - I i k e m a t e r i a l . O n the o t h e r h a n d , C G R P seems n o t to be involved in electrical s t i m u l a t i o n e v o k e d ileum c o n t r a c t i o n s o r in the r e g u l a t i o n o f s p o n t a n e o u s s m o o t h muscle m o v e m e n t s . This s t u d y was s u p p o r t e d b y the H u n g a r i a n R e s e a r c h G r a n t s O T K A 84, 287, 307 a n d E T T 287, T 562, T 563, as well as b y the A u s t r i a n Scientific R e s e a r c h F u n d s (no. 7845). T h a n k s are d u e to Dr. E l v a r T h e o d o r s s o n ( K a r o linska H o s p i t a l , S t o c k h o l m , Sweden) a n d to Dr. S. G i u liani (A. M e n a r i n i P h a r m a c e u t i c a l s , F l o r e n c e , Italy) for their v a l u a b l e help in statistical processing o f d a t a a n d in p r e p a r i n g the m a n u s c r i p t .

1 Barth6, L., Calcitonin gene-related peptide and capsaicin inhibit the circular muscle of the guinea-pig ileum, Reg. Peptides, in press. 2 Barth6, L., Lembeck, F. and Holzer, P., Calcitonin gene-related peptide is a potent relaxant of intestinal muscle, Eur. J. Pharmacol., 135 (1987) 449-451. 3 Barth6, L., Peth6, G., Antal, A., Holzer, P. and Szolcs~nyi, J., Two types of relaxation due to capsaicin in the guinea-pig isolated ileum, Neurosci. Lett., 81 (1987) 146-150. 4 Chiba, T., Yamaguchi, A., Yamatani, T., Nakamura, A., Morishita, T., Inui, T., Fukase, M., Noda, T. and Fujita, T., Calcitonin gene-related peptide receptor antagonist human CGRP (8-37), Am. J. Physiol., 256 (1989) E331-E334.

5 Costa, M., Furness, J.B. and Llewellyn-Smith, I., Histochemistry of the enteric nervous system. In L.R. Johnson, J. Christensen, M.J. Jackson, E.D. Jacobson, J.H. Walsh (Eds.), Physiology of the Gastrointestinal Tract, Raven, New York, 1987, pp. 1--40. 6 Dannis, T., Fournier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F.B., St.-Pierre, S. and Quirion, R., hCGRPs_37, a calcitonin generelated peptide antagonist revealing calcitonin gene-receptor heterogeneity in brain and periphery, J. Pharmac. Exp. Ther., 254 (1990) 123-128. 7 Holzer, P., Lembeck, F. and Donnerer, J., Caerulein, substance P, serotonin and cholinomimetics induce rhythmic contractions of the intestinal circular muscle, Naunyn-Schmiedeberg's Arch. Pharmacol., 312 (1980) 131-137. 8 Maggi, C.A., Chiba, T. and Giuliani, S., Human ct-calcitonin generelated peptide-(8-37) as an antagonist of exogenous and endogenous calcitonin gene-related peptide, Eur. J. Pharmacol., 192 (1991) 85-88. 9 Maggi, C.A., Patacchini, R., Santicioli, P., Theodorsson, E. and Meli, A., Several neuropeptides determine the visceromotor response to capsaicin in the guinea-pig isolated ileal longitudinal muscle, Eur. J. Pharmacol., 148 (1988) 43-49. I0 Szolcs/tnyi, J., Sz/tllfisi, A., Sz~illfisi,Z., Jo6, F. and Blumberg, P.M., Resiniferatoxin: an ultrapotent selective modulator of capsaicin-sensitive primary afferent neurons, J. Pharmacol. Exp. Ther., 255 (1990) 923-928. 11 Takaki, M., Jin, J.-G. and Nakayama, S., Possible involvement of calcitonin gene-related peptide (CGRP) in non-cholinergic, nonadrenergic relax~ion induced by mesenteric nerve stimulation in the guinea-pig ileum, Brain Res., 478 (1989) 199-203. 12 Takaki, M., Jin, J.-G. and Nakayama, S., Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum, Acta Med. Okayama, 43 (1989) 353-357.