- + ! ,' Br. J. Pharmac. (1978), 64, 293-300. F'(O t.,," e reab- ,ellular PHARMACOLOGICAL CHARACTERIZATION OF THE PRESYNAPTIC _-ADRENOCEPTORS REGULATING CHOLINERGIC ACTIVITY IN THE GUINEA-PIG ILEUM 1978) G.M. Departmentof Pharmacology,Allen and HzmburysResearchLimited, Ware, Hertfordshire,SG12 ODJ I The presynaptic ct-adrenoceptors located on the terminals of the cholinergic nerves of the guinea- pig myenteric plexus have been characterized according to their sensitivities to at-adrenoceptor agonists and antagonists. 2 Electrical stimulation of the cholinergic nerves supplying the longitudinal muscle of the guinea-pig ! ileum caused a twitch response. Clonidine caused a concentration-dependent inhibition of the twitch i response; the maximum inhibition obtained was 80 to 95_o of the twitch response. Oxymetazoline and xylazine were qualitatively similar to clonidine but were about 5 times less potent. Phenylephrine and methoxamine also inhibited the twitch response but were at least 10,000 times less potent than clonidine. 3 The twitch-inhibitory effects of clonidine, oxymetazoline and xylazine, but not those of phenyl- ephrine or methoxamine, were reversed by piperoxan (0.3 to 1.0 lag/ml). 4 Lysergic acid diethylamide (LSD) inhibited the twitch response, but also increased the basal tone of the ileum. Mepyramine prevented the increase in tone but did not affect the inhibitory action of LSD. Piperoxan or phentolamine only partially antagonized the inhibitory effect of LSD. 5 Phentolamine, yohimbine, piperoxan and tolazoline were potent, competitive antagonists of the inhibitory effect of clonidine with pA2 values of 8.51, 7.78, 7.64 and 6.57 respectively. 6 Thymoxamine was a weak antagonist of clonidine; it also antagonized the twitch-inhibitory effect of morphine. Thus, its effect against clonidine is probably not mediated specifically at presynap- tic _t-adrenoceptors. 7 Labetalol, itself, depressed the twitch response but did not antagonize the inhibitory effect of clonidine on the residual twitch. 8 The results demonstrate that the presynaptic _t-adrenoceptors in the guinea-pig ileum are or +, the same type as those located presynaptically in sympathetically innervated tissues. They are ct2-adrenoceptors and are different from those located postsynaptically. Introduction The guinea-pig ileum receives parasympathetic and adrenergic-inhibitory effect is reduced by phentola- sympathetic innervations. The preganglionic, para- mine (Krtineberg & Oberdorf, 1974). sympathetic nerves synapse primarily in the myenteric The twitch response of the guinea-pig ileum to low (Auerbach's) plexus, a dense network of short, mainly frequency stimulation of the cholinergic nerves is also cholinergic postganglionic fibres. Stimulation of these inhibited by adrenaline, noradrenaline and isoprena- nerves releases acetylcholine which, in turn, causes line. The maximum inhibition caused by adrenaline contraction of the longitudinal muscle fibres. The or noradrenaline is greater than that produced by iso- longitudinal muscle receives only a sparse adrenergic prenaline and differs from it in being accompanied innervation; instead, the adrenergic nerve terminals by a reduction in the release of acetylcholine from form a network around the intramural neurones (for the cholinergic nerves, fl-Adrenoceptor blockade references see reviews by Furness & Costa {1974) and greatly reduces or abolishes the twitch inhibition Wilkberg (1977)). Stimulation of the adrenergic nerves' caused by isoprenaline but has little or no effect on inhibits cholinergic nerve activity and thus reduces either the twitch-inhibition or the reduction in acetyl- responses to cholinergic nerve stimulation: the choline release caused by adrenaline. On the other 294 G.M. DREW hand, _t-adrenoceptor blockade prevents both actions 95%, 02. Intramural nerves were stimulated electri- of adrenaline, but has little effect on isoprenaline, cally with supramaximal square wave pulses, ! ms Noradrenaline-induced twitch inhibition is reduced in duration, delivered at a frequency of 0.1 Hz from by _t- or fl-adrenoceptor blockade, but its effects on a Farnell physiological stimulator connected to two acetylcholine release are prevented only by _t-anta- platinum electrodes placed on either side of the ileum. gonists. From these observations it has been con- Contractions of the longitudinal muscle were eluded that, in the guinea-pig ileum, twitch-inhibition recorded isometrically, with a Dynamometer UF 1 caused by fl-adrenoceptor stimulation is the result of 2 oz strain gauge, and displayed on a Devices chart a direct inhibitory action on the longitudinal muscle, recorder. whereas the twitch inhibition caused by _t-adrenocep- tor stimulation is primarily the result of a reduction in the release of acetylcholine, although a small com- Measurement of ¢t-ayonist potency ponent of the inhibition may be mediated via ct-adrenoceptors on the smooth muscle (Anderson & When twitch responses to transmural stimulation had Lees, 1976). The _t-adrenoceptors involved in regulat- become constant, one of the agonists was added to ing acetylcholine release are thought to be located the bathing fluid in a cumulative-concentration sche- presynaptically at the postganglionic cholinergic dule. The interval between successive doses was nerve terminals (Paton & Vizi, 1969; Kosterlitz, adjusted to allow the effect of each dose to develop Lydon & Watt, 1970; Knoll & Vizi, 1971). This in- fully. The concentration of each agonist required hibitory mechanism is analogous to that which modu- to reduce the twitch responses by 50% was deter- lates the release of noradrenaline from sympathetic mined. At the end of each experiment piperoxan (0.3 nerve terminals (Langer, 1974). to I I_g/ml; 1.3 to 4.3 p.moi/1) was added to the It has recently been shown that pre- and postsynap- bathing fluid. If piperoxan reversed the twitch-inhibi- tic _t-adrenoceptors in sympathetically innervated tis- tion it was assumed that the agonist had exerted its sues are different. In particular, presynaptic a-adreno- effect via _t-adrenoceptors. eeptors are less sensitive than postsynaptic receptors to the agonist actions of phenylephrine and methoxa- Effects of clonidine on contractile responses to acetyl- i mine (Starke, 1972; Starke, Endo & Taube, 1975; choline Steinsland & Nelson, 1975; Drew, 1976; 1977a), and ! to the antagonist actions of phenoxybenzamine In three unstimulated preparations the effect of cloni- ! (Dubocovich & Langer, 1974), labetalol (Blakely & dine on contractile responses to acetylcholine was Summers, 1977) and thymoxamine (Drew, 1976; examined. A concentration-response curve to acetyl- i 1977a). Langer (1974) has suggested the notation _tz choline was first established; then a submaximal dose for post- and _t2 for presynaptic a-adrenoeeptors. The (30 ng/ml; 0.2 lamol/I) producing about 80% of the present experiments were carried out to characterize maximum attainable response, was administered at the _t-adrenoceptors present in the guinea-pig ileum. 4 min intervals until tissue sensitivity became con- _ A preliminary account of the results has been pre- stant. Clonidine (1 to 10 ng/ml; 4.3 to 43 nmol/1) was sented to the British Pharmacological Society (Drew, then added to the bathing fluid and the response to 1977b). acetylcholine was measured 2 min later. Methods Measurement of g-antagonist potency , Male or female guinea-pigs (Duncan Hartley-Por- Initial experiments showed that the inhibitory effects ! ceUus) weighing 300 to 400g were killed by cervical of repeated administration of clonidine (0.3 to 30 dislocation and the small intestine was removed. The ng/ml: 1.3 to 130 nmol/I) were reproducible over .'l i 10cm nearest to the ileocaecal junction was dis- period of 3 to 4 h. Therefore the following experimen- carded. Alter carefully washing out the luminal con- tal protocol was observed in the subsequent experi- tents, segments of ileum, 2 to 3 cm long were selected ments. A concentration-response curve to clonidine from the terminal portion and suspended under an was first established. Exposure to clonidine was then initial tension of 0.5 to 1.0g in Krebs solution at 37°C discontinued; when the twitch responses had re- in a 50ml gut bath. The composition of the Krebs covered, the antagonist was added to the bathing solution was (mmol/I): Na* 143.4, K _ 5.9, Mg 2_ 0.6, fluid. The concentration response curve to clonidine Ca 2÷ 1.3, CI 124.5, H2PO4 1.2, SO z 0.6, HCO3- was repeated 15 min later in the presence of the an- 25; and glucose 11.1. The solution contained pro- tagonist. This procedure was repeated on two further pranolol (0.3 lag/ml; 1.2 lamol/i) to block fl-adrenocep- occasions with progressively higher concentrations of tors and was bubbled continuously with 5_o CO2 and antagonist. Clonidine dose-ratios were determined PRESYNAPTIC o_-ADRENOCEPTORSIN GUINEA-PIG ILEUM 295 ectri- from the concentrations causing 50% of the maximum methonium (10 to 30 lag/ml; 50 to 150 _tmol/i) 1 ms inhibition of the twitch response in the absence and reduced the responses by only about 10% showing from in the presence of each concentration of antagonist, that the nerves involved were mainly postganglionic, i ) two Results were expressed in the form of a Schild plot leum. (Arunlakshana & Schild, 1959), and the pA2 and slope Effects of a-adrenoceptor agonists on twitch responses were ol the regression line were calculated. JF 1 In some experiments, morphine was used instead Clonidine (0.1 to 30 ng/ml)produced a concentration- " chart