Two Types Ofreceptors for 5-Hydroxytryptamine on The

Br. J. Pharmac. (1985), 85, 529-539

Two types of receptors for 5-hydroxytryptamine on the cholinergic nerves ofthe guinea-pig myenteric plexus

H. Kilbinger & I. Pfeuffer-Friederich Pharmakologisches Institut der Universitat Mainz, Obere Zahlbacher StraBe 67, D-6500 Mainz, F.R.G.

1 The effects of 5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked release of[3H]- acetylcholine (ACh) from guinea-pig myenteric plexus preparations preincubated with [3H]-choline have been investigated in the absence of cholinesterase inhibitors. 2 5-HT caused a transient increase in spontaneous release and an inhibition ofthe electrically-evoked release of[3H]-ACh. The 5-HT-induced contractions of the longitudinal muscle were clearly related to the increase in spontaneous release. The inhibitory effect was not due to activation ofa-adrenoceptors since it was also observed in the presence of tolazoline and on strips from reserpine-pretreated guinea- pigs. 3 After desensitization of the excitatory 5-HT receptors with 5-HT or metoclopramide the effects of 5-HT on spontaneous [3H]-ACh release were largely reduced. A variety of established antagonists at neuronal 5-HT receptors (i.e. metitepine 0.1-1 jtM; methysergide 1 gM; ketanserin 0.1-1 jiM; MDL 72222 0.1 jiM; tropacocaine 1 gM) failed to block the excitation. 4 The inhibition by 5-HT of the electrically evoked [3H]-ACh release was competitively antagonized by metitepine (pA27.6) and methysergide (pA27.0) but not by ketanserin. Tachyphylaxis to the inhibitory action of 5-HT did not occur. 5 The results suggest that the excitatory 5-HT receptor ('M'-receptor) differs in its pharmacological properties from other neuronal 5-HT receptors. The presynaptically located inhibitory receptor may roughly correspond to the 5-HT, receptor subtype but probably differs from the 5-HT autoreceptor.

Introduction It has been suggested that 5-hydroxytryptamine (5- differential effects of 5-HT on the release ofACh. The HT) is an enteric neurotransmitter which is involved in objective of the present study was, therefore, to the control ofgastrointestinal motility (Gershon et al., examine in detail the effects of 5-HT on spontaneous 1983). Intestinal motility can both be inhibited and and electrically evoked release of ACh from guinea- stimulated by exogenous 5-HT. Thus, 5-HT contracts pig myenteric neurones. In addition, we have tried to the resting ileum and, in addition, inhibits the twitch- characterize with some established antagonists the 5- like contractions of the ileum induced by electrical HT receptors modulating ACh release. ACh release stimulation of cholinergic nerves (Gintzler & Mu- was measured in the absence of cholinesterase inhibi- sacchio, 1974). As the contractions were partly in- tion from preparations that had been preloaded with hibited by tetrodotoxin or antimuscarinic drugs it was [3H]-choline. Preliminary reports of these findings concluded that the contractile action of 5-HT is have been presented to the German Pharmacological mediated by release of acetylcholine (ACh) (Gaddum Society (Kilbinger & Pfeuffer-Friederich, 1982). & Picarelli, 1957; Brownlee & Johnson 1963; Costa & Furness, 1979). Direct evidence for an ACh releasing effect of 5-HT was presented by Adam-Vizi & Vizi (1978). When 5-HT is applied to the serosal surface of Methods the small intestine the peristaltic reflex is inhibited but 5-HT stimulates peristalsis when applied to the Release experiments mucosal surface (see review by Gershon et al., 1983). Since cholinergic nerves are critically involved in the Guinea-pigs of either sex weighing 300-400g were control of intestinal motility, the above mentioned stunned by a blow to the head and bled. Unless excitatory and inhibitory actions could be due to otherwise stated, longitudinal muscle strips with the i) The Macmillan Press Ltd 1985 530 H. KILBINGER & I. PFEUFFER-FRIEDERICH myenteric plexus attached were prepared from the was used to correct for counting efficiency. The proximal half of the small intestine. Two strips recovery for ['4Cl-choline was 40 ± 2% (n = 22) and weighing approximately 50mg were suspended the values for (3H]-choline were corrected accordingly. isotonically under a tension of 1 g in a 2 ml organ bath and superfused with Tyrode solution (composition in Contraction experiments mM: NaCI 137, KCI 2.7, CaCI2 1.8, MgCI2 1.0, NaH2PO4 0.4, NaHCO3 11.9 and glucose 5.6), that A piece (about 2 cm in length) of longitudinal muscle contained in addition 1 pM choline. The solution was strip was suspended isometrically under a tension of gassed with carbogen and warmed to a constant 0.7 g in a 7 ml organ bath, containing Tyrode solution temperature of 37C. After a 30 min incubation with (37C, gassed with carbogen). The strips were connec- [3H]-choline (5 JCi ml- ) during which the tissue was ted to a force-displacement transducer and the con- stimulated with square wave pulses (0.2 Hz, 1 ms), the tractions displayed on a Hellige recorder. After 60 min strips were superfused (1 ml min I) with Tyrode solu- of equilibration, during which the strips were challen- tion containing 10 pM hemicholinium-3 to prevent re- ged 4 times with 1 gLM 5-HT, a non-cumulative concen- uptake of choline. After a washout period of 70 min tration-response curve for 5-HT was established. 5- the superfusate was collected in 3 min fractions. The HT was added to the organ bath in increasing strips were stimulated by field stimulation at frequen- concentrations and was left in contact with the tissue cies of0.1 or 1 Hz. Biphasic square wave pulses (1 ms) for 1 min. Thereafter, the organ bath was washed out 3 were applied by 2 platinum electrodes, which were times and the next higher concentration applied positioned parallel to the strips. The potential drop 15 min later. between the electrodes was 11 V cm-' giving a current In some experiments the strips were stimulated of 380 mA. Tritium content ofthe superfusate samples continuously at 0.1 Hz. When the twitch size was was measured by liquid scintillation spectrometry and constant, 5-HT was added to the bath fluid. The effect the counting efficiency was determined using external of 5-HT on twitch height was expressed as a percen- standardization. The outflow of tritium evoked by tage ofthe contraction immediately before addition of electrical stimulation or by 5-HT was obtained from 5-HT. the difference of the total tritium outflow and the spontaneous outflow calculated by interpolation from Reserpine-pretreatment the first 3 and the last 3-5 unstimulated samples as described previously (Kilbinger & Wessler, 1980). Guinea-pigs received reserpine 1.5 mg kg-' subcutan- eously and were killed 3 days later. The ileum was Extraction and separation of radiolabelled choline homogenized in 15 ml trichloroacetic acid (5%) and the noradrenaline present in the tissue extract deter- Basically, the procedure described by Kilbinger & mined fluorimetrically with the trihydroxyindole Wessler (1980) was used. [3H]-choline was extracted method according to Muscholl (1966). from 5 ml of the superfusate into 2 ml allylcyanid containing 10 mg of sodium tetraphenylborate. The Statistics organic phase was evaporated to dryness and the residue redissolved in 0.5 ml methanol. The methanol Results are expressed as means ± s.e. means. Student's solution was applied to an anion-exchange column t test was used to assess the significance of differences (Dowex 1 x 8 Cl- ) and eluted with 6 ml methanol. between two mean values. If one control group was The eluate was again evaporated to dryness and the compared with more than one group of treatments, a residue redissolved in 100 pl methanol containing one-way analysis of variance was performed followed unlabelled carrier choline and ACh Qmol(2 each). Ten by Dunnett's test (Dunnett, 1964). To quantify 1tl of this solution was spotted on cellulose thin layer antagonism, apparent pA2 values were calculated chromatography sheets and developed in butan-1-ol: according to: pA2= pAx+ log (dose ratio - 1) methanol: glacial acetic acid: H20 (8:2:1:3 by vol.). (Schild, 1947). pD'2 values were calculated according The choline and ACh bands were detected using to van Rossum (1963). Dragendorff's reagent. The thin layer chromatograms were cut into 5 mm sections which were added to vials Drugs containing I ml ofa mixture ofmethanol and 1 M HCI (19:1 by vol.). The samples were counted in 7 ml of a The following were used: [methyl-3H]-choline (NEN; Triton-toluene-scintillator. Recoveries were deter- 80 Ci mmol-' Dreieich, F.R.G.); choline chloride mined by adding ['4C]-choline (2-1OnCi) as internal (Fluka, Neu-Ulm; F.R.G.); hemicholinium-3 (EGA- standard to each 5 ml superfusate sample. Radioac- Chemie, Steinheim, F.R.G.); 5-hydroxytryptamine tivity was measured in a Packard Tricarb Model 460 C creatinine sulphate (Merck, Darmstadt, F.R.G.); scintillation spectrometer. External standardization ketanserin (Janssen, Beerse, Belgium); MDL 72222 5-HT RECEPTORS ON MYENTERIC NEURONES 531 Si S2 (IaH, 3a, 5o H-tropan-3-yl-3,5-dichlorobenzoate) (gift from Dr J.R. Fozard, Merrell, Strasbourg, France); metoclopramide dihydrochloride mono- 400 - hydrate (Dolorgiet, Bad Godesberg, F.R.G.); meth- 300- ysergide hydrogen maleate (Sandoz, Basel, Swit- zerland); metitepine (Hoffmann-La Roche, Basel, _ 200- 0. Switzerland); reserpine (Serpasil injection 2.5 mg ml', Ciba-Geigy, Basel, Switzerland); C *1A\ A tetrodotoxin; tolazoline hydrochloride (both Sigma, 400- St Louis, MO, U.S.A.); tropacocaine (benzoylp- I; I seudotropine) hydrochloride (Aldrich, Nettetal, 300 - 911'E ji F.R.G.). V 200- \ Results o *1 T-HT 10 Xm E Facilitation ofspontaneous, and inhibition ofelectrical- .2 300- ly evoked outflow of[3HJ-acetylcholine Z 200- 0*** *i \Ai, Longitudinal muscle strips which had been pre- B 100 incubated with [3H]-choline were stimulated twice (SI; 0 TTX 300 nM S2), 5-HT being added to the superfusion medium o 3400- 30 min before S2. Figure 1 shows that 5-HT (1OriM) caused a transient increase in outflow of tritium 3001 whereas the stimulation-evoked outflow was inhibited 200- (by 62 ± 2%; n = 6). The reduction by 5-HT of the stimulated outflow might be due to depletion of the TTX 300 nM 5-HT 10 M available store oftritiated transmitter by the preceding superfusion with 5-HT. In order to test this possibility experiments were performed in which the excitatory 0 30 60 90 effect of 5-HT was prevented by tetrodotoxin. Figure I Time (min) shows that during and after perfusion with tetrodotoxin (300 nM), 5-HT no longer increased the Figure 1 Effects of 5-hydroxytryptamine (5-HT) in the outflow of tritium. Yet, the electrically-evoked out- absence and presence of tetrodotoxin on spontaneous flow was significantly reduced (by 45 ± 1 %; n = 6) and electrically evoked outflow of [3H]-acetylcholine. by 5-HT. Thus, the inhibition by 5-HT of the evoked Strips preincubated with [3H]-choline were subsequently outflow of tritium is independent of the preceding superfused (I ml min ') with Tyrode solution containing facilitatory action of 5-HT. IOJM hemicholinium-3. Electrical stimulation (0.1 Hz, 60 In order to identify the nature of the 3H-radio- pulses) was performed 9 min (SI) and 66 min (S2) after the end of the washout period as indicated at the top of activity present in the medium before and during the the figure. Abscissa scale starts at the end of the washout superfusion with 5-HT, choline and ACh were isolated period. Horizontal bars indicate superfusion with 5-HT from the Tyrode solution by ion-pair extraction (n = 6), tetrodotoxin (TTX, n = 3) and TTX plus 5-HT followed by thin layer chromatography. [3H]-ACh was (n = 6). Upper panel, control experiments (n = 6). Means- not detected in the samples. The spontaneous outflow ± s.e.means are shown in this and subsequent figures. Table 1 Comparison of the outflow of tritium and [3H1-choline from the myenteric plexus preparation Collection period Tritium [31H-choline (min) (103 d.p.m. g- ) (% of tritium) n Spontaneous 0- 9 609 ± 25 60± 8 3 0.1 Hz, 60 pulses 9-27 368±64 101 ± 19 3 Spontaneous 27-36 425 ± 22 57± 6 8 5-HT, 10#M 36-60 780 ± 98 118 ± 15 8 Strips were incubated with [3H]-choline and superfused subsequently (1 ml min- '). At the end of the 60 min washout period (zero time) the superfusate was collected in 3 min intervals. Tritium and [3H]-choline were determined in aliquots of a superfusate collected immediately before, and in aliquots collected during and after stimulation (0.1 Hz) or exposure to 5-HT. The outflow evoked by electrical stimulation or by 5-HT was calculated from the difference of the total outflow and the preceding spontaneous outflow. 532 H. KILBINGER & I. PFEUFFER-FRIEDERICH

of tritium consisted of 50-60% [3H]-choline. The a -100 : c remaining 40-50%, not detected by ion-pair extrac- * 800- 0 tion and thin layer chromatography probably re- -C, present labelled choline metabolites (Szerb, 1976). The < 'ao 600- Q outflow of tritium evoked by electrical stimulation or I E by 5-HT consisted of 100% tritiated choline (Table 1). 2 . -50 ._E - a 400- Previous experiments (Szerb, 1976; Kilbinger & Wes- E sler, 1980) have shown that the tetrodotoxin-sensitive O:0 0° outflow of tritium from strips preincubated with [3H]- = 200- .RE choline represents exclusively release of neuronal [3H]- 0 0 ACh. We suggest that by analogy, the tetrodotoxin- 0- -0 sensitive outflow of [3H]-choline induced by 5-HT originates from the release of [3H1-ACh that is hy- drolyzed by cholinesterases. Subsequently, outflow of D [3H]-choline evoked by 5-HT or by electrical stimula- 1.0,1 outflow. 0 tion is therefore designated [3H]-ACh 0 The excitatory and inhibitory effects of different * ° concentrations of 5-HT are shown in Figure 2. Com- parison of the concentration-effect curves indicates "" * S2 0.5- ** Rf* that 5-HT is somewhat more potent in inhibiting the Si \, ** electrically-evoked outflow (EC50 0.63 gM) than in ** facilitating the spontaneous outflow (EC50 2.3IM). The inhibitory effect of 5-HT depended on the stimulation frequency. At 1 Hz (60 pulses) the reduc- O- tion by 5-HT of the evoked outflow was smaller than I . I C 0.1 1 10 at 0.1 Hz stimulation. 5-HT (>M) The increase by 5-HT of the spontaneous outflow was paralleled by its effect on longitudinal muscle contraction (Figure 2). The maximal effective concen- Figure 2 Concentration-response curves for the effects tration and the ECo value for the increase in resting of 5-hydroxytryptamine (5-HT). (a) Effects on spontan- tension (1.27 ± 0.26 gM 5-HT; n = 11) were similar to eous outflow of [3H]-acetylcholine ([3H]-ACh, left ordin- the corresponding values obtained in release ex- ate scale, and (0) n = 4-13), and on isometric tension of the longitudinal muscle (right ordinate scale, and A). periments, which suggests that the contractile effect of Contractions were measured in separate experiments 5-HT is related to the release of ACh. On the other (n = 11) and on each strip a complete concentration- hand, there was no such clear correlation between the response curve to 5-HT was established. (b) Effects on effects of 5-HT on the electrically-evoked outflow of electrically-evoked outflow of [3H]-ACh. The strips were [3H]-ACh and the twitch response to 0.1 Hz stimula- stimulated twice (SI, S2) with trains of 60 pulses. tion. The twitch height was reduced only by 10 IAM 5- Stimulation frequencies were 0.1 Hz (0; n = 4-6) and HT (by 43 ± 6%; n = 16), whereas 0.1 and 1 iM 5-HT I Hz (E; n = 4 for each value). 5-HT was added to the ± and ofevoked slightly increased the twitch height by 7 1, medium 30 min before S2. Ordinate scale, ratio = outflow during S2 and S1. C, control experiments. 12 ± 2%, respectively (both n 10). Significance of difference from corresponding control The stimulation-evoked release of ACh from the (Dunnett's test): * P < 0.05; ** P < 0.01. guinea-pig myenteric plexus can be inhibited by

Table 2 Effects of5-hydroxytryptamine (5-HT) on spontaneous and electrically evoked outflow of[3H]-acetylcholine in the presence of 1 gAM tolazoline (group A), or from strips of reserpine pretreated guinea-pigs (group B)

Group 5-HT Si 52/Si 5-HT induced outflow n (gm) (103 d.p.m.g-') (103 d.p.m.9- )

A 530 ± 98 0.85 ± 0.04 - 4 A 10 775 ± 138 0.44 ± 0.09* 835 ± 121 4 B 2861 ± 382 0.67 ± 0.03 4 B 10 1166 ± 167 0.21 ± 0.01* 1574 ± 232 4

Strips preloaded with [3H]-choline were stimulated at 0.1 Hz (60 pulses) 9 min (Sl) and 66 min (S2) after the end of the washout period. 5-HT was added 30min before S2, and tolazoline (group A) 30min before Sl. * P< 0.01. 5-HT RECEPTORS ON MYENTERIC NEURONES 533 noradrenaline (Paton & Vizi, 1969). In order to Si S2 exclude an indirect (noradrenaline releasing) action of 5-HT on the electrically evoked outflow of [3HJ-ACh, the effects of 5-HT were investigated in the presence of the a-adrenolytic drug tolazoline. Tolazoline was applied to the superfusion medium 30 min before S1 in 500- a concentration of IM which has been shown to block the effects of x-adrenoceptor agonists in the ileum (Drew, 1978; G6rich et al., 1982). Table 2 shows that the facilitatory effect of 10 FM 5-HT was not affected oX 400- by tolazoline, and that 5-HT significantly reduced (by CD, 49 ± 10%) the evoked outflow of [3H]-ACh in the 0 K A presence of tolazoline. X 300- In other experiments the actions of 5-HT were studied on strips obtained from four reserpine- 6. pretreated guinea-pigs. Release experiments were per- -6 formed on strips from the proximal half of the small 200-1 intestine, whilst the distal halfwas used for determina- tion of the endogenous noradrenaline content. After .0 reserpine treatment the noradrenaline content of the '%41 I~ ~~ ... distal ilea was 0.13 ± 0.03 nmol g- ', which is 4% of MTP 1 IM the noradrenaline content of distal ilea from four control animals (3.31 ± 0.79 nmol g '). Electrical 5-HiT 10 tM stimulation as well as 5-HT caused a much larger I I outflow of [3HJ-ACh from strips from reserpinized o 30 60 90 guinea-pigs (Table 2) compared to controls (cf. Time (min) Figure 2). 5-HT significantly depressed the electrically-evoked outflow by 69 ± 1% (Table 2). Thus, it can Figure 3 Effects of 5-hydroxytryptamine (5-HT) on be concluded that the inhibitory action of 5-HT is not spontaneous and electrically evoked (0.1 Hz) outflow of ['HI-acetylcholine in the presence of 1 JAM metitepine. mediated via a-adrenoceptor stimulation. Metitepine (MTP) was added to the medium 30 min Costa & Furness (1979) observed that the hyoscine- before SI in 4 experiments. For further details see legend sensitive contractile effects of 5-HT were more marked to Figure 1. in the oral than in the anal parts ofthe guinea-pig small intestine. In eight experiments we found that the outflow of[3H]-ACh elicited by I tiM 5-HT was slightly response curve for the inhibitory effect of 5-HT is but significantly (P < 0.05, paired t test) larger in the shifted in parallel to the right, and there is no proximal part (first 25 cm behind the duodeno-jejunal statistically significant attenuation of the maximum junction; 325 + 34 x 103 d.p.m. g- 1) than in the distal (Figure.4). The apparent pA2 value for metitepine part (last 25 cm before the ileo-caecal junction; calculated from the shift in the curve to 5-HT was 7.6. 231 ± 27 xl03 d.p.m. g'). In the presence of 1 pM metitepine, 5-HT no longer caused any inhibition of the evoked outflow, in Antagonism of the inhibitory effects of5-hydroxytryp- contrast, 3pM 5-HT significantly enhanced evoked tamine by metitepine and methysergide outflow of[3H]-ACh (Figure 4). Neither concentration of metitepine (0.1 and I M) significantly affected the Strips were stimulated twice (0.1 Hz, 60 pulses) and increases by 5-HT (0.1-30 JAM) of [3H]-ACh outflow. metitepine was added to the superfusion medium Methysergide alone added to the medium 30min 30 min before S2. Neither of the two concentrations before S2 significantly reduced the electrically evoked tested (0.1 and 1 pLM) significantly affected the spon- outflow of [3H]-ACh at concentrations of 10JM (by taneous outflow or the outflow evoked by electrical 32±5%, n=4, P<0.05) and 30JM (by 54±4%, stimulation (S2/S1: 0.91 ± 0.13, n = 4, and n = 4, P < 0.01), but not at a concentration of I laM. 0.99 ± 0.12, n = 5). For interaction experiments The spontaneous outflow of tritium was not changed metitepine was added to the medium 30 min before SI by either concentration of methysergide. For interac- and 5-HT 30 min before S2. Figure 3 shows that 1 ptM tion experiments between methysergide and 5-HT the metitepine completely prevented the inhibition by protocol was similar to that shown in Figure 3. 10 pM 5-HT ofthe evoked outflow of[3H]-ACh, whilst Methysergide (1 gM), like metitepine, antagonized the the facilitatory action of5-HT remained unaffected. In inhibitory action of 5-HT but did not affect the 5-HT- the presence of 0.1IAM metitepine, the concentration- induced increase in [3H]-ACh outflow. The concentra- 534 H. KILBINGER & I. PFEUFFER-FRIEDERICH

a 800 1

1.0- I *c 600- 4 LOE I 400- S2 0.5- Si -6 'D 200- E FiM 5-HT 600- 0- ._.7 r/ AII 4- 0 so~~~~~~~ 1 . 400- 1.0 - 6 6 0 t~*"' 2001 69o*-6 `1 1 p.M 5-HT s ** S2 **0 91 0.5 - 0 30 60 90 Si Time (min) b **K 100-

0 0- 00< - WA-/i. . . a C 0.1 1 10 50 - 5-HT (>M) Figure 4 Concentration-response relationship of the I inhibitory effect of 5-hydroxytryptamine (5-HT) on the 0- I I outflow of [3H]-acetylcholine evoked by 0.1 Hz stimula- 0.01 0.1 1 tion and the effects of metitepine (a) or methysergide (b). 5-HT was added to the medium 30 min before S2 (0). 5-HT (>M) Metitepine (0.1 FIM, 0; 1 LM, A) or methysergide (1 jAM, Figure 5 Effects of sustained exposure to 5-hydrox- 0) were added 30 min before SI. Each value is the mean ytryptamine (5-HT) on [3H]-acetylcholine ([3H]-ACh) of 4-6 experiments, vertical lines show s.e.means. Sig- outflow evoked by 1OM 5-HT. (a) Time course of the nificance ofdifference from corresponding control (Dun- experiments. Paired strips from the same small intestine netts' test): *P<0.05; **P<0.01. were exposed to 10 jAM 5-HT either in the presence of 1 AM 5-HT, or after the effect of 1 AM 5-HT had been washed out (control). Samples were collected in 5 min intervals. n = 4 for each experiment. (b) Inhibition by a desensitiz- tion-response curve to 5-HT was shifted in parallel to ing concentration of 5-HT (0.01-1 jM) of the 13H]-ACh the right and an apparent pA2 value of 7.0 was outflow evoked by 10 AM 5-HT expressed as percentage of calculated for methysergide (Figure 4). the outflow in corresponding control experiments. Each value is the mean of4 experiments; vertical lines show s.e. Antagonism ofthe excitatory effects of5-HT by S-HT means. Significance of difference from control: and metoclopramide * P <0.01. A typical feature observed in contraction experiments on the guinea-pig ileum is that 5-HT produces rapid tion had been washed out. In the presence of 100 nM and reversible tachyphylaxis (see e.g. Huidobro-Toro 5-HT the excitatory action of 5-HT was slightly but & Foree, 1980). We have studied the effects of 10 M not significantly reduced. However, I gLM 5-HT in- 5-HT on [3H]-ACh outflow in the presence of smaller, hibited by about 70% the [3H]-ACh releasing effect of desensitizing concentrations of 5-HT (Figure 5). In 10 gM 5-HT. The release experiments, thus, confirm control experiments the response to 10 IAM 5-HT was the previous data indicating that 5-HT desensitizes the studied after the effects of the desensitizing concentra- receptors after having excited them. 5-HT RECEPTORS ON MYENTERIC NEURONES 535

SI S2 presence of 10 #M metoclopramide (S2/Si: 0.38 ± 0.01; n = 3). With l00IM metoclopramide present 5-HT also caused a significant inhibition ofthe evoked outflow (S2/SI: 0.47 ± 0.02; n = 4) although 300- the degree of inhibition was somewhat less than in control experiments with 10tiM 5-HT alone (S2/SI: 0.35 ± 0.05; n = 4). The ratios S2/S1 in the presence of 10 and 100 IuM metoclopramide alone were 0.80 ± 0.02 200- (n = 4) and 0.83 ± 0.02 (n = 3). The antagonism by metoclopramide of the facilitatory action of 5-HT was studied in more detail *g100- in experiments in which 5-HT was added in increasing E uu concentrations as a bolus into the superfusion stream m MCP 10 I'M to give final concentrations of 1-30 jM in the 2 ml 0 5-HT 10 FM organ bath. Metoclopramide shifted the concentration-response curve for 5-HT to the right, but at higher x concentrations both the slopes and the maxima of the 5-HT curve were progressively decreased (Figure 7) Q300 - jI*I A pD' value of5.40 ± 0.15 (CI = 1) was calculated for this effect of metoclopramide. Effects of ketanserin, MDL 72222 and tropacocaine - 4*jI~ 200 II !1|. \1 1!1 Ketanserin The effects of 1 pM 5-HT on spontaneous I~~~~~~~~~~~~~~~~~~I and electrically-evoked outflow of [3H]-ACh were '1o4 -1..1,.....0 100- I ftIV4CP 100 KM 5I-HT 10 FLM

I I 0 30 60 90 600 Time (min)

0 Figure 6 Effects of 5-hydroxytryptamine (5-HT) on spontaneous and electrically (0.1 Hz) evoked outflow of X 400- [3H]-acetylcholine in the presence of metoclopramide (MCP). Metoclopramide was added to the superfusion C6 medium 30 min before SI. Means of3 (MCP 10 pM) and 4 (100 JM) experiments; vertical lines show s.e.means. For 200- further details see legend to Figure 1.

Metoclopramide, like 5-HT, stimulates the ex- 0- citatory 5-HT receptors of the guinea-pig ileum and causes an increase in spontaneous ACh release (Kil- l- 3 10I 30 binger et al., 1982). We have studied whether meto- 5-HT (>M) clopramide affects the excitatory or inhibitory action Figure 7 Effects of metoclopramide on [3H]-acetyl- of 5-HT. Strips were stimulated twice at 0.1 Hz (60 choline outflow evoked by 5-hydroxytryptamine (5-HT). pulses) and metoclopramide was added to the medium Strips preincubated with [3HJ-choline were superfused 30 min before SI. The transient increase in [3H]-ACh with Tyrode solution. After a 60 min washout period outflow occurring immediately after the administra- 5-HT was injected (100 Ill) four times at 30 min intervals tion of metoclopramide (Kilbinger et al., 1982) was into the superfusion stream to give final concentrations of 1-30;LM. Metoclopramide was superfused from 30 min not studied in these experiments. Figure 6 shows that before the application of the first concentration of 5-HT the excitatory effect of 5-HT (10 fM) on [3H]-ACh until the end of the experiment. Cumulative concentra- ± = ± outflow was reduced by 66 4 (n 3), and 94 3 tion response curves for 5-HT alone (-, n = 4) or in the (n = 4) %, respectively, in the presence of 10 and presence of the following concentrations of meto- 100 pM metoclopramide. On the other hand, the clopramide (gM): 0.1 (0), I (A), 3 (0), (each n = 4), 10 inhibitory action of 5-HT was not affected in the (* ) (n = 3)- 536 H. KILBINGER & I. PFEUFFER-FRIEDERICH

Table 3 Effects of5-hydroxytryptamine (5-HT) on spontaneous and electrically evoked outflow of[3H]-acetylcholine ([3H]-ACh) in the presence of ketanserin

Ketanserin 5-HT Si S2/SJ 5-HT induced outflow n (M) (jm) (103 d.p.m. g 1) (103 d.p.m. g- ') 468±64 0.87 ± 0.051 8 352 ± 62 0.54 ± 0.05 ** 300 32 6

0.1 570 ± 99 0.86±0.10l ** 4 0.1 I 414±69 0.55±0.05J 261 ± 59 8 1.0 481± 71 0.79±0.06l 6 1.0 393 ± 96 0.53 ± 0.03 1 217 ± 38 4 Ketanserin was added to the medium 30 min before SI. For further details see Table 2. * P <0.02; ** P <0.01.

Table 4 Effects of5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked outflow of[3H]-acetylcholine ([3H]-ACh) in the presence of MDL 72222

MDL 72222 5-HT Si S2/SJ 5-HTinduced outflow n CuM) (uM) (103 d.p.m. g-') (103 d.p.m.g-') 450 ± 118 0.78 ± 0.07 } ** 6 3 486 ± 129 0.44 ± 0.03 J 516 ± 86 9 0.1 534 ± 170 0.73 ± 0.08 - 1** 4 0.1 3 377 ± 75 0.48 ± 0.08 J 405 ± 142 6 1.0 282 ± 37 0.84 ± 0.05 ** 4 1.0 3 424 ± 69 0.38 ± 0.05 J 65 ± 20 5 MDL 72222 was added to the medium 30 min before S1. For further details see Table 2. * P <0.05; ** P <0.01. tested in the presence of the 5-HT antagonist, ketan- Discussion serin. Table 3 shows that neither effect of 5-HT was significantly changed in the presence of 0.1 or I JM The results demonstrate that 5-HT exerts a dual effect ketanserin. The spontaneous outflow of tritium was on the cholinergic neurones of the guinea-pig ileum: not affected by either of the concentrations of ketan- increase of spontaneous and inhibition of the elec- serin. trically-evoked release of ACh. MDL 72222, tropacocaine Both drugs are selective Excitatory effects of 5-hydroxytryptamine and potent antagonists at neuronal 5-HT receptors of postganglionic sympathetic fibres (Fozard et al., 1979; The similarity between concentration-response curves Fozard, 1984). The effects of MDL 72222 on the for 5-HT-induced contraction and ACh release sug- facilitatory and inhibitory actions of 5-HT are given in gests that the increase in muscle tension is mainly due Table 4. MDL 72222 (0.1 and 1 gsM) did not antagon- to the release of ACh. A similar conclusion was ize the 5-HT-induced inhibition of [3H]-ACh outflow. reached by Costa & Furness (1979) who found that in On the other hand, 1 gLM MDL 72222 markedly the proximal ileum the indirect action of 5-HT is reduced the facilitatory effect of 5-HT. The outflow dominant whereas the direct effects of 5-HT on the evoked by electrical stimulation during SI was not longitudinal muscle are almost insignificant. significantly changed in the presence of 0.1 or 1 lM In order to characterize the excitatory receptor (the MDL 72222. Tropacocaine (I gM) affected neither the so-called 'M' receptor) we have studied the effects of facilitatory nor the inhibitory actions of 3 JM 5-HT several 5-HT antagonists. Both 5-HT and meto- (data not shown). Neither MDL 72222 (0.1 and 1 jaM) clopramide antagonized the facilitatory action of nor tropacocaine changed the spontaneous outflow of 5-HT, which probably reflects desensitization after the tritium. initial excitation of the receptor. The non-parallel 5-HT RECEPTORS ON MYENTERIC NEURONES 537

displacement of the 5-HT curves with depression of myenteric plexus preparations from reserpine-treated the slope and the maximum in the presence of guinea-pigs responded to electrical stimulation (Sl) metoclopramide (Figure 7) is typical for a compound and to superfusion with 5-HT with a much higher ACh having both agonist and antagonist activities (Ariens release than control preparations. Such an increased & Simonis, 1964). Metoclopramide has been shown to release after catecholamine depletion has also been act as a competitive antagonist upon excitatory 5-HT found by Paton & Vizi (1969) and is probably due to receptors on postganglionic sympathetic nerves of the the loss ofan endogenous adrenergic inhibition. Thus, rabbit heart (Fozard & Mobarok Ali, 1978), as have both electrically-evoked release and also 5-HT-evoked tropacocaine and nanomolar concentrations of MDL release of ACh may be modulated through inhibitory 72222 (Fozard et al., 1979; Fozard, 1984). The lack of x-adrenoceptors. In the presence of tolazoline the effect of tropacocaine and 0.1I1M MDL 72222 in the electrically-evoked ACh release also appeared to be present experiments together with the failure of meto- slightly higher than under control conditions (cf. SI clopramide to antagonize competitively the effects of values ofTable 2 with SI values from Tables 3 and 4), 5-HT confirms the view (Wallis, 1981; Fozard, 1984) but these differences were not significant because of that the excitatory 5-HT receptors on sympathetic the large variations of the absolute amounts of ACh nerve terminals are pharmacologically different from released. those on myenteric cholinergic neurones. Since the inhibitory action of 5-HT was measured However, micromolar concentrations of MDL after a 30 min superfusion of the tissue with 5-HT, the 72222 blocked excitatory 5-HT receptors ofmyenteric inhibitory receptor is not readily desensitized in nerves (Table 4). On the other hand it is known that marked contrast to the strong desensitization similar concentrations fail to antagonize the indirect observed for the excitatory effect of 5-HT. (i.e. neuronally-mediated), 5-HT contractions of the Electrophysiological studies in which exogenous longitudinal muscle (Fozard, 1984). An explanation 5-HT induced membrane depolarization of myenteric for this discrepancy might be the existence of spare neurones (Wood & Mayer, 1979; Johnson et al., 1980), receptors for ACh on the longitudinal muscle (Sastry and a reduction ofthe amplitude ofthe cholinergic fast & Cheng, 1972; Birdsall et al., 1978). The amount of e.p.s.p. (North et al., 1980) may aid in the interpreta- ACh liberated by 5-HT in the presence of MDL 72222 tion of our results. The studies of North et al. (1980) may be sufficient to cause a maximal contraction indicate that 5-HT acts presynaptically at the termin- although the release of transmitter is reduced. als of cholinergic interneurones. From our results it Ketanserin is a selective ligand for central 5-HT2 seems unlikely that the cholinergic nerves endowed binding sites (Leysen et al., 1981). In our study with excitatory 5-HT receptors also bear inhibitory ketanserin failed to antagonize the excitatory effects of 5-HT receptors. If this were so, one would expect 5- 5-HT (Table 3). Likewise, metitepine and methyser- HT-evoked ACh release to be enhanced when the gide (I pM) which bind to both 5-HT, and 5-HT2 sites inhibitory response is blocked by metitepine or meth- (Leysen et al., 1981) did not affect the 5-HT-induced ysergide, whereas it was not affected. The inhibition of ACh release, suggesting that the excitatory 5-HT the electrically-evoked ACh released by 5-HT was not receptor does not belong to either the 5-HT, or 5-HT2 paralleled by a simultaneous reduction of the twitch subtype. contraction. The twitch height was significantly reduced only by 1O1AM 5-HT, whilst 0.1 and I 1AM 5-HT Inhibitory effects of 5-hydroxytryptamine enhanced the contractions. It is known that 5-HT increases the longitudinal muscle contraction to ex- 5-HT caused a concentration-dependent inhibition of ogenous ACh or nicotine (Bulbring & Crema, 1958). the electrically-evoked release of ACh. As with other Thus, only when large concentrations of 5-HT are agonists that diminish the release of ACh from used is the twitch response reduced through reduction myenteric nerves (oxotremorine, noradrenaline, mor- of the evoked ACh release. phine; for references see Kilbinger, 1982), the in- Metitepine and methysergide, but not ketanserin hibitory effect was more marked at a lower than at a antagonized the inhibition by 5-HT of electrically- higher stimulation frequency. 5-HT applied to the evoked ACh release. This suggests that the inhibitory serosa easily reaches the myenteric plexus (Gershon et 5-HT receptor may roughly correspond to the 5-HT, al., 1983) and inhibition of the peristaltic reflex is subtype. In the presence of 100 nM metitepine or 1 1AM probably due to reduced ACh release, mediated via methysergide the concentration-response curve was inhibitory 5-HT receptors. A possible direct or indirect shifted in parallel to the right and there was no action of 5-HT at inhibitory a-adrenoceptors can be depression of the maximum response, characteristic excluded because the inhibition ofevoked ACh release for an interaction between an agonist and a com- was also observed in the presence of tolazoline and petitive antagonist. With 1 1AM metitepine a reduction after a 96% reduction of the endogenous noradren- by 5-HT of the electrically-evoked ACh release was aline content by reserpine treatment. It was noted that not observed, but whether this is indicative of a non- 538 H. KILBINGER & I. PFEUFFER-FRIEDERICH competitive mode of interaction only additional differs in its pharmacological properties from the studies will reveal. From the shift of the 5-HT 5-HT autoreceptor. concentration-response curve in the presence of In conclusion, we have demonstrated that 5-HT 100 nM metitepine an apparent pA2 value of 7.6 was increases spontaneous and inhibits electrically-evoked calculated. This is considerably higher than pA2 values release ofACh from myenteric nerves. Both effects are obtained for metitepine at central 5-HT autoreceptors mediated through stimulation of specific 5-HT recep- (6.6, Schlicker & G6thert, 1981; 7.0, Engel et al., 1983; tors which differ in their pharmacological properties. 6.9, Middlemiss, 1984). Since methysergide was also an antagonist in the present experiments (pA2 7.0) and We thank Miss Doris Wolf for competent technical assis- in electrophysiological studies (North et al., 1980) but tance and the pharmaceutical companies for donation of is inactive at the central 5-HT autoreceptor (Cerrito & drugs. This work was supported by the Deutsche Forschungs- Raiteri, 1979; Baumann & Waldmeier, 1981; Martin & gemeinschaft. The paper contains part of the Dr. rer. nat. Sanders-Bush, 1982), we assume that the 5-HT recep- Thesis of I.P.-F., Naturwissenschaftliche Fachbereiche der tor inhibiting ACh release from myenteric neurones Universitiit Mainz.

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