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Br. J. Pharmac. (1978), 64, 293-300. F'(O t.,," e reab- ,ellular PHARMACOLOGICAL CHARACTERIZATION OF THE PRESYNAPTIC _-ADRENOCEPTORS REGULATING 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 and antagonists. 2 Electrical stimulation of the cholinergic nerves supplying the longitudinal muscle of the guinea-pig ! ileum caused a twitch response. caused a concentration-dependent inhibition of the twitch i response; the maximum inhibition obtained was 80 to 95_o of the twitch response. and were qualitatively similar to clonidine but were about 5 times less potent. and 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 (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 only partially antagonized the inhibitory effect of LSD. 5 Phentolamine, , piperoxan and 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 . Thus, its effect against clonidine is probably not mediated specifically at presynap- tic _t-adrenoceptors. 7 , 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 -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 , 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 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 (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 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 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 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 of clonidine to inhibit the twitch response. Morphine, dependent reduction in the twitch response of the like adrenaline, inhibits the release of acetylcholine ileum, but rarely abolished it; the maximum inhibi- from the cholinergic nerves of the myenteric plexus tion obtained was 80 to 95% (Figure 1). Twitch re- _! (Paton, 1957; Paton & Aboo Zar, 1968) but its effects sponses recovered rapidly after the tissue was washed are mediated specifically via presynaptic recep- with clonidine-free Krebs solution. Tissue sensitivity ! had tors, (Kosterlitz & Watt, 1968; Vizi, 1974). In prelimi- to repeated administration ofclonidine remained con- ., •.d to nary experiments it was found that the second of two stant over a period of 3 to 4 h: in seven experiments, sche- consecutive concentration-response curves to mor- the mean concentrations of clonidine producing 50% was phine (0.1 to 100 ng/ml; 0.35 to 350 nmol/1) was of the maximum inhibition in four consecutive con- velop always displaced about 3 to 5 fold to the right of centration-response curves were 1.62, 1.70, 1.76 and uired the first curve (n = 6). Thereafter there was little or 1.56 ng/ml respectively (similar results have been il ]eter- no further shift of repeated morphine dose-response reported recently by Hughes, Kosterlitz, Robson & n (0.3 curves. Accordingly, in subsequent experiments, the Waterfieid, 1978). The maximum twitch inhibition :_ the first curve was ignored and only the second to the produced by clonidine remained constant. In two ex- ' : , lhibi- fifth concentration-response curves were used to periments hexamethonium (10 and 30 lag/ml) did not i :d its determine the specificity of the antagonists, alter the responsiveness to clonidine, which indicates that its site of action is probably the postganglionic _ _'t Drugs cholinergic nerve terminal. '_-_,, Oxymetazoline and xylazine also reduced the _ cetyl- The following drugs were used; acetylcholine chloride twitch response. The maximum inhibition produced i (BDH), sulphate (BDH), clonidine hydro- by these compounds was similar to that produced .,_i! chloride (Boehringer lngelheim), iabetalol {5-[1- by clonidine but these agents were about 5 times less i i cloni- hydroxy-2-[(1-methyl-3-phenylpropyi)amino]ethyl]- potent than clonidine. Phenylephrine and methoxa- !, .' was hydrochloride; AH 5158, Allen and mine were much less effective in reducing the twitch ', cetyl- Hanburys), lysergic acid diethylamide tartrate (San- response, being at least 10,000 times less potent than 'ii I dose doz), (+)-methoxamine hydrochioride (Burroughs clonidine. At the highest concentration tested (300 '_i )f the Wellcome), mepyramine maleate (May and Baker), ltg/ml; 1.5 mmoi/1) methoxamine produced about the _ ed at morphine hydrochloride (MacFarlan Smith), napha- same maximum inhibition as clonidine, whereas con- zoline nitrate (Ciba), oxymetazoline hydrochioride phenylephrine reduced responses only by 60 to 7(Y_/o. !I I) was (Merck), phentolamine mesylate (Ciba), (-)-phe- Piperoxan (0.3-1 _tg/mi) reversed the inhibitory _! _se to .... nylephrine hydrochloride (Koch-Light), piperoxan effects of cionidine (Figure 1), oxymetazoline and :_! hydrochioride (May and Baker), tolazoline hy- xylazine, but not those of phenylephrine or methoxa- drochioride (Ciba), thymoxamine hydrochloride mine. The concentration of each agonist producing ,._ (Warner), xylazine (2-(2,6--dimethylphenylamino)-4- 5_/o reduction in the twitch response is given in Table i:* H-5,6-dihydro-l,3-thiazin hydrochloride; BAY-1470, 1. Results are expressed in tool/! to enable accurate AG) and yohimbine hydrochloride (Sigma). All comparison of drug potencies. ,_ffects drugs were dissolved in 0.9°/0 w/v NaCi solution Lysergic acid diethylamide (LSD, 0.01 to 3 lag/ml; i) to 30 () or distilled water immediately before use. 0.03 to 10 i_mol/I) exerted two effects; it increased i l )ver a Concentrations mentioned in the text refer to the the basal tone of the ileum and reduced the size of _! imen- base. the twitch response by up to 60% (Figure 2). The _ xperi- effects occurred at similar concentrations but pre- t nidine Results treatment with mepyramine (10 to 100 ng/ml; 35 to " then 350 nmol/l) prevented the LSD-induced increase in ,d re- Electrical stimulation of the guinea-pig ileum at 0.1 basal tone without affecting its twitch-inhibitory [ nidineathing veHzlopproded uinceedacha trewitspochnsreespowasnsegene. Therallypeabetwk teeennsioni ande-d seiffecmpt.ly Thia csonsshoequwsencethatofththee twitincrcehas-inhied bbasaitionl towasne. Thnoet !:!i : ae an- 3 g, and there was little variation in individual prep- inhibition of the twitch elicited by LSD in the pres- i urther arations over 3 to 4 h. Atropine (I lag/mi; 3.5 lamol/i) ence of mepyramine was only partially antagonized ! ons of abolished the responses, confirming that they were by piperoxan (0.3 to 1 lag/mi) or phentolamine I i mined produced by cholinergic nerve stimulation. Hexa- (1 _tg/ml; 3.6 ltmoi/1). [

t t i J '_i_i_i

296 G.M. DREW [ IlllllIIIIII[[LLIILIILLIILL!LLJlLLhLIII!,,,,,,,,,,,,,,,,,,,,, lmln 0.1 0.3 1 3 10 30 1

Clonidine (ng/ml) Piperoxan (pglml) Figure 1 The twitch-inhibitory effect of clonidine in the transmurally stimulated guinea-pig ileum (0.1 Hz; 1 ms, supramaximal voltage), and its reversal by piperoxan. Clonidine was added to the bathing fluid in a cumulative-concentration schedule.

The effect of clonidine on contractile responses to Interactions between clonidine and ct-adrenoceptor acetylcholine antagonists

Acetylcholine (30 ng/ml) caused a rapid and sustained Phentolamine (O.01 to 1 p,g/ml; 0.036 to 3.6 lamol/I), contracture of the ileum, with a peak tension similar piperoxan (0.03 to 0.3 tug/ml; 0.13 to 1.3 tamol/I) and to that produced by electrical stimulation, about 1.5 tolazoline (0.1 to 1 yg/ml; 0.62 to 6.2 I_mol/I) given to 2.5g. Clonidine (1 to 10 ng/ml) did not alter the alone produced a small (up to 30".,_,)but sustained responsiveness of the ileum to acetylcholinc, increase in the size of the twitch response. Thymoxa-

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I ! o'o,o'o3 o', 0"3 t _ 2rain b l!J

o'o,0"03 _, _3 _ ; ;

LSD (l_glml) Piperoxan (pglml)

Figure 2 The effect of lysergic acid diethvlamide (LSD) on the twitch response of the transmurally stimulated guinea-pig ileum. LSD was added to the bathing fluid in a cumulative concentration schedule. (a) LSD alone; (b) LSD in the presence of mepyramine (10 ng/ml). Note the poor reversal by piperoxan. PRESYNAPTIC =-ADRENOCEPTORS IN GUINEA-PIG ILEUM 297

mine (1 to 10 pg/ml; 3.6 to 36 I_moi/1) caused larger other than blockade of presynaptic =-adrenoceptors. increases in the twitch response (up to 50°/_)and this This suspicion was confirmed, in other experiments, effect was also sustained. In contrast, yohimbine (0.03 by the finding that thymoxamine antagonized the to 0.3 gLg/ml; 0.085 to 0.85 i.tmol/1) caused only a con- twitch-inhibition caused by morphine. Thymoxamine centration-dependent reduction of the twitch re- (1, 3 and 10 pg/ml) caused the morphine (0.1 to 100 sponse; labetaloi (0.3 I_g/ml; 0.9 Izmol/I) also inhibited ng/ml) concentration-response curve to be displaced the twitch. The effects of the antagonists on the twitch 2.45-, 3.49- and 3.92-fold to the right (n = 5): for response are summarised in Table 2. clonidine-induced inhibition the values were 2.60, 4.93 Phentolamine, piperoxan, yohimbine and tolazine and 8.25 respectively (n = 9). Thus, only the highest caused parallel displacements to the right of the cloni- concentration caused a greater shift of the concentra- dine concentration-response curve: the maximum in- tion-response curve to clonidine than to morphine. ! hibitory effect of clonidine was unaltered. Schild plots In contrast, neither piperoxan (0.03 to 0.3 I_g/ml) nor , i gave linear regressions, and their slopes were close phentolamine (0.01 to 1.0 _tg/ml) antagonized the to unity, suggesting that these compounds exert corn- effects of morphine. petitive antagonism at the presynaptic _-adrenoeep- Although labetalol, 0.3 pg/ml, itself reduced the tors in the ileum, twitch response by 19 to 43% (see Table 2), the resi- In contrast, thymoxamine only weakly antagonized dual twitch was inhibited by clonidine over the same the responses to clonidine and the slope of the Schiid concentration range that was effective in untreated plot was significantly less than unity, which suggested preparations. It is likely that the twitch-inhibitory eptor this effect of thymoxamine might involve an action effect of labetaloi alone, was the result of the com- i bination of the membrane stabilizing effect of iabeta- 1ol with that exerted by the already in iol/i), the Krebs solution (see Discussion). Thus, in order ) and Table 1 Concentrations of =-adrenoceptor to investigate the interaction between clonidine and agonists required to reduce by 50% (ICoo) the given twitch responseof the electricallystimulatedguinea- a higher concentration of labetalol (1 I_g/ml; 3 fined pig ileum l_mol/I), propranolol was omitted from the Krebs mxa- solution in an attempt to minimize the inhibitory effectof labetalol on the twitch response. Under these Agonists n IC,o (95% confidence limits) conditions, labetalol reduced the twitch by 20 to 57_o Clonidine 7 9.6 (7.4-12,6) nmol/I (n = 3), but the effect of clonidine on the residual Xylazine 6 48.2 (40.9-56.8) nmol/I twitch response was unaffected. Oxymetazoline 6 43.4 (22,3-84.1) nmol/I The pAz values for the antagonists and the slopes Phenylephrine" 4 216.7 (2'9.8-1575) iJmol/I of regression lines of the Schild plots are given in i Methoxamine" 5 576.7 (390-853) tamol/I Table 3.

• Inhibitory effect not reversed by piperoxan (see

- text)Table 2 Effectsof =-adrenoceptorantagonists,alone, on the size of the twitch responseof the electrically 1 stimulatedguinea-pig ileum t

i Antagonist n Mean % changer in twitch-size in the presence of antagonist at the i stated concentrations

Phentolamine6 0.0+51 0.03 +0.91 0.3 +21 3 10 #g/ml _i Yohimbine 6 -9 -13 -25 _: Piperoxan 5 +12 +22 +29 1 Tolazoline 7 + 10 + 15 + 29 1 Thymoxamine 9 + 42 + 48 + 26 ii Labetalol 6 - 31 - 37"

In seven control preparations,in which no antagonist was administeredthe twitch size immediately I prior to each of 4 consecutiveclonide concentration-responsecurvesvaried by lessthan +5%. t • in the absenceof propranolol,n = 3. t mean changeexpressedas % of averagetwitch amplitude before exposureto antagonist ,298 G.M. DREW

Disctmsion 1976) and rabbit pulmonary (Starke eta/., 1975; Borowski, Starke, Ehrl & Endo, 1977), and of The twitch response of the guinea-pig ileum to low the motor nerves supplying the rat vas deferens frequency stimulation of the intramural nerves was (Drew, 1977a). inhibited by all the a-adrenoceptor agonists exam- The actions of LSD at c¢-adrenoceptors are compli- ined. The inhibitory effect of clonidine was clearly cated. It has been shown to be a potent agonist at mediated presynaptically because it did not alter the presynaptic =2-adrenoceptors in the rat heart (Drew, responsiveness of the ileum to exogenous acetylcho- 1976), rat vas deferens (Ambache, Dunk, Verney & line. Thus, clonidine probably reduces the twitch re- Zar, 1973; Hughes, 1973), rat anococcygeus muscle sponse to nerve stimulation by reducing acetylcholine and dog retractor penis muscle (Ambache, Killick, release, as has been shown previously for xylazine Srinivasan & Zar, 1975), but a weak agonist, or even (Vizi, 1974). Oxymetazoline probably causes twitch- an antagonist, at postsynaptic at-adrenoceptors inhibition via the same mechanism because piperoxan (Ambache et al., 1975; Drew, 1976). Hughes 11973) antagonized the inhibitory effect of all three agonists, has reported that LSD inhibits the twitch response The very low potency of phenylephrine and methoxa- of the guinea-pig ileum by a presynaptic agonist mine at inhibiting the twitch is consistent with their action, which suggests that a2-adrenoceptors are in- ineffectiveness at reducing acetylcholine release volved. However, Ambache et al. (1975) found that (Paton & Vizi, 1969) and the inhibition caused by LSD blocked presynaptic =-adrenoceptors in the very high concentrations of both drugs is unlikely to ileum, which suggests they are of the a_-variety. In be mediated via presynaptic _-adrenoceptors since the present experiments LSD was found to be a weak piperoxan did not antagonize their effects. Thus, the inhibitor of the twitch response and the inhibition order of potency of the agonists in causing twitch was only partly antagonized by at-adrenoceptor antag- inhibition, clonidine > xylazine-----oxymetazoline _ onists. Further work is obviously needed to clarify phenylephrine > methoxamine, clearly indicates that the mechanism whereby LSD reduces the twitch re- presynaptic ¢-adrenoceptors in the guinea-pig ileum spottse of the guinea-pig ileum; a 'neurone blocking' are of the _2-type. This was confirmed by the finding action similar to that observed in the rat vas deferens that phentolamine, yohimbine, piperoxan and tolazo- and anococcygeus muscle (Gillespie & McGrath, line were potent antagonists of the twitch-inhibitory 1975) may be responsible. action of clonidine, whilst thymoxamine and labetalol The contractile effect of LSD noted in these experi- were weak antagonists. The low potency of labetalol ments has also been observed by Ambache et al. in blocking the presynaptic ,t2-adrenoceptors is in (1975), and it seems to be mediated via Hi-receptors contrast to its effect at postsynaptic =_-adrenoceptors since it was prevented by pretreatment with mepyra- where it exerts profound blockade at the concen- mine in concentrations previously shown to have little trations used in the present experiments (Kennedy & or no effect on muscarinic or tryptaminergic receptors Levy, 1974). Furthermore, the antagonist action of in this tissue (Cambridge & Holgate, 1955; Harry, thymoxamine seems to be largely non-specific because 1963; Brown & QuiUiam, 19651. It is not known the inhibitory effect of morphine was also anta- whether the effect of LSD on Ht-receptors is direct gonized. In summary, the orders of potency of the or indirect. 0t-agonists and antagonists show that the presynaptic In the present experiments, some of the =-adreno- ¢-adrenoceptors on the cholinergic nerves of the ceptor antagonists potentiated the twitch response, an guinea-pig myenteric plexus are of the same type as effect which might be thought to reflect the abolition those on the terminals of the sympathetic nerves sup- of presynaptic inhibition caused by endogenous nor- plying the rabbit heart (Starke, 1972), rat heart (Drew, adrenaline released from the sympathetic nerve ter-

Table 3 Potencies of =-adrenoceptorantagonistsagainst clonidine at the presynaptic _-adrenoceptors in the electricallystimulatedguinea-pig ileum

Antagonist n pA= (95% confidence limits) Slope (95% confidence limits)

Phentolamine 6 8.51 (8264,70) 1.23 (1.14-1.32) Yohimbine 6 7.78 (7.52-8.05) 1,02 (0.80--1.23) Piperoxan 5 7.64 (7.43-7.84) 1.11 (0.99-1.22) Tolazoline 7 6.57 (631-6.81) 1.04 (0.90--1.19) Thymoxamine 9 6.12 (5.61-6,63) 0,60 (0,38-0,82) Labetalol 6 < 5.5 -- y ,

PRESYNAPTIC =-ADRENOCEPTORS IN GUINEA-PIG ILEUM 299

et al., minals during transmural stimulation. This seems un- responses of the guinea-pig ileum to acetylcholine and and of likely, however, because the effects of the antagonists histamine (Raper & Wale, 1968) and labetalol does leferens on the twitch did not correlate with their abilities the same (Kennedy, personal communication). to antagonize responses to cionidine, and because The failure of labetaiol to block the presynaptic :ompli- Dzoljic (1967) found that the potentiating effect of 0t-adrenoceptors may explain why it does not cause mist at tolazoline persisted in preparations removed from diarrhoea in man at therapeutic dose levels. In con- : (Drew, reserpine-treated animals. He attributed the potentia- trast, phentolamine and tolazoline block the pre- rney & tion to an anticholinesterase action, which might be synaptic =-adrenoceptors and both compounds are i muscle valid for tolazoline but is unlikely to hold for the reported to cause diarrhoea in man (Nickerson, 1970). Killick, other agonists since their potentiating effects were This effect may result from the loss of a physiological ] _r even apparent at concentrations below those required to modulatory action of endogenous , [ ceptors inhibit significantly cholinesterase activity (Boyd, mediated via the presynaptic a-adrenoceptors in the (1973) Chang & Rand, 1960). Whatever the mechanism in- ileum, with a resultant enhancement of vagal activity :sponse volved,' it may be the same as that which causes and increase in intestinal motility. agonis potentiation of the twitch responses of the rat vas are in- deferens to motor nerve stimulation (Drew, 1977a). id that The twitch-inhibitory effect of labetalol may result The author wishes to thank Mr D. Baker for excellent in the from a membrane-stabilizing action on smooth technical assistance and Bayer AG., Boehringer lngelheim , ety. In muscle ceils, because in concentrations above 10-6 Limited, May and Baker Limited and Warner Limited for a weak M, /]-adrenoceptor antagonists such as propranolol their generous gifts of xylazine, clonidine, piperoxan and , dbition and are known to reduce the contractile thymoxamine respectively. ; antag- clarify tch re- References ocking' eferens AMBACHE, N., DUNK, L.P., VERNEY, J. & ABOO ZAR, M. DREW, G.M. (1977a). Pharmacological characterisation of :Grath, (1973). An inhibition of post-ganglionic motor the presynaptic a-adrenoceptor in the rat vas deferens. transmission in the mammalian vas deferens by Eur. J. Pharmac.,42, 123-130. '; experi- D-lysergic acid diethylamide. J. Physiol., 231, 251-270. DREW,G.M. (1977b). Pharmacological characterisation of et al. AMBACHE, N., KILLICK, S.W., SRINIVASAN, V. & ABOO presynaptic =-adrenoceptors which regulate cholinergic ZAR,M. (1975). Effects of lysergic acid diethylamid¢ on activity in the guinea-pig ileum. Br. J. Pharmac., 59. :eptors autonomic post-ganglionic transmission. J. Physiol., 513P. _epyra- e little 246, 571-593. DUBOCOVICH,M.L.& LANGER,S.Z. 0974). Negative feed- ANDERSON,A.A. & LEES, G.M. (1976). Investigation of back regulation of noradrenaline release by nerve eptors occurrence of tolerance to bronchodilator drugs in stimulation in the perfused cat's spleen: differences in ! Harry, chronically pretreated guinea-pigs. Br. J. Pharmac, 56, potency of phenoxyhenzamine in blocking the pre- and : _nown 331-338. post-synaptic adrenergic receptors. J. Physiol., 237, direct-- ARUNLAKSHANA,O. & SCH1LD,H.O.(1959). Some quanti- 505-519. tative uses of drug antagonists. Br. J. Pharmac. Che- DZOLJIC, M. (1967). Stimulatory effect of tolazoline on :lreno- mother, 14, 48-58. smooth muscle. Br. J. Pharmac. Chemother, 30, ise, an BLAKELY,A.G.H. & SUMMERS,RJ. (1977). The effects of 203-212. i 31ition labetalol (AH 5158) on adrenergic transmission in the FURNESS,JB. & COSTA,M. (1974). The adrenergic inner- _ s nor- cat spleen. Br. J. Pharmac., 59, 64.3-650. vation of the gastrointestinal tract. Rev. Physiol., 69, BOROWSKI, E., STARKE, K., EHRL, H. & ENDO, T. (1977). 1-51. _e ter- A comparison of pre- and postsynaptic effects of GILLESPIE,J.S. & MCGRATH, J.C. (1975). The effects of a-adrenolytic drugs in the pulmonary artery of the rab- lysergic acid diethylamide on the responses to field bit. Neurosclence, 2, 285-296. stimulation of the rat van deferens and the rat anococ- BOYD. H.. CHANG,V. & RAND,M..I.(1960). The anticho- cygeus muscles. Br. d. Pharmae, 54, 481-488. 3rs iinesterase activity of some antiadrenaline agents. Br. HARRY, J. (1963). The action of drugs on the circular J. Pharmac. Chemother, 15, 525-531. muscle strip from the guinea-pig isolated ileum. Br. J. BROWN,D.A.& QUILLIAM,J.P.(1965). Some effects of sta- Pharraac. Chemother, 20, 399-417. phylococcal a-toxin on isolated mammalian smooth HUGHES,J. (1973). Inhibition of noradrenaline release by muscle preparations. Br. J. Pharmac. Chemother, 25, lysergic acid diethylamide. Br. J. Pharmac., 49, 706--708. 781-789. HUGHES,J., KOSTERLITZ,H.W.,ROBSON.L.E. & WATER- CAMBRIOGF..G.W. & HOLGATE.J.A. (1955). Superfusion FIELD. A.A.11978). The inhibitory effects of clonidine as a method for the study of drug antagonism. Br. J. on the contractions of the guinea-pig ileum in the mor- Pharmac. Chemother, 10, 326--335. phine-dependent and withdrawn states. Br. J. Pharmac., DREW,G.M. (1976)• Effects of =-adrenoceptor agonists and 62, 388-389P. antagonists on pre- and postsynaptically located KENNEDY,!. & LEVY, G.P. (1974). Combined _- and t ct-adrenoceptors. Eur. J. Pharmac., 36, 313-320. fl-adrenoceptor blocking drug. AH 5158; furtherstudies II 300 G.M. DREW

on specificity and 0t-adrenoceptor blocking action. Proc. put by guinea-pig ileum longitudinal muscle strip. Br. Aus. physiol, pharmac. Sac., 5, 216-217. J. Pharmac., 35, 10-28. KNOI,L, J, & VIZI. E.S. (1971). Effect of frequency or stimu- PATON, W.D.M. & ABOO ZAR, M, (1968). The origin of ace- lation on the inhibition by noradrenaline of the acetyl- tylcholine released from guinea-pig intestine and Iongi- choline output from parasympathetic nerve terminals, tudinal muscle strips. J. Physiol., 194, 13-33. Br. J. Pharmac., 4!, 263.-272. RAPER, C. & WALE, J. (1968). Specificity of fl-receptor an- KOSTERLITZ, H.W., LYDON, R.J. & WATT, A.J. (1970). The tagonists. Eur. J. Pharmac., 3, 279-281. effects of adrenaline, noradrenaline and isoprenaline on STAKE, K. (1972). Alpha sympathomimetic inhibition of inhibitory 0t- and fl-adrenoceptors in the longitudinal adrenergic and cholinergic transmission in the rabbit muscle of the guinea-pig ileum. Br. J. Pharraac., 39, heart. Naunyn-Schmiedebergs Arch. Pharmac., 274_ 398--413. 18--45. KOSTERLITZ, H.W. & WATT, A.J. (1968). Kinetic par- STARKE, K., ENDO, T.E. & TAUBE, H.J. (1975). Relative pre- ameters of narcotic agonists and antagonists, with par- and post-synaptic potencies of a-adrenoceptor agonists ticular reference to N-allylnoro-xymorphone (Nalox- in the rabbit pulmonary artery. Naunyn- one). Br. J. Pharmac. Chemother, 33, 266-276. Schmiedebergs Arch. Pharmac., 291, 55-78. KRONEBERG, G. & OBERDORF, A. (1974). Inhibition of STEINSLAND, O.S. & NELSON, SH. (1975). "Alpha adrener- acetylcholine release and acetylcholine action in the gic" inhibition of the response of the isolated rabbit guinea-pig ileum by sympathetic =- and fl-receptor ear artery to brief intermittent sympathetic stimulation. stimulation. In Proceedings, First congress of the Hun- Blood vessels, 10, 378-379. garian Pharmacological Society. ed. Knoll, J. & Vizi, VlZI, E.S. (1974). Inhibitory effect of morphine and norad- E. S. pp 3%48. Budapest: Akademiai Kiado. renaline on neurochemical transmission. In Proceed- LANGER, S.Z. (1974). Presynaptic regulation of catechol- ings, First congress of the Hungarian Pharmacological release. Biochem. Pharmac., 23, 1793-1800. Society. ed. Knoll, J. & Vizi, E.S. pp 1%38, Budapest: NICKERSON, M (1970). Drugs inhibiting adrenergic nerves Akademiai Kiado. and structures innervated by them. In The Pharmacolo- W1LKBERG, J. (1977). Localisation of adrenergic receptors gical Basis Of Therapeutics, Fourth Edition. ed. Good- in guinea-pig ileum and rabbit jejunum to cholinergic man, L.S. & Gilman, A. New York: Macmillan Co. neurons and to smooth muscle cells. Acta physiol. PATON, W.D.M. (1957). The action of morphine and related scand., 99, 190-207. substances on contraction and on acetylcholin¢ output of coaxially stimulated guinea-pig ileum. Br. d. Phur- mac. Chemother, 12, 119-127. PATON, W.D.M. & VIZI, E.S. (1969). The inhibitory action (Received March I0, 1978. of noradrenaline and adrenaline on acetylcholine out- Revised May 1, 1978,)