Inhibition of cholinergic neurotransmission in isolated guinea-pig main bronchi by SR 48968

European Journal of Pharmacology, 243 (1993) 309-312 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

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EJP 21354

Short communication

Inhibition of cholinergic neurotransmission in isolated guinea-pig main bronchi by SR 48968 C o r i n n e A . E . M a r t i n *'a, X a v i e r E m o n d s - A l t b a n d C h a r l e s A d v e n i e r a Laboratoire de Pharmacologie, Facult~ de M~decine Paris-Ouest, 15, Rue de l'Ecole de Mddecine, F-75270 Paris Cedex 06, France, and b Sanofi Recherche, Avenue du Pr Blayac, F-34184 Montpellier Cedex, France Received 31 August 1993, accepted 2 September 1993

SR 48968 (10 6 to 10 -5 M) inhibited the cholinergic response of the isolated guinea-pig main bronchus to electrical field stimulation. Since this effect was reversed by naloxone 10 -5 M and since SR 48968 had no effect on the contractile response to exogenous acetylcholine, we conclude that SR 48968 acts at a prejunctional level and that opioid receptors are involved. This effect was observed at concentrations approximately 75 000 times higher than those needed for blockade of tachykinin NK 2 receptors. SR 48968; Tachykinin NK 2 receptors; Opioid receptors

1. Introduction

SR 48968 is a new potent and selective non-peptide antagonist of tachykinin N K 2 receptors. It inhibits the binding of [125I]neurokinin A to its receptors from rat d u o d e n u m m e m b r a n e s with an inhibition constant ( K i) of 0.51 _+ 0.09 nM (Emonds-Alt et al., 1992) and exerts a potent competitive antagonism against tachykinin N K 2 receptor agonists in tissues obtained from rabbits, guinea-pigs, rats and humans, with a pA 2 value of 9.4-10.5 (Advenier et al., 1992b; Maggi et al., 1993). SR 48968 has also been shown to inhibit the microvascular leakage induced by neurokinin A in guinea-pig airways (Qian et al., 1993; Tousignant et al., 1993) and to abolish the non-adrenergic non-cholinergic response of the guinea-pig main bronchus to electrical field stimulation, with an ECs0 of 0.056 nM (Martin et al., 1992). However, while SR 48968 is clearly devoid of tachykinin NK1 receptor antagonistic activity (EmondsAlt et al., 1992; Boyle et al., 1993), at high concentrations this drug has been reported to interact with guinea-pig tachykinin N K 3 cortical binding sites (ECs0 = 0.1-1 tzM) (Petitet et al., 1993; Boyle et al., 1993) and with /z-opioid receptors of the guinea-pig ileum in response to electrical field stimulation (ECs0 = 3 /zM) (Boyle et al., 1993).

* Corresponding author. Tel. 33-1-43.26.09.52, fax 33-1-44.07.13.52.

The aim of this study was to investigate whether SR 48968 reduced the cholinergic responses of the isolated guinea-pig main bronchus to electrical field stimulation and if stimulation of opioid receptors was involved in its inhibitory effects.

2. Materials and methods

2.1. Tissue preparations Main bronchi rings obtained from tricoloured guinea-pigs of either sex (250-350 g) anaesthetized with urethane (1.25 g . k g -1 i.p.) were equilibrated under an initial tension of 2.0 g in Krebs solution at 37°C gassed with 95% 0 2 - 5 % CO 2. Tension was measured isometrically with Pioden strain gauges (UF-1) and amplifiers (EMKA, France) and displayed on recorders (Linseis L6512, Germany). The composition of the Krebs solution was (mM): NaCI 118.0, KC1 5.4, CaC12 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, N a H C O 3 25.0, and glucose 11.7. After a 1-h resting period, bronchial rings were contracted to maximal tension with acetylcholine (1 mM) and then allowed to equilibrate for 60 min while they were washed with Krebs solution every 15 rain. The resting tension was adjusted to 1.5-2.0 g. In all experiments, propranolol ( 1 0 - 6 M ) and indomethacin ( 1 0 - 6 M) were added to the buffer solution to avoid the influence of adrenergic nerve stimulation and indi-

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rect effects of prostaglandins on the neuronal responses (Martin et al., 1992). After tension had returned to the baseline tone, the preparations were stimulated every 8-15 rain at increasing frequency, 0.5-32 Hz, 1 ms, and 320 mA current for 10 s by using a stimulator (EMKA, France) with the voltage output adjusted to give a constant current and biphasic rectangular pulse of alternating polarity. These procedures were repeated in the absence or presence of morphine (10 -6 M), SR 48968 (10 -6 to 10 -5 M) and naloxone (10 -5 M). Drugs were added 30 min (morphine and naloxone) or 120 min (SR 48968) before transmural stimulation. The results are expressed as percentages of the maximal contraction induced by acetylcholine (1 mM). Control experiments (n = 6) showed no significant fading of the response to field stimulation during the experimental period. The stimulus parameters caused an optimal, reproducible biphasic contraction consisting of a fast cholinergic contraction followed by a sustained contraction due to the release of sensory neuropeptides from C-fibre endings. In this study, we only investigated the effects of SR 48968 and morphine on the cholinergic response, the effects of SR 48968 on the NANC response being described elsewhere (Martin et al., 1992).

rophenyl-butyl]benzamide (Sanofi Recherche, Montpellier, France); indomethacin (Merck); naloxone, propranolol (Sigma, St. Louis, USA). All drugs were dissolved in distilled water and then diluted in Krebs solution, except for indomethacin and SR 48968, which were dissolved in ethanol and then diluted in Krebs solution. The maximal amount of ethanol used (0.4%) did not alter the response to acetylcholine.

3. Results

Figs. 1A and 2A show that morphine (10 -6 M) exerted an inhibitory effect on the cholinergic response induced by electrical fel d stimulation of the isolated guinea-pig main bronchus. This inhibition was higher for low frequencies of neuronal discharge and was reversed by naloxone (10 -5 M). Figs. 1B and 2B-D show that SR 48968 (3 × 10 -6 M and 10 -5 M) was also able to inhibit the cholinergic response induced by electrical field stimulation of the isolated guinea-pig main bronchus whereas at 10 -6 M it did not significantly influence the cholinergic contraction. As with morphine, the phenomenon of frequency dependence was observed and the effects of SR 48968 were totally (3 × 10 -6 M) or partially (10 -5 M) reversed by naloxone (10 -5 M). For 4 and 8 Hz, the ECs0 values of SR 48968 were 4.5 and 4 ~M, respectively.

2.2. Data analysis Data are expressed as means + S.E.M. ECs0 values represent the concentrations producing 50% of the maximal effect. The results were analysed statistically by variance analysis a n d / o r Student's t-test.

4. Discussion

Our results show that morphine (10 -6 M) exerted an inhibitory effect on the cholinergic response induced by electrical field stimulation of the isolated guinea-pig main bronchus and that this inhibition was reversed by naloxone (10 -5 M). Under similar condi-

2.3. Materials The drugs used were acetylcholine HC1, morphine HC1 (PCH, Paris, France); SR 48968: (S)-N-methylN[4-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlo-

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Fig. 1. Representative traces showing the responses of the isolated guinea-pig main bronchus to electrical field stimulation (. : 1-8 Hz, 1 ms, 320 m A for 10 s; in the presence of propranolol (10 -6 M) and indomethacin (10 - 6 M) in the bath), the effects of morphine 10 - 6 M (upper trace, A) and of SR 48968 3 × 10 - 6 M (lower trace, B) and their reversal by naloxone 10 -5 M.

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Fig. 2. Histograms illustrating the effects of morphine 10 -6 M (A) and SR 48968 10 -6 M (B), 3 × 10 - 6 M (C) and 10 -5 M (D) on the cholinergic response to electrical field stimulation (0.5-32 Hz, 1 ms, 320 mA, for 10 s) of the isolated guinea-pig main bronchus. Control, morphine 10 6 M, SR 48968 10 - 6 M, 3 × 10 - 6 M and 10 -5 M, and responses in the presence of naloxone 10 - s M. Columns represent contractions expressed as percentages of the maximal contraction induced by acetylcholine (1 mM). Experiments were performed in the presence of propranolol (10 - 6 M) and indomethacin (10-6 M). M e a n s + S.E.M. for 4 - 6 animals• Significant differences from control: * P < 0.05; * * P < 0.01; * P < 0.001.

tions, Belvisi et al. (1990) reported that the selective agonist of /~ opioid receptors, D A M G O ([DAlaZ,NMePhea,Gly-olS]enkephalin), inhibited both the cholinergic and non-adrenergic non-cholinergic response to electrical field stimulation of the guinea-pig main bronchus, with ECs0 values of 36 nM and 113 nM, respectively. Since naloxone has no effect on the cholinergic neurotransmission when given alone, and since morphine and opioid agonists do not antagonize the contractile response to exogenous acetylcholine in vitro, this effect is thought to be prejunctional (Barnes, 1992). SR 48968 (3 × 10 -6 M and 10 -5 M) was also able to inhibit significantly the cholinergic response induced by electrical field stimulation of the isolated guinea-pig main bronchus. As with morphine, the effects of SR 48968 were totally (3 x 10 -6 M) or partially (10 -5 M) reversed by naloxone. Our results are, therefore, concordant with those of Boyle et al. (1993) who have demonstrated that SR 48968 has an agonistic effect on /z-opioid receptors of the isolated guinea-pig ileum, with an ECs0 of 3 #M, and interacts with the same receptors in the guinea-pig brain. It is possible that SR 48968 acts at a prejunctional level since this drug is

devoid of anticholinergic effects at concentrations lower than 10 -5 M (Advenier et al., 1992a). It must be stressed that these results were obtained at very high concentrations, since the ECs0 values of SR 48968 were 4.5 and 4 /zM at 4 and 8 Hz, respectively. In contrast, we have previously shown that the ECs0 of SR 48968 for inhibition of the non-adrenergic non-cholinergic component of the electrical field stimulation of the isolated guinea-pig bronchus was 0.056 nM. Consequently, the agonistic effect of SR 48968 on opioid receptors was observed in concentrations that were 75 000 times higher than those reported as being required to inhibit the NK 2 receptors of tachykinins.

References Advenier, C., E. Naline, L. Toty, H. Bakdach, X. Emonds-Alt, P. Vilain, J.-C. Breli~re and G. Le Fur, 1992a, Effects on the isolated h u m a n bronchus of SR 48968, a potent and selective non-peptide antagonist of the neurokinin A (NK2)-receptor , Am. Rev. Respir. Dis. 146, 1177. Advenier, C., N. Rouissi, Q.T. Nguyen, X. Emonds-Alt, J.C. Breli~re, G. Neliat, E. Naline and D. Regoli, 1992b, Neurokinin A (NK 2)

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Martin, C.A.E., E. Naline, Xavier Emonds-Alt and C. Advenier, 1992, Influence of (_+)-CP-96,345 and SR 48968 on electrical field stimulation of the isolated guinea-pig main bronchus, Eur. J. Pharmacol. 224, 137. Petitet, F., J.-C. Beaujouan, M. Saffroy, Y. Torrens and J. Glowinski, 1993, The nonpeptide NK2-antagonist SR 48968 is also a NK~ antagonist in guinea pig but not in the rat, Biochem. Biophys. Res. Commun. 191, 180. Qian, Y., X. Emonds-Alt and C. Advenier, 1993, Effects of capsaicin, (_+)-CP-96,345 and SR 48968 on the bradykinin-induced airways microvascular leakage in guinea-pigs, Pulm. Pharmacol. 6, 63. Tousignant, C., C.-C. Chan, D. Guevremont, C. Brideau, J.J. Hale, M. MacCoss and I.W. Rodger, 1993, NK 2 receptors mediate plasma extravasation in guinea-pig lower airways, Br. J. Pharmacol. 108, 383.