The Guinea-Pig Distal Colon - a Sensitive Preparation for the Investigation of 5-HT4 Receptor-Mediated Contractions 'Kay A

'." 0 Macmillan Press Ltd, 1993 Br. J. Pharmacol. (I993), 110, 1593 1599 Macmillan Press Ltd, 1993 The guinea-pig distal colon - a sensitive preparation for the investigation of 5-HT4 receptor-mediated contractions 'Kay A. Wardle & Gareth J. Sanger

SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex CM 19 5AD 1 Experiments were designed to characterize pharmacologically the contractile responses to 5-hydroxytryptamine (5-HT) in the guinea-pig isolated distal colon longitudinal muscle-myenteric plexus preparation (LMMP). 2 In the presence of methiothepin (100 nM) and granisetron (1 0tM), 5-HT (10 pM- 10 nM) produced concentration-dependent contractile responses of the guinea-pig distal colon LMMP, with a pEC50 of 9.2 ± 0.08. 3 Responses to 5-HT were mimicked by a series of tryptamine analogues, with the following rank order of potency; 5-HT> 5-MeOT> >5-CT> tryptamine> 2-Me-5-HT. All were found to be full agonists. 4 Responses to 5-HT were also mimicked by a series of substituted benzamide analogues. Their rank order of potency was 5-HT > renzapride > cisapride > (S)-zacopride > (R)-zacopride > metoclopramide. All were full agonists relative to 5-HT. 5 The benzimidazolone derivatives, BIMU 1 and BIMU 8 were approximately equipotent partial agonists (intrinsic activities of 0.8 ± 0.07 and 0.5 ± 0.08 respectively) in the guinea-pig distal colon. 6 Tropisetron produced a rightward displacement of the 5-HT concentration-effect curve, yielding an apparent pA2 of 6.4 ± 0.1. The slope of the Schild plot (1.3 ± 0.1) was significantly greater than unity. 7 SDZ 205 557 produced a concentration-dependent shift to the right of the 5-HT concentration- response curve, yielding an estimated pA2 of 7.8 ± 0.1 and a slope which did not significantly deviate from unity. SDZ 205 557 produced similar pKB estimates (7.3-7.9) when tested against 5-MeOT, renzapride and 5-CT, indicating a common site of action. 8 The pharmacological profile of the 5-HT-evoked contractions of the guinea-pig distal colon LMMP are consistent with activity at the 5-HT4 receptor. Furthermore, of the models of this receptor described in the literature, the guinea-pig distal colon appears to be the most sensitive model to date, making it a useful tool in the investigation of 5-HT4 receptor-mediated events. Keywords: Guinea-pig colon; 5-HT4 receptors; 5-HT (5-hydroxytryptamine); tryptamine analogues; substituted benzamide analogues; benzimidazolone derivatives; tropisetron; SDZ 205 557

Introduction Based on an elegant series of experiments carried out in the 5-HT4 receptor, namely the guinea-pig distal colon long- mouse embryo colliculi neurones and guinea-pig hippocam- itudinal muscle myenteric plexus (LMMP). pus (Dumuis et al., 1988a,b; Bockaert et al., 1990) it has been Some of these results have been presented to the British proposed that at least some of the reported 'atypical' res- Pharmacological Society (Wardle & Sanger, 1992). ponses to 5-hydroxytryptamine (5-HT) can be attributed to stimulation of a novel receptor, termed 5-HT4. Thus, the 5-HT4 receptor is characterized by a high sensitivity to both Methods 5-HT and 5-methoxytryptamine (5-MeOT) and is stimulated by certain substituted benzamides such as renzapride, cisa- Preparation of tissues pride and metoclopramide. Antagonists at this site include high concentrations of tropisetron (ICS 205 930, Dumuis et Distal colon (approximately 7-8 cm from the anus) was al., 1988a), SDZ205 557 (Buchheit et al., 1991; 1992) and removed from young male Dunkin Hartley guinea-pigs (200- DAU 6285 (Dumuis et al., 1992). 300 g) and placed in Krebs solution of the following com- 5-HT4 receptor-mediated functional responses have been position (mM): NaCl 121.5, CaCl2 2.5, KH2PO4 1.2, KCI 4.7, detected in a variety of tissues including the guinea-pig ileum MgSO4 1.2, NaHCO3 25.0, glucose 5.6 (Craig & Clarke, 1990) and ascending colon (Elswood et al., Sections of colon (2-3 cm in length) were slipped over a 1991), rat oesophagus (Baxter et al., 1991), frog and human glass rod. With a scalpel blade, the longitudinal muscle layer adrenocortical cells (Idres et al., 1991; Lefebvre et al., 1992), was cut along the mesenteric border, taking care not to cut sheep pulmonary vein (Cocks & Arnold, 1992) and in both through all the muscle layers. The longitudinal muscle- pig (Villalon et al., 1990) and human heart (Kaumann et al., myenteric plexus was then stripped off with a piece of moist 1990a,b). The sensitivity of each of these preparations to cotton wool and mounted under a 0.5 g load in a 10 ml tissue 5-HT is in the sub-micromolar range, with pECm values bath. Tissues were bathed with Krebs solution at 37°C gassed ranging from 6.7 in the piglet heart (Lorrain et al., 1992) to with 5% CO2 in °2 and containing granisetron (1 ;M) and 8.6 in the guinea-pig ileum (Craig & Clarke, 1990). In the methiothepin (100 nM) to inhibit responses mediated by 5- current investigation we describe a highly sensitive model of HT3 receptors and 5-HTIl.ik. and 5-HT2 receptors respectively. Responses were recorded isotonically and displayed on a Lectromed MT8P multitrace chart recorder. Tissues were allowed to stabilize for 45 min (washing every 15 min) prior ' Author for correspondence. to starting the experiment. 1594 K.A. WARDLE & G.J. SANGER

In a few experiments, sections of guinea-pig ileum, 10 cm hydrochloride, acetylcholine perchlorate, 1,l-dimethyl-4-phe- proximal to the caecum were removed, dissected and set up nyl-piperazinium iodide (DMPP) (Sigma), methiothepin hy- in the same way as described for the distal colon. drochloride (Roche), 5-methoxytryptamine hydrochloride (5- MeOT, Aldrich), cisapride (Janssen; free base converted into Experimental protocol and concentration-response curves a hydrochloride salt in house), granisetron, renzapride, metoclopramide (SmithKline Beecham), 2-methyl 5-hydroxytryp- Tissues were first exposed to a supramaximal concentration tamine (2-Me-5-HT), SDZ 205 557 (2-methoxy-4-amino-5- of acetylcholine (ACh, 40 1M) to establish the maximum chloro-benzoic acid 2-diethylamino ethyl ester), tropisetron response evoked by each tissue. Initial responses to 5-HT (ICS 205 903), (R)- and (S)-zacopride, 5-hydroxy-indalpine were found to be small, but increased on repeated dosing. (5-OH-IP), 5-carboxyamidotryptamine (5-CT), (± )-8-hy- Hence it was deemed necessary to sensitize each tissue to droxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT, syn- 5-HT before constructing the first concentration-response thesized in house). BIMU 1 (endo-N-(8-methyl-8-azabi- curve. This was done by dosing the preparations every cyclo[3.2. 1]oct-3-yl)-2,3-dihydro-3-ethyl-2-oxo- IH-benzimi- 15 min with a supramaximal concentration of 5-HT (1I ,M) dazole-l-carboxamide hydrochloride) and BIMU 8 (endo-N- four to five times until consistent responses were produced. (8-methyl-8-azabicyclo[3.2. 1]oct-3-yl)-2,3-dihydro-(1-methyl) Following sensitization, agonist-concentration response cur- ethyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride) ves were constructed non-cumulatively, by adding increasing were a kind gift from Dr Carlo Rizzi, Boehringer Ingelheim, concentrations of agonist at 15 min time intervals. The Italy. agonist was left in contact with the tissue until a maximum All drugs were dissolved in 0.9% saline and diluted to response was obtained (usually 30 s). In each tissue, two appropriate concentrations with Krebs solution. With tropi- agonist concentration-response curves were constructed. In setron and cisapride (free base only), a minimum amount of all experiments, the first curve was to 5-HT itself. In ex- tartaric acid was used to dissolve the compounds. periments to investigate 5-HT4 receptor agonist activity, the second curve was constructed to either 5-HT (in time control experiments) or to a test agonist. In time control experi- Results ments, the two concentration-response curves to 5-HT were super-imposable (pEC50 and Emax values for first and second Preliminary experiments curves were respectively 9.2 ± 0.1, 100 ± 0 and 9.2 ± 0.2, 98 ± 4, n = 6). For antagonist experiments, the second curve In the absence of granisetron (but in the presence of meth- was constructed to 5-HT in the presence of the appropriate iothepin (100 nM)), concentration-response curves to 5-HT concentration of antagonist, added immediately after com- (10 pM- 100 liM, n = 4) in the guinea-pig ileum and distal pletion of the first curve. In all experiments, a minimum of colon were biphasic in nature (Figure 1). In the distal colon, 45 min was left between successive curves. Responses were the first phase of the curve had an estimated pEC50 of expressed as a percentage of the maximum 5-HT-evoked 9.2 ± 0.2 and plateaued at 52.5 ± 3.5% of the 5-HT-evoked contraction in the first concentration-response curve in each maximum (n =4). This compares with an estimated pEC50 tissue. value of 7.9 ± 0.04 and maximum of 31.8 ± 1.6% of the All agonist concentration-response curves were fitted using 5-HT maximum in the ileum (n = 4). The second phase of the a non-linear iterative fitting programme (Kaleidagraph, Syn- curves were selectively antagonized by granisetron (1 ,AM, ergy Software, PCS Inc., Reading, Pa) according to the results not shown), leaving a simple monophasic curve. following relationship (Parker & Waud, 1971): In the guinea-pig colon, the concentration-response curve to 5-HT was E = M[A]P/([A]p + [K]P) low concentrations of (10 pM- 10 nM) This relationship describes a curve with a maximum response M, an EC50 equal to K and a slope determined by the power p. [A] represents agonist concentration and E is the response.

Agonist potency c 0 The ability of agonists to evoke contraction of the guinea-pig (U isolated distal colon was expressed in terms of a pEC_% value C (relative to their own maxima) and in terms of a maximum 0- response, relative to 5-HT (intrinsic activity, where intrinsic 0o activity of 5-HT = 1.0). The potency relative to 5-HT was 0 calculated from experiments in which two concentration- 0 response curves were constructed in each preparation; the L._ first curve to 5-HT itself, the second curve to the test agonist. x Antagonist potency CU E pA2 values for a variety of antagonists versus 5-HT were determined according to the method of Arunlakshana & Schild (1959). Two concentration-response curves were constructed in each tissue, the first in the absence, the second in the presence of the appropriate concentration of antagonist. Apparent pKB values for SDZ 205 557 versus all other -8 -7 -5 -4 agonists were determined by the method of Furchgott (1972) using a single concentration of antagonist (100 nM). log [5-HT] M Figure 1 Concentration-response curves to 5-hydroxytryptamine (5- Drugs HT) in isolated longitudinal-muscle myenteric plexus (LMMP) strips from guinea-pig ileum (0) and colon (@). Experiments were carried The following were used: 5-hydroxytryptamine creatinine out in the presence of methiothepin (100 nM), but in the absence of sulphate (5-HT), tryptamine hydrochloride, cocaine hydro- granisetron. Each point represents the mean ± s.e.mean of 4 observa- chloride, tetrodotoxin (TTX), atropine sulphate, pargyline tions. 5-HT4 RECEPTORS IN THE GUINEA-PIG DISTAL COLON 1595

Table 1 Agonist potency in the guinea-pig distal colon longitudinal muscle myenteric plexus preparation Intrinsic Agonist Concentrations pEC50 activity n 5-HT 10 pM-10 nM 9.2 ± 0.08 1.0 38 5-MeOT 100 pM-iILM 8.7 ± 0.1 1.0 ± 0.02 9 5-CT 10 nM-100tsM 5.7 ± 0.08 1.0 ± 0.02 13 2-Me-5-HT 1 gsM-100 AM 3.8 ± 0.07 1.0 ± 0.03 4 Tryptamine 100 nM-300 IgM 4.7 ± 0.1 0.9 ± 0.1 14 Renzapride 1 nM- 10 pM 7.2 ± 0.1 1.0 ± 0.02 13 (R)-zacopride 10 nM-303M 5.8 ± 0.1 1.0 ± 0.05 12 (S)-zacopride 10 nM-30j1M 6.3 ± 0.08 1.0 ± 0.02 12 Cisapride base 10 nM-33M 6.9 ± 0.2 0.4 ± 0.1 4 Cisapride HCI 10 nM-3 pM 6.9 ± 0.1 0.9 ± 0.06 4 Metoclopramide 10 nM-30 jiM 5.7 ± 0.1 0.9 ± 0.05 11 BIMU 1 10 nM-3 jiM 6.7 ± 0.1 0.8 ± 0.07 4 BIMU 8 10 nM-3 pM 6.9 ± 0.08 0.5 ± 0.08 8 8-OH-DPAT OlnM-10pM Inactive NA 4 5-OH-indalpine 10 nM-1OpiM Inactive NA 4 Each value represents the mean ± s.e.mean of a number (n) of preparations. unaffected by granisetron (1 giM). Thus, the pEC_o values were identical in the absence and presence of granisetron 120 (9.2 ± 0.2 (n = 4) and 9.2 ± 0.08 (n = 38) respectively). Con- sequently, granisetron was added routinely in all subsequent experiments to block the second phase of the 5-HT curve. Under these conditions, the contractions evoked by 5-HT 0 (10 pM- 10 nM) were prevented by atropine (1 jiM, n = 6) and TTX (300 nM, n = 4), suggesting that responses were predominantly due to stimulation of cholinergic neurones C.) (results not shown). o 0 Responses to 5-HT (10 pM-10 nM) were not significantly affected by cocaine (300 nM-30 jM). The respective pEC_0 60- and E.. values for 5-HT were 9.3 ± 0.1, 100 ± 0 (control), 9.3 ± 0.06, 100 ± 2 (300 nM cocaine), 9.2 ± 0.08, 97 ± 3 (3 gM V-O A 0 S cocaine) and 9.3 ± 0.07, 102 ± 2 (30 cocaine); n = 4 each. E lAM m 40 Concentration-response curves to both 5-HT (1O pM-10 nM) E -1 -10 -8 - 4 - and 5-methoxytryptamine (5-MeOT, 100 pM- 100 nM) were unaffected by a 30 min pre-incubation with the irreversible monoamine oxidase inhibitor pargyline (100IM). The pECio 20- and Em.1, values for 5-HT in the absence and presence of pargyline were 9.3±0.1, 100±0 and 9.4±0.1, 98±3 respectively. The equivalent values for 5-MeOT were 8.1 ± 0.08, -1 97 ± 2 and 8.2 + 0.05, 99 + 4; n = 4 each. Thus, in the -12 -10 -8 -6 _-4 -2 guinea-pig distal colon, neither neuronal uptake nor mono- log [Agonist] M amine oxidase metabolism were believed to affect significantly the responses to these agonists. Hence, it was Figure 2 Concentration-response curves to 5-hydroxytryptamine (5- not deemed necessary to include these agents in subsequent HT, 0), 5-methoxytryptamine (0), 5-carboxamidotryptamine (0), experiments. tryptamine (-) and 2-methyl-5-hydroxytryptamine (A) in guinea-pig distal colon LMMP. Experiments were carried out in the presence of methiothepin (100 nM) and granisetron (1 pM). Each point represents Agonist profile the mean ± s.e.mean of between 4 and 38 observations. For n values In the guinea-pig distal colon, 5-HT evoked concentration- for each compound, see Table 1. dependent contractions with a mean pEC,0 of 9.2 ± 0.08. Responses were slow in onset, reaching a plateau after approximately 30 s. Preliminary experiments with animals of 6.9 and 5.7 respectively (Figure 3, Table 1). When cisapride a different age demonstrated that the sensitivity of the was used in its original form (free base), it was a partial preparations to 5-HT was highly dependent on the size of the agonist (pEC50 of 6.9 and an intrinsic activity of 0.4). guinea-pigs used. Thus, with very small animals (150-200 g) The benzimadazolone derivatives, BIMU 1 and BIMU 8 the pEC50 could be as low as 10.1, although dissection of the were also examined. Both were partial agonists relative to tissue often proved difficult due to the thinness of the 5-HT with intrinsic activities of 0.5 and 0.8 respectively. The LMMP. With large guinea-pigs (450-550 g), the pECm of pEC50 for BIMU 1 was estimated to be 6.9, and for BIMU 8, 5-HT dropped to 7.8. For this reason, the size of the guinea- 6.7. (Figure 4, Table 1). pigs was tightly controlled in all further experiments (200- The 5-HTIA receptor agonist, 8-OH-DPAT and the puta- 300 g). tive 5-HTIp receptor agonist 5-OH-indalpine were found to Four tryptamine analogues namely, 5-MeOT, 5-CT, tryp- be inactive. The results for all compounds tested are sum- tamine itself and 2-Me-5-HT acted as full agonists in this marized in Table 1. model. Their pEC_% values were 8.7, 5.7, 4.7 and 3.8 respectively. Results are shown in full in Figure 2 and Table 1. Antagonist profile Five substituted benzamides were examined. Renzapride, (S)-zacopride (R)-zacopride, cisapride (HCI) and metoclo- Tropisetron (ICS 205-930, 1-10 jiM, n = 7) produced a paral- pramide were each full agonists with pEC50s of 7.2, 6.3, 5.8, lel rightward shift of the 5-HT concentration-response curve, 1596 K.A. WARDLE & G.J. SANGER

120 120

c_100 0 0 0 0 L._o 0 a) 0 0 0c C.)

60- Lg) I E :3 E E 40-1 -10 -8 - 4 - Ex E E) X0 20-

0 -12 -10 -8 -6 -4 -2 -11 -10 -9 -8 -7 -6 -5 -4 log [5-HT] M log [5-HT] M Figure 3 Concentration-response curves to 5-hydroxytryptamine (5- Figure 5 Concentration-response curves to 5-hydroxytryptamine (5- HT, 0), renzapride (a), (R)-zacopride (0), (S)-zacopride (U), HT) in the absence (0) and presence of 100 nM (M), 300 nM (0) cisapride (HCl, A) and metoclopramide (A) in guinea-pig distal and 1 gtM (M) SDZ 205 557 in guinea-pig distal colon LMMP. colon LMMP. Experiments were carried out in the presence of Experiments were carried out in the presence of methiothepin (100 methiothepin (100 nM) and granisetron (1 gM). Each point represents nM) and granisetron (1 JuM). Each point represents the mean ± s.e. the mean ± s.e.mean of between 4 and 38 observations. For n values mean of 6 observations. for each compound, see Table 1.

pA2 value of 7.8 ± 0.1. At the concentrations tested, SDZ 205 557 had no effect on DMPP (100nM-100 iM)-evoked contractions in the same preparation. * 100- 0 Estimates of the pKB value for SDZ 205 557 against several other 5-HT4 receptor agonists were calculated to be 7.3 ± 0.2 C (n =4) for 5-MeOT, 7.5 ± 0.09 (n =4) for renzapride and 0o 80- 7.9 ± 0.2 (n = 4) for 5-CT. ~0 60 Discussion The present study clearly demonstrates that, in the presence E4o of 5-HT,, 5-HT2 and 5-HT3 receptor antagonists, 5-HT E evokes cholinergically-mediated contractions of the guinea- pig distal colon LMMP which are mediated by the 5-HT4 E 20- receptor. A similar preparation, dissected from the ileum, has long been used to study the effects of 5-HT on the 'atypical' 5-HT 0 receptor in the guinea-pig gastrointestinal tract (Buchheit et -12 -11 -10 -9 -8 -7 -6 -5 al., 1985; Sanger, 1987). The receptor mediating this response log [Agonist] M is believed to be of the 5-HT4 subtype (Eglen et al., 1990). Although clearly identifiable, the response in the guinea-pig Figure 4 Concentration-response curve to 5-hydroxytryptamine (5- ileum is very small, making accurate analysis of its pharma- HT, 0), BIMU 1 (@) and BIMU 8 (0), in guinea-pig distal colon cology difficult. The present paper identifies a similar res- LMMP. Experiments were carried out in the presence of methio- ponse in the guinea-pig distal colon. Relative to the 5-HT3 thepin (100 nM) and granisetron (1 ytM). Each point represents the mean ± s.e.mean of between 4 and 38 observations. For n values for receptor-mediated contraction in either the ileum or colon, each compound, see Table 1. both responses are slow in onset and are well maintained, suggesting that they are mediated by a similar mechanism. The response in the colon, however, is significantly larger than that in the ileum, making it a better preparation in yielding an apparent pA2 of 6.4 ± 0.1 and a slope of the which to investigate the pharmacology of the 5-HT4 receptor. Schild plot of 1.3 ± 0.1. This value was significantly (P < In the rat oesophagus (Baxter et al., 1991) and human 0.05) greater than unity, but may be attributed to the fact atrial (Kaumann et al., 1990a,b) models of the 5-HT4 recep- that, at 10IOM, tropisetron reduced the maximum DMPP- tor, cocaine caused a concentration-dependent shift to the evoked contraction (by 29 ± 5%; P <0.05) in the same pre- left of the 5-HT concentration-effect curves. Since cocaine paration. has been shown to block neuronal uptake mechanisms (Ver- SDZ 205 557 (100 nM- 1 4M, n = 6) produced a parallel beuren et al., 1983), such an observation is consistent with rightward shift of the 5-HT concentration-response curve the idea that exogenously added 5-HT is being 'lost' through (Figure 5). The slope of the Schild plot (1.06 ± 0.09) was not uptake systems. However, since cocaine, at a concentration significantly different from unity and yielded an estimated ranging from 300 nM to 30 gM, had no significant effect on 5-HT4 RECEPTORS IN THE GUINEA-PIG DISTAL COLON 1597 the concentration-effect curves to 5-HT in the guinea-pig identification of 5-HT4 receptor-mediated responses (Dumuis distal colon, it would appear that neuronal uptake mechan- et al., 1989; Craig & Clarke, 1990; Baxter et al., 1991). isms play no significant role in this preparation. Whereas in the colliculi neurones, renzapride, cisapride and In certain isolated gastrointestinal smooth muscle prepara- zacopride have a greater intrinsic activity than 5-HT itself, in tions (e.g. rat stomach), contractions evoked by tryptamine the guinea-pig ileum and rat oesophagus their intrinsic act- and 5-MeOT were potentiated by inhibitors of the enzyme ivities range from around 0.8 to 1.0. Metoclopramide, on the monoamine oxidase (MAO), whereas those produced by 5- other hand, in all models tested to date, is a partial agonist HT were unaffected (Vane, 1959; Handschumacher & Vane, (Dumuis et al., 1989; Baxter et al., 1991; Elswood et al., 1967). Such an observation is consistent with the idea that 1991). In the guinea-pig distal colon, all substituted ben- the lipophilic tryptamine and 5-MeOT molecules entered the zamides tested (including metoclopramide) were full agonists cell and were metabolized, whereas the polar 5-HT molecule relative to 5-HT. The high intrinsic activity of metoclo- was excluded from the cell and hence from metabolism by pramide in the present study may be attributed to either the the cytoplasmic MAO enzyme. In view of this observation, effective coupling mechanism in this preparation or to a high the irreversible monoamine oxidase inhibitor, pargyline, was receptor density. The rank order of potency of agonists for investigated in the guinea-pig distal colon model. At a con- this group of compounds was: 5-HTi> renzapride> centration of 100 LM, pargyline had no effect on the concen- cisapride > (S)-zacopride > (R)-zacopride > metoclopramide. tration-response curves to either 5-HT or the more lipophilic Such a rank order of potency of agonists is consistent with analogue, 5-MeOT, suggesting that a MAO barrier does not that reported in the literature for 5-HT4 receptor activation. preclude the access of 5-HT or 5-MeOT to the 5-HT4 recep- In particular, it is significant that the 5-HT receptor in the tors in this model. Since this is a stripped LMMP prepara- guinea-pig distal colon discriminates between (R)- and (S)- tion, however, these results do not preclude the existence of zacopride, with the (S)-isomer being about 3 times more such a barrier in another layer of the guinea-pig gastrointes- potent than the (R)-form. This finding is consistent with that tinal tract. Indeed, histochemical studies in guinea-pig intes- in the guinea-pig ileum (Eglen et al., 1990) and rat oesoph- tine have previously demonstrated strong MAO staining in agus (Baxter et al., 1991). the mucosa, but weak or negative activity in the muscle The final set of agonists investigated in the guinea-pig layers (Penttila, 1968). Thus, the lack of mucosa in the distal colon were the benzimidazolone derivatives (Nicola et present preparation probably explains the lack of activity of al., 1990; Dumuis et al., 1991). BIMU 1 and BIMU 8 had pargyline. very similar potencies and were both partial agonists relative Contractions evoked by 5-HT in the guinea-pig distal to 5-HT. At high concentrations (> 10 tLM), BIMU 1 and colon were prevented by both atropine and TTX. Such an BIMU 8 revealed bell-shaped concentration-response curves. observation suggests that, as is the case in the guinea-pig The partial agonism seen with both of these compounds ileum (Buchheit et al., 1985; Sanger & Nelson, 1989), the suggests that their high potency stems from their high 5-HT4 receptors in the colon are located on cholinergic affinity, rather than from a high intrinsic activity at the neurones within the myenteric plexus. Thus, 5-HT4 receptor- 5-HT4 receptor. BIMU 1 and BIMU 8 have also been obser- mediated contractions are evoked indirectly via acetylcholine ved to be highly potent agonists in the mouse embryo col- release. liculi neurones (Dumuis et al., 1991), rat oesophagus (Baxter In comparison with other published 5-HT4 receptor models & Clarke, 1992), guinea-pig ileum (Rizzi et al., 1992) and (see Introduction for references), the guinea-pig distal colon guinea-pig proximal colon (Grossman et al., 1993). In rat is a very sensitive model to 5-HT. Thus, in the distal colon, oesophagus, both were partial agonists, whereas in the col- 5-HT evokes contractions with a pEC,; of 9.2. This compares liculi neurones and guinea-pig ileum, BIMU 8 displayed a with published values of 7.4 (human atria), 8.2 (rat oeso- higher intrinsic activity than 5-HT and 5-MeOT respectively. phagus), 7.5 (guinea-pig ascending colon), 7.5 (human In the brain, at least, this high intrinsic activity observed with adrenocortical tissue) and 8.4 (sheep pulmonary vein) in BIMU 8 may be due to an additional action in the CNS other isolated preparations. The exact mechanism underlying assay system (see Baxter & Clarke, 1992). Bearing in mind the high potency of 5-HT in the guinea-pig distal colon can the apparent effective coupling or high receptor density in the only be speculated upon. It may be attributed to either a guinea-pig distal colon model, it would have been expected high density of 5-HT4 receptors, or to good amplification that BIMU 1 and BIMU 8 would have behaved as full capabilities of the tissue. Since the response to 5-HT in the agonists. The fact that this was not the case is difficult to guinea-pig distal colon is mediated indirectly via ACh release, explain, but may again be due to an additional action of a multi-step process would lend itself to a large degree of these compounds at high concentrations (e.g. anticholinergic amplification. This contrasts with the less well coupled rat activity), opposing the 5-HT4 receptor-mediated contraction. oesophagus model of the 5-HT4 receptor, where responses are Comparing agonist potencies and intrinsic activities with mediated directly via activation of a receptor located on the those in the guinea-pig proximal colon (Elswood et al., 1991), smooth muscle. it is clear that differences are observed. While this may reflect The rank order of potency of agonists has long been used regional differences in the receptor density or coupling as a means of characterizing receptors. For tryptamine mechanisms, an alternative explanation may be .the size of analogues, the rank order of potency in the guinea-pig distal the animals used. In the present study it was found that the colon was; 5-HTi> 5-MeOT> > 5-CT> tryptamine>2-Me- sensitivity of the preparation to 5-HT was highly dependent 5-HT. While the rank order is the same as that reported in on the age of the guinea-pigs. With very young animals, both the guinea-pig ileum (Craig & Clarke, 1990) and the rat pEC50 values could be as low as 10.1, compared with 7.8 in oesophagus (Baxter et al., 1991), subtle differences do exist very old animals. Similar findings have been reported by when results are compared with those from the other models. Buchheit et al. (1985) in experiments involving longitudinal For example, the potency of 5-MeOT, relative to 5-HT muscle strips of guinea-pig ileum. In the Elswood study, appears to vary from one model to another. In the rat larger (300-400 g) animals were used, perhaps reflecting the oesophagus, 5-HT and 5-MeOT were approximately equipo- lower sensitivity of their preparations to 5-HT and other tent (Baxter et al., 1991). In the present study 5-HT was 3.4 agonists. times more potent than 5-MeOT. In the guinea-pig ileum From the agonist studies it can be concluded that the (Craig & Clarke, 1990) and guinea-pig ascending colon (Els- receptor mediating 5-Hi-evoked contractions in the guinea- wood et al., 1991) however, the potency ratios went up to 8 pig isolated distal colon is not significantly different from and 25 respectively. The reason for these discrepancies is that ip other 5-HT4 receptor models. Further evidence for the unknown but may reflect varying attempts to block factors existence of the 5-HT4 receptor in the guinea-pig distal colon such as uptake and metabolism of the tryptamine analogues. stems from studies with tropisetron and SDZ 205 557. In the Substituted benzamides have been used as tools in the present study tropisetron produced a rightward displacement 1598 K.A. WARDLE & G.J. SANGER of the 5-HT concentration-effect curve, yielding an apparent independent, ranging from 7.5 to 7.9. Although these values pA2 of 6.4 and a slope of the Schild plot significantly greater are slightly higher than those quoted in the literature (Buch- than unity. This value compares well with that reported in heit et al., 1991; 1992; Lorrain et al., 1992) they are consis- the literature for the colliculi neurones (6.0-6.3; Dumuis et tent with activity at the 5-HT4 receptor. al., 1988a), rat oesophagus (6.7; Baxter et al., 1991) and In summary, the present results suggest that the guinea-pig guinea-pig ascending colon (6.4; Elswood et al., 1991). distal colon contains neuronal receptors with a pharmacology SDZ 205 557 has been identified as a selective competitive similar to that of the 5-HT4 receptor. Furthermore, this antagonist of the 5-HT4 receptor-mediated contractions in appears to be a highly sensitive 5-HT4 receptor model and the guinea-pig ileum, with a 30 fold lower affinity for the may be a useful tool in the investigation of 5-HT4 receptor- 5-HT3 receptor (Buchheit et al., 1991; 1992). In the present mediated events. study, SDZ 205 557 acted as a potent competitive antagonist of 5-HT-evoked contractions in the guinea-pig distal colon, yielding a pA2 of 7.8. Furthermore, when examined against other 5-HT4 receptor agonists (both tryptamine analogues We wish to thank Dr Carol Rizzi for his generous gift of BIMU I and benzamides), the pKB values were found to be agonist- and BIMU 8.

References

ARUNLAKSHANA, 0. & SCHILD, H.O. (1959). Some quantitative uses EGLEN, R.M., SWANK, S.R., WALSH, L.K.M. & WHITING, R.L. of drug antagonism. Br. J. Pharmacol. Chemother., 14, 48-58. (1990). Characterisation of 5-HT3 and the 'atypical' 5-HT recep- BAXTER, G.S. & CLARKE, D.E. (1992). Benzimidazolone derivatives tors mediating guinea-pig ileal contractions in vitro. Br. J. Phar- act as 5-HT4 receptor ligands in rat oesophagus. Eur. J. Phar- macol., 101, 513-520. macol., 212, 225-229. ELSWOOD, C.J., BUNCE, K.T. & HUMPHREY, P.P.A. (1991). BAXTER, G.S., CRAIG, D.A. & CLARKE, D.E. (1991). 5-Hydroxy- Identification of putative 5-HT4 receptors in guinea-pig ascending tryptamine4 receptors mediate relaxation of the rat oesophagus colon. Eur. J. Pharmacol., 196, 149-155. tunica muscularis mucosae. Naunyn-Schmied. Arch. Pharmacol., FURCHGOTT, R.F. (1972). The classification df adrenoceptors (ad- 343, 439-446. renergic receptors). An evaluation from the standpoint of recep- BOCKAERT, J., SEBBEN, M. & DUMUIS, A. (1990). Pharmacological tor theory. In Handbook of Experimental Pharmacology. Vol. 33, characterisation of 5-hydroxytryptamine4 (5-HT4) receptors posi- pp. 283-335. Berlin, Heidelberg, New York: Springer. tively coupled to adenylate cyclase in adult guinea-pig hippocam- GROSSMAN, C.J., KILPATRICK, G.J. & BUNCE, K.T. (1993). Develop- pal membranes: effect of substituted benzamide derivatives. Mol. ment of a radioligand binding assay for 5-HT4 receptors in Pharmacol., 37, 408-411. guinea-pig and rat brain. Br. J. Pharmacol., 109, 618-624. BUCHHEIT, K.H., ENGEL, G., MUTSCHLER, E. & RICHARDSON, B. HANDSCHUMACHER, R.E. & VANE, J.R. (1967). The relationship (1985). Study of the contractile effects of 5-hydroxytryptamine between the penetration of tryptamine and 5-hydroxytryptamine (5-HT) in the isolated longitudinal muscle strip from guinea-pig into smooth muscle and the associated contractions. Br. J. Phar- ileum. Evidence for two distinct release mechanisms. Naunyn- macol., 29, 105-118. Schmied. Arch. Pharmacol., 329, 36-41. IDIRES, S., DELARUE, C., LEFEBVRE, H. & VAUDRY, H. (1991). BUCHHEIT, K.-H., GAMSE, R. & PFANNKUCHE, H.-J. (1991). SDZ Benzamide derivatives provide evidence for the involvement of a 205 557, a selective antagonist at 5-HT4 receptors in the isolated 5-HT4 receptor type in the mechanism of action of serotonin in guinea-pig ileum. Eur. J. Pharmacol., 200, 373-374. frog adrenocortical cells. Mol. Brain Res., 10, 251-258. BUCHHEIT, K.-H., GAMSE, R. & PFANNKUCHE, H.-J. (1992). SDZ KAUMANN, A.J., SANDERS, L., BROWN, A.M., MURRAY, K.J. & 205 557, a selective, surmountable antagonist for 5-HT4 receptors BROWN, M.J. (1990a). Human atrial 5-HT receptors: similarity to in the isolated guinea-pig ileum. Naunyn-Schmied. Arch. Phar- rodent neuronal 5-HT4 receptors. Br. J. Pharmacol., 100, 319P. macol., 345, 387-393. KAUMANN, A.J., SANDERS, L., BROWN, A.M., MURRAY, K.J. & COCKS, T.M. & ARNOLD, P.J. (1992). 5-Hydroxytryptamine (5-HT) BROWN, M.J. (1990b). A 5-hydroxytryptamine receptor in human mediates potent relaxation in the sheep isolated pulmonary vein atrium. Br. J. Pharmacol., 100, 879-885. via activation of 5-HT4 receptors. Br. J. Pharmacol., 107, 591- LEFEBVRE, H., CONTESSE, V., DELARUE, C., FFEUILLOLEY, M., 596. HERY, F., GRISE, P., RAYNAUD, G., VERHOFSTAD, A.A.J., CRAIG, D.A. & CLARKE, D.E. (1990). Pharmacological characterisa- WOLF, L.M. & VAUDRY, H. (1992). Serotonin-induced stimulation of a neuronal receptor for 5-hydroxytryptamine in guinea- tion of cortisol secretion from human adrenocortical tissue is pig ileum with properties similar to the 5-hydroxytryptamine4 mediated through activation of a serotonin4 receptor subtype. receptor. J. Pharmacol. Exp. Ther., 252, 1378-1386. Neuroscience, 47, 999-1007. DUMUIS, A., BOUHELAL, R., SEBBEN, M. & BOCKAERT, J. (1988a). LORRAIN, J., GROSSET, A. & O'CONNOR, S.E. (1992). 5-HT4 recep- A 5-HT receptor in the central nervous system, positively coupled tors, present in piglet atria and sensitive to SDZ 205 557, are with adenylate cyclase, is antagonised by ICS205-930. Eur. J. absent in papillary muscle. Eur. J. Pharmacol., 229, 105-108. Pharmacol., 146, 187-188. NICOLA, M., TURCONI, M., SCHIAVONE, A., RIZZI, C.A., CESANA, DUMUIS, A., BOUHELAL, R., SEBBEN, M., CROY, R. & BOCKAERT, R., MICHELETrI, R., MONFERINI, E. & LADINSKY, H. (1990). A J. (1988b). A nonclassical 5-hydroxytryptamine receptor posi- new class of non benzamide 5-HT3 antagonists and 5-HT4 tively coupled with adenylate cyclase in the central nervous sys- agonists. XIth International Symposium on Medicinal Chemistry, tem. Mol. Pharmacol., 34, 880-887. Jerusalem, Israet, Abstract P4. DUMUIS, A., GOZLAN, H., SEBBEN, M., ANSANAY, H., RIZZI, C.A., PARKER, R.B. & WAUD, D.R. (1971). Pharmacological estimation of TURCONI, M., MONFERINI, E., GIRALDO, E., SCHIANTARELLI, drug receptor dissociation constants. Statistical evaluation: I. P., LADINSKY, H. & BOCKAERT, J. (1992). Characterisation of a Agonists. J. Pharmacol. Exp. Ther., 177, 1-12. novel 5-HT4 receptor antagonist of the azabicycloalkyl ben- PENTTILA, A. (1968). Distribution and intensity of monoamine zimidazolone class: DAU6285. Naunyn-Schmied Arch. Pharma- oxidase activity in the mammalian duodenum. Acta Physiol. col., 345, 264-269. Scand., 73, 121-127. DUMUIS, A., SEBBEN, M. & BOCKAERT, J. (1989). The gastrointes- RIZZI, C.A., COCCINI, T., ONORI, L., MANZO, L. & TONINI, M. tinal prokinetic benzamide derivatives are agonists at the non- (1992). Benzimadazolone derivatives: a new class of 5-hydroxy- classical 5-HT receptor (5-HT4) positively coupled to adenylate tryptamine4 receptor agonists with prokinetic and acetylcholine cyclase in neurons. Naunyn-Schmied. Arch. Pharmacol., 340, 403- releasing properties in the guinea-pig ileum. J. Pharmacol. Exp. 410. Ther., 261, 412-419. DUMUIS, A., SEBBEN, M., MONFERINI, E., NICOLA, M., TURCONI, SANGER, G.J. (1987). Increased gut cholinergic activity and antag- M., LADINSKY, H. & BOCKAERT, J. (1991). Azabicycloalkyl ben- onism of 5-hydroxytryptamine M-receptors by BRL 24924: po- zimidazolone derivatives as a novel class of potent agonists at the tential clinical importance of BRL 24924. Br. J. Pharmacol., 91, 5-HT4 receptor positively coupled to adenylate cyclase in brain. 77-87. Naunyn-Schmied. Arch. Pharmacol., 343, 245-249. 5-HT4 RECEPTORS IN THE GUINEA-PIG DISTAL COLON 1599

SANGER, G.J. & NELSON, D.R. (1989). Selective and functional 5- VILLALON, C.M., DEN BOER, M.O., HEILIGERS, J.P.C. & SAXENA, hydroxytryptamine3 receptor antagonism by BRL 43694 (granise- P.R. (1990). Mediation of 5-hydroxytryptamine-induced tachycar- tron). Eur. J. Pharmacol., 159, 113-124. dia in the pig by the putative 5-HT4 receptor. Br. J. Pharmacol., VANE, J.R. (1959). The relative activities of some tryptamine 100, 665-667. analogues on the isolated rat stomach strip preparation. Br. J. WARDLE, K.A. & SANGER, G.J. (1992). Guinea-pig distal colon; a Pharmacol. Chemother., 14, 87-98. sensitive model of the putative 5-HT4 receptor. Br. J. Pharmacol., VERBEUREN, T.J., JORDAENS, F.H. & HERMAN, A.G. (1983). Ac- 105, 279P. cumulation and release of [3H]-5-hydroxytryptamine in saphenous veins and cerebral arteries of the dog. J. Pharmacol. Exp. Ther., (Received May 26, 1993 226, 579-588. Revised August 23, 1993 Accepted August 24, 1993)