Br. J. Pharmacol. (1990), 101, 908-912 Effects of putative neurotransmitters and related drugs on withdrawal contractures of guinea-pig isolated ileum following brief contact with [Met5]enkephalin Loris A. Chahl Neuropharmacology Laboratory, Faculty of Medicine, University of Newcastle, N.S.W. 2308, Australia 1 Brief exposure for 2 min of guinea-pig isolated ileum to [Met5]enkephalin (MEnk) and noradrenaline has been shown previously to produce withdrawal contractures on washout of the agonist or addition of naloxone (MEnk) or phentolamine (noradrenaline). 2 The present study was undertaken to investigate firstly, whether other putative neurotransmitters and/or related drugs which inhibit transmitter release also produced withdrawal responses following 2 min contact with the ileum and secondly, whether they affected the opioid withdrawal response. 3 Adenosine (1-5 uM), but not U-50,488H (1-5 pM), somatostatin (0.01-5 pM), ocreotide (1-5 UM), baclofen (1-25uMm) or dopamine (5, 5O#M), produced a contracture on washout following 2min contact with the ileum. The adenosine (5,UM) washout contracture, in common with MEnk and noradrenaline washout contractures, was inhibited by the substance P antagonist, spantide (10pM). 4 Added 30s before washout at a concentration of 5pgM, noradrenaline, U-50,488H, adenosine, somato- statin and ocreotide significantly inhibited the washout withdrawal response following 2 min contact of the ileum with MEnk, 1 M. A higher concentration of baclofen, 250,UM, also inhibited this response. 5 The naloxone (1 pM)-precipitated withdrawal response following contact of the ileum with MEnk, 1 ,UM, for 2 min, was inhibited only by noradrenaline (5pM) and U-50,488H (5pM). 6 It is concluded that during naloxone-precipitated opioid withdrawal an additional population of enteric motor neurones is recruited which is not involved in the washout withdrawal response and these neurones have less diversity of presynaptic receptors mediating inhibition of transmitter release than cholinergic motor neurones. Introduction Brief exposure for 2 min to [Met5]enkephalin (MEnk), several enkephalin analogues and noradrenaline produced depen- dence in guinea-pig ileum as measured by withdrawal contrac- tures on washout (Chahl, 1983; 1985; 1986). Withdrawal responses were also precipitated by naloxone following 2 min contact with several opioids including morphine, fi-endorphin and dynorphin A- (1-13), and by phentolamine following contact with clonidine (Chahl, 1985). Furthermore it has been shown that the a2-adrenoceptor agonist clonidine (Chahl, 1985), and both p and K-opioid receptor agonists inhibited the opioid withdrawal response of the guinea-pig ileum (Chahl, 1986). Opioids and a-adrenoceptor agonists have in common an action to inhibit release of acetylcholine (ACh) and substance P (SP) from enteric neurones (see Bartho & Holzer, 1985). Several other putative enteric neurotransmitters, including adenosine (see Fredholm & Dunwiddie, 1988), somatostatin (Guillemin, 1976; Yau et al., 1986) and y-aminobutyric acid (GABA) (Cherubini & North, 1984), have also been reported to inhibit transmitter release from enteric neurones. In order to obtain further information on possible mechanisms involved in withdrawal responses, the present study was undertaken to investigate firstly, whether these putative neurotransmitters and/or related drugs produced dependence as manifest by a withdrawal response following brief contact with the ileum, and secondly whether they affected the opioid withdrawal response. Methods Adult guinea-pigs of either sex were killed by a blow to the head and a 2 cm segment of ileum was removed and sus- pended under 1 g tension in a 2 ml organ bath containing oxy- genated Tyrode solution at 37°C. Responses were recorded with a force transducer and Grass polygraph. At the start of each experiment a maximum response to ACh (5 pM) was obtained so that all responses could be expressed as percent- ages of the ACh maximum. Standard responses were then obtained to ACh (0.1 pM), SP (2.5 nM), and to washout follow- ing 2 min contact with MEnk, 1 pM. Effects of agonists and antagonists Neurotransmitters and related drugs, were each tested for their effects on the ileum on contact, and on washout follow- ing 2 min contact. The effect of the SP antagonist, spantide, 1pOjM, added 30s before washout, was tested on the washout response to adenosine. The effect of the somatostatin antagonist, cyclo(7- aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl]) (Fries et al., 1982) added 2 min after addition of somatostatin to the organ bath was tested. The effect of naloxone, 1 pM, was tested following 2 min contact of the ileum with the K-opioid agonist, U-50, 488H. Likewise the effect of the GABAB antagonist, phaclofen (Kerr et al., 1987), was tested following 2 min contact of the ileum with baclofen. Effects of agonists and related drugs on the opioid withdrawal response The effects of the neurotransmitters and related drugs were tested on the washout withdrawal response to MEnk. In these experiments MEnk, 1,um was added to the bath for 2 min before washout and the substance to be tested was added 0.5 min before washout (1.5 min after addition of MEnk). In some experiments ocreotide was added 5 min before addition of MEnk. Control responses to MEnk washout were obtained on the same preparations. In some experiments the effects of the substances were tested as above on the naloxone-precipitated withdrawal response to MEnk. In these experiments MEnk, Ip^ was added to the bath and naloxone, 1 pm, was added 2 min later. Since only one response to naloxone was obtained on each preparation, control responses were obtained on other prep- arations from the same animals. C Macmillan Press Ltd, 1990